METHOD FOR CONTROLLING VIRAL INFECTIONS THROUGH ADOPTIVE TRANSFER OF A CELL PRODUCT COMPRISING AN EXPANDED AND ENRICHED POPULATION OF SUPERACTIVATED CYTOKINE KILLER CELLS
20220401477 · 2022-12-22
Assignee
Inventors
- Helen Hao (Piscataway, NJ, US)
- Lilit Garibyan (Newton, MA, US)
- Beverly W. Lubit (Kinnelon, NJ, US)
- Sean O'Connell (Budd Lake, NJ, US)
Cpc classification
A61K35/17
HUMAN NECESSITIES
A61K2035/124
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
A61K38/14
HUMAN NECESSITIES
A61K38/14
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
International classification
Abstract
The invention of the present disclosure provides a method for treating a viral infection in a recipient subject suffering from or at risk of a viral infection including administering to the recipient subject a pharmaceutical composition comprising a therapeutic amount of superactivated cytokine killer T cells (SCKTCs) and a pharmaceutically acceptable carrier, and mobilizing an immune response of the recipient subject to the viral pathogen. When tested in vitro, the SCKTCs are characterized by a predominant production of T.sub.H1 dominant cytokines including IFN-γ; an IFN-γ:IL-4 ratio of at least 500:1; and at least 50% killing of target A549 cells at an effector:target ratio of 20:1. The present disclosure further provides a method of preparing a pharmaceutical composition comprising an enriched population of superactivated cytokine killer T cells (SCKTCs) wherein pulsing steps with monocyte-derived dendritic cells (DCs) loaded with alpha-GalCer achieve at least an 80% pure population of SCKTCs without positive or negative cell separation methods.
Claims
1. A method for treating a viral infection in a recipient subject suffering from or at risk of the viral infection comprising a. administering to the recipient subject a pharmaceutical composition comprising a cell product containing a therapeutic amount of superactivated cytokine killer T cells (SCKTCs) and a pharmaceutically acceptable carrier, and b. mobilizing an immune response of the recipient subject to the viral pathogen; wherein the therapeutic amount is at least 0.2×10.sup.9 SCKTCs per 30 day treatment cycle; and wherein when tested in vitro, the SCKTCs predominantly produce T.sub.H1 dominant cytokines including IFN-γ; or an IFN-γ:IL-4 ratio of the SCKTC population when tested in vitro is at least 500:1 with IL-12 stimulation; and at an effector:target ratio of 20:1, cytotoxicity against A549 target cells is >50%.
2. The method according to claim 1, wherein the immune response of the recipient subject comprises stimulating activation of one or more immune cell population of the recipient subject.
3. The method according to claim 2, wherein the immune cell population of the recipient subject comprises one or more of a dendritic cell population; a CD8+ T cell population; an NK cell population; or an MHC-restricted T cell population.
4. The method according to claim 3, wherein the MHC-restricted T cell population comprises an invariant NKT population.
5. The method according to claim 3, wherein the therapeutic amount stimulates an effector function of the immune cells of the recipient subject.
6. The method according to claim 5, wherein the effector function includes one or more of cytokine secretion, cytotoxicity, or antibody-mediated clearance of the pathogen.
7. The method according to claim 1, wherein the viral infection is characterized by virus-infected cells.
8. The method according to claim 7, wherein the therapeutic amount destroys virus-infected cells through direct lysis, by effecting destruction of the infected cells indirectly or both.
9. The method according to claim 8, wherein destruction of the infected cells indirectly comprises mobilizing attracting cell cytotoxicity agents through secretion of cytokines.
10. The method according to claim 1, wherein the virus infection is an infection with a respiratory virus.
11. The method according to claim 10, wherein the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, a West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a coxsackie virus, a norovirus, or an encephalomyocarditis virus (EMCV).
12. The method according to claim 11, wherein the coronavirus is SARS-CoV-1, SARS-CoV-2 or MERS.
13. The method according to claim 1, wherein a. the therapeutic amount reduces risk of the virus infection; or b. the therapeutic amount reduces signs, symptoms, or both signs and symptoms of the viral infection; or c. the therapeutic amount reduces extent of the viral infection where symptoms are not yet clinically recognized; or d. the therapeutic amount reduces worsening or progression of the viral infection; or e. the therapeutic amount reduces severity of the viral infection, compared to an untreated subject; or f. the therapeutic amount improves progression-free survival; or g. the therapeutic amount improves overall survival.
14. The method according to claim 1, wherein a. the superactivated cytokine killer T cells (SCKTCs) are derived from blood; or b. The SCKTCs are derived from a leukapheresis; or c. The SCKTCs are derived from hematopoietic stem cells; or d. The SCKTCs are derived from hematopoietic stem cells derived from adult bone marrow, umbilical cord, umbilical cord blood, placental tissue or fetal liver.
15. The method according to claim 1, wherein the pharmaceutical composition further comprises an enriched differentiated and expanded population of NK cells.
16. The method according to claim 1, a. wherein the population of SCKTCs is autologous to the recipient subject; or b. wherein the population of SCKTCs is allogeneic to the recipient subject.
17. The method according to claim 15, wherein the NK cells are derived from CD34+ hematopoietic stem cells of a donor.
18. The method according to claim 15, wherein the population of NK cells is depleted of CD3+ T cells, CD19 B cells or both.
19. The method according to claim 17, (a) wherein the population of NK cells of the donor is autologous to the recipient subject. or (b) wherein the population of NK cells of the donor is allogeneic to the recipient subject.
20. The method according to claim 1, further comprising administering the pharmaceutical composition comprising the cell product containing the population of SCKTCs with a supportive therapy or an additional compatible therapeutic agent.
21. The method according to claim 20, wherein the supportive therapy reduces viral load.
22. The method according to claim 20 wherein the additional compatible therapeutic agent is one or more of an immunomodulatory agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
23. The method according to claim 22 wherein a. the immunomodulatory agent comprises one or more of methotrexate; a glucocorticoid, cyclosporine, tacrolimus and sirolimus; a recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNFα inhibitor/antagonist; an IL-1β inhibitor; a chimeric IL-1Ra; an IL-6 inhibitor; an IL-12/IL-23 inhibitor selected from ustekinumab, briakinumab; an IL-23 inhibitor selected from guselkumab, tildrakizumab; a compound that targets TLR4 signaling; a p38 MAPK inhibitor, a Janus kinase signaling inhibitor; a compound that targets cell adhesion molecules to reduce leukocyte recruitment; a checkpoint inhibitor, or a recombinant anti-inflammatory cytokine; or b. the anti-infective agent is amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem; or c. the anti-viral agent is selected from acyclovir, gancidovir, foscamet; ribavirin; amantadine, azidodeoxythymidine/zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine; or d. the anti-fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
24. The method according to claim 22, wherein the immunomodulatory agent comprises recombinant IL-37, recombinant CD24, or both.
25. The method according to claim 23, the anti-viral agent is an agent that inhibits viral entry and decreases viral load.
26. The method according to claim 23, wherein the checkpoint inhibitor is YERVOY™ (Ipilimumab; CTLA-4 antagonist), OPDIVO™ (Nivolumab; PD-1 antagonist) or KEYTRUDA™ (Pembrolizumab; PD-1 antagonist).
27. A method for preparing a pharmaceutical composition comprising an enriched population of superactivated cytokine killer T cells (SCKTCs) comprising, in order (a) isolating a population of mononuclear cells (MCs) comprising a population of cytokine killer T cells (CKTCs); (b) transporting the preparation of (a) to a processing facility under sterile conditions; (c) on day 0, placing the population of MCs in a suspension culture system comprising a serum-free culture medium; (d) on day 6, contacting the culture system of step (c) with the serum-free culture medium containing IL-2 and IL-7, wherein the contacting stimulates CKTC activation; (e) on day 7, pulsing the CKTCs of step (d) with an enriched population of CD1d-expressing antigen presenting cells (APCs) derived from the MCs in (a) loaded with α-GalCer; (f) replenishing the serum-free culture medium every 1-3 days from day 7 to day 14; (g) on day 14, adding CD1d expressing APCs loaded with α-GalCer; (h) replenishing the serum-free culture medium of the cells every 1-3 days; (i) On day 14+7 days, replenishing the culture medium of the culture and pulsing with CD1d expressing APCs loaded with α-GalCer; (j) On day 14+14 days, a replenishing the culture medium of the culture and pulsing with CD1d-expressing APCs loaded with α-GalCer; (k) On day 14+21 days, replenishing the culture medium of the culture and adding IL-12; (l) On Day 14+22 harvesting the amplified enriched superactivated population of SCKTCs from the culture system to form a SCKTC cell product; and (m) filling and finishing the SCKTC cell product into a container; and (n) optionally cryopreserving the SCKTC cell product in the vapor phase of a liquid nitrogen freezer in a serum-free cryo freezing medium.
28. The method according to claim 27, wherein the population of MCs comprising the population of CKTCs: (a) is derived from hematopoietic stem cells derived from adult bone marrow, umbilical cord, umbilical cord blood, placental tissue, or fetal liver; or (b) is derived from leukapheresis of a donor subject allogeneic to a recipient subject; or (c) is derived from leukapheresis of a donor subject autologous to a recipient subject.
29. The method according to claim 27, wherein in step (a) frequency of the population of CKTCs from the donor represents <0.5% of the total MNC population.
30. The method according to claim 27, wherein the population of MCs comprises subpopulations of T lymphocytes, NK cells, B lymphocytes, and monocytes.
31. The method according to claim 30, wherein the subpopulation of T lymphocytes comprises NKT cells, CD4+ T cells, and CD8+ T cells.
32. The method according to claim 27, wherein a) the CD1d− expressing antigen presenting cells (APCs) derived from the MCs comprise CD14+ monocytes; or b) the CD1d− expressing antigen presenting cells (APCs) derived from the MCs comprise an irradiated population of PBMCs.
33. The method according to claim 27, wherein the CD1d-expressing population of APCs loaded with alpha-GalCer is a population of monocyte-derived dendritic cells.
34. The method according to claim 33, wherein at least 30% of the monocyte derived population of DCs constitutively expresses CD1d.
35. The method according to claim 27, wherein the pulsing steps with DCs loaded with alpha-GalCer achieve at least an 80% pure population of SCKTCs without positive or negative cell separation methods.
36. The method according to claim 33, wherein the population of dendritic cells loaded with αGalCer is prepared by a method comprising (i) isolating a population of mononuclear cells (MCs) comprising CD14+ monocytes; (ii) inducing differentiation of the CD14+ monocytes into dendritic cells by culturing the population of CD14+ monocytes in a culture system; and (iii) contacting the culture system with αGalCer, wherein the contacting is sufficient to load the monocyte-derived dendritic cells with αGalCer.
37. The method according to claim 27, wherein minimum acceptable specifications of the SCKTC cell product when tested in vitro include: (i) cytokine production comprising IL-4 low, IL-5 low, IL-6 low, IL-10 low, IFNγ high, and (ii) a ratio of IFN-γ:IL-4 in culture supernatants of at least 500: 1; and (iii) at an effector:target cell ratio of 20:1 greater than or equal to 50% cytotoxicity against A549 cells; and (iv) a therapeutic dose of the cell product per treatment cycle of 30 days comprising about 0.2×10.sup.1 activated SCKTCs.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0229] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0243] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
[0244] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0245] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer. According to some embodiments, to A without B (optionally including elements other than B); According to some embodiments, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0246] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0247] As used herein, the phrase “integer from X to Y” means any integer that includes the endpoints. That is, where a range is disclosed, each integer in the range including the endpoints is disclosed. For example, the phrase “integer from X to Y” discloses 1, 2, 3, 4, or 5 as well as the range 1 to 5.
[0248] As used herein, when used to define products, compositions and methods, the term “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide “comprises” an amino acid sequence when the amino acid sequence might be part of the final amino acid sequence of the polypeptide. Such a polypeptide can have up to several hundred additional amino acids residues (e.g. tag and targeting peptides as mentioned herein). “Consisting essentially of” means excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. A polypeptide “consists essentially of” an amino acid sequence when such an amino acid sequence is present with eventually only a few additional amino acid residues. “Consisting of” means excluding more than trace elements of other components or steps. For example, a polypeptide “consists of” an amino acid sequence when the polypeptide does not contain any amino acids but the recited amino acid sequence.
[0249] As used herein, “substantially equal” means within a range known to be correlated to an abnormal or normal range at a given measured metric. For example, if a control sample is from a diseased patient, substantially equal is within an abnormal range. If a control sample is from a patient known not to have the condition being tested, substantially equal is within a normal range for that given metric.
[0250] The terms “activate,” “stimulate,” “enhance” “increase” and/or “induce” (and like terms) are used interchangeably to generally refer to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. “Activate” refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. Further, the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule. Thus, ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cellular responses.
[0251] As used herein, the terms “activating or activated cytokine killer T cells” or “CKTC1 activation” is meant to refer to a process causing or resulting in one or more cellular responses of CKTCs, including: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. As used herein, an “activated cytokine killer T cell” refers to a cytokine killer T cell that has received an activating signal, and thus demonstrates one or more cellular responses, including proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The activating of the CKTC can comprise one or more of inducing secretion of a cytokine from the CKTC, stimulating proliferation of the CKTC, and upregulating expression of a cell surface marker on the CKTC. The cytokine can be one or more of IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-15, TNF-α, TNF-β, and IFN-γ. According to certain embodiments, activating of a CKTC can comprise secretion of one or more of, IL-4, IL-5, Il-6, IL-10, or IFN-γ. Suitable assays to measure CKTC activation are known in the art and are described herein.
[0252] The term “active” refers to the ingredient, component or constituent of the pharmaceutical compositions of the described invention responsible for an intended therapeutic effect.
[0253] The term “administration” and its various grammatical forms as it applies to a mammal, cell, tissue, organ, or biological fluid, as used herein is meant to refer without limitation to contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition to the subject, cell, tissue, organ, or biological fluid, and the like. “Administration” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration” also encompasses in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. It should be understood that “administration” includes co-formulation (meaning formulated together) as well as administration via one or more pharmaceutical compositions administered concurrently (meaning at the same time, including, e.g., co-administration) or sequentially (meaning coming after in time or order).
[0254] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI and its more severe form, ARDS, are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation. ALI/ARDS is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders, including severe acute pancreatitis, drug over dose, and blood products [Ware, L. and Matthay, M., N Engl J Med, (2000) 342:1334-1349,]. Most patients require assisted ventilation with positive pressure. The primary physiologic abnormalities are severe arterial hypoxemia as well as a marked increase in minute ventilation secondary to a sharp increase in pulmonary dead space fraction. Patients with ALI/ARDS develop protein-rich pulmonary edema resulting from exudation of fluid into the interstitial and airspace compartments of the lung secondary to increased permeability of the barrier. Additional pathologic changes indicate that the mechanisms involved in lung edema are complex and that edema is only one of the pathophysiologic events in ALI/ARDS. One physiologic consequence is a significant decrease in lung compliance that results in an increased work of breathing [Nuckton T. et al., N Engl J Med. (2002) 346:1281-1286,], one of the reasons why assisted ventilation is required to support most patients. It has been reported that soon after onset of respiratory distress from COVID, patients initially retain relatively good compliance despite very poor oxygenation. [Marini, J J and Gattinoni, L., JAMA Insights (2020) doi: 10.1001/jama.2020.6825, citing Grasselli, G. et al., JAMA (2020) doi: 10.1001/jama.2020.5394; Arentz, M. et al. JAMA (2020) doi: 10.1001/jama.2020.4326]. Minute ventilation is characteristically high. Infiltrates are often limited in extent and, initially, are usually characterized by a ground-glass pattern on CT that signifies interstitial rather than alveolar edema. Many patients do not appear overtly dyspneic. These patients can be assigned, in a simplified model, to “type L,” characterized by low lung elastance (high compliance), lower lung weight as estimated by CT scan, and low response to PEEP. (Id., citing Gattinoni, L. et al. Intensive Care Med. (2020) doi: 10.1007/s00134-020-06033-2). For many patients, the disease may stabilize at this stage without deterioration while others, either because of disease severity and host response or suboptimal management, may transition to a clinical picture more characteristic of typical ARDS. These can be defined as “type H,” with extensive CT consolidations, high elastance (low compliance), higher lung weight, and high PEEP response. Types L and H are the conceptual extremes of a spectrum that includes intermediate stages, in which their characteristics may overlap.
[0255] As used herein, the term “adaptive cellular therapy” or “adaptive transfer” refer to a treatment used to help the immune system fight diseases by which T cells collected from a patient are expanded (grown in a laboratory in culture) to increase the number of T cells able to fight the disease. These T cells then are given back to the patient.
[0256] The term “adaptor molecule” as used herein refers to a specialized protein that links protein components of a signaling pathway, thereby aiding intracellular signal transduction.
[0257] As used herein the term “allogeneic” is meant to refer to being derived from two genetically different individuals.
[0258] The term “alveolar type II cells (AT2 cells)” as used herein refers to the progenitors for alveolar type I cells. Alveolar type I cells cover 95 percent of the alveolar surface of the lung; they comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. AT2 cells are the only pulmonary cells that synthesize, store, and secrete all components of pulmonary surfactant important to regulate surface tension, preventing atelectasis and maintaining alveolar fluid balance within the alveolus.
[0259] The terms “amino acid residue” or “amino acid” or “residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids. The amino acids may be L- or D-amino acids. An amino acid may be replaced by a synthetic amino acid, which is altered so as to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide.
[0260] The single letter designation for amino acids is used predominately herein. As is well known by one of skill in the art, such single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine.
[0261] The following represents groups of amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0262] The term “anergy” as used herein refers to a state of lymphocyte nonresponsiveness to specific antigen induced by an encounter of the lymphocyte with cognate antigen under less than optimal conditions, such as in the absence of costimulation.
[0263] The term “angiotensin-converting enzyme 2” or “ACE2” as used herein refers to a type 1 integral membrane glycoprotein [Tikellils, C. and Thomas M C. Intl J. Peptides (2012) 256294, citing Tipnis, S R et al. J. Biol. Chem. (2000) 275 (43): 33238-43] that is expressed and active in most tissues. The highest expression of ACE2 is observed in the kidney, the endothelium, the lungs, and in the heart [Id., citing Donoghue, M. et al. Cir. Res. (2000) 87 (5): E1-E9, Tipnis, S R et al. J. Biol. Chem. (2000) 275 (43): 33238-43]. The extracellular domain of ACE2 enzyme contains a single catalytic metallopeptidase unit that shares 42% sequence identity and 61% sequence similarity with the catalytic domain of ACE [[Id., citing Donoghue, M. et al. Cir. Res. (2000) 87 (5): E1-E9]. However, unlike ACE, it functions as a carboxypeptidase, rather than a dipeptidase, and ACE2 activity is not antagonized by conventional ACE inhibitors [Id., citing Rice, G I et al. Biochemical J. (2004) 383 (1): 45-51]. The major substrate for ACE2 appears to be (Ang II) [Id., citing Donoghue, M. et al. Circulation Res. (2000) 87 (5): E1-E9; turner, AJ and Hooper N M, Trends in Pharmcological Sci. (2002) 23 (4): 177-83; Rice, G I et al. Biochemical J. (2004) 383 (1): 45-51], although other peptides may also be degraded by ACE2, albeit at lower affinity. For example, ACE2 is able to cleave the C-terminal amino acid from angiotensin I, vasoactive bradykinin, des-Arg-kallidin (also known as des-Arg10 Lys-bradykinin), Apelin-13 and Apelin-36 [Id., citing Kuba, K. et al. Circulation Res. (2007) 101 (4): e32-e42] as well as other possible targets [Id., citing Vickers, C. et al. J. Biol. Chem. (2002) 277 (17): 14838-43]. The noncatalytic C-terminal domain of ACE2 shows 48% sequence identity with collectrin [Id., citing Zhang, H. et al. J. Biol. Chem. (2001) 276 (20): 17132-39], a protein shown to have an important role in neutral amino acid reabsorption from the intestine and the kidney [Id., citing Kowalczuk, S. et al. The FASEB J. (2008) 22 (8): 2880-87]; the removed amino acid then becomes available for reabsorption. The cytoplasmic tail of ACE2 also contains calmodulin-binding sites [Id., citing D W Lambert, et al. FEBS Letters (2008) 582 (2): 385-90] which may influence shedding of its catalytic ectodomain. In addition, ACE2 has also been associated with integrin function, independent of its angiotensinase activity.
[0264] As used herein, the term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal antibodies and monoclonal antibodies, and fragments thereof. Furthermore, the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
[0265] As used herein, the term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, antibody fragments, chimeric antibodies and wholly synthetic antibodies as long as they exhibit the desired antigen-binding activity. In nature, antibodies are serum proteins the molecules of which possess small areas of their surface that are complementary to small chemical groupings on their targets. These complementary regions (referred to as the antibody combining sites or antigen binding sites) of which there are at least two per whole antibody molecule, and in some types of antibody molecules ten, eight, or in some species as many as 12, may react with their corresponding complementary region on an antigen (the antigenic determinant or epitope) to link several molecules of multivalent antigen together to form a lattice. The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.
[0266] The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.
[0267] Human antibodies show two kinds of light chains, κ and λ; individual molecules of immunoglobulin generally are only one or the other. In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain. All five immunoglobulin classes differ from other serum proteins in that they show a broad range of electrophoretic mobility and are not homogeneous. This heterogeneity—that individual IgG molecules, for example, differ from one another in net charge—is an intrinsic property of the immunoglobulins.
[0268] The principle of complementarity, which often is compared to the fitting of a key in a lock, involves relatively weak binding forces (hydrophobic and hydrogen bonds, van der Waals forces, and ionic interactions), which are able to act effectively only when the two reacting molecules can approach very closely to each other and indeed so closely that the projecting constituent atoms or groups of atoms of one molecule can fit into complementary depressions or recesses in the other. Antigen-antibody interactions show a high degree of specificity, which is manifest at many levels. Brought down to the molecular level, specificity means that the combining sites of antibodies to an antigen have a complementarity not at all similar to the antigenic determinants of an unrelated antigen. Whenever antigenic determinants of two different antigens have some structural similarity, some degree of fitting of one determinant into the combining site of some antibodies to the other may occur, and that this phenomenon gives rise to cross-reactions. Cross reactions are of major importance in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity makes possible the detection of small amounts of impurities/contaminations among antigens.
[0269] Monoclonal antibodies (mAbs) can be generated by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line to yield established mouse hybridoma clones that grow in selective media. A hybridoma cell is an immortalized hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell with a myeloma cell. In vitro immunization, which refers to primary activation of antigen-specific B cells in culture, is another well-established means of producing mouse monoclonal antibodies.
[0270] Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes also can be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single chain Fv or scFv) can be made by randomly combining heavy and light chain V-genes using PCR. A combinatorial library then can be cloned for display on the surface of filamentous bacteriophage by fusion to a minor coat protein at the tip of the phage.
[0271] The technique of guided selection is based on human immunoglobulin V gene shuffling with rodent immunoglobulin V genes. The method entails (i) shuffling a repertoire of human V.sub.L chains with the heavy chain variable region (V.sub.H) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting half-human Fabs on that antigen (iii) using the selected V L genes as “docking domains” for a library of human heavy chains in a second shuffle to isolate clone Fab fragments having human light chain genes; (v) transfecting mouse myeloma cells by electroporation with mammalian cell expression vectors containing the genes; and (vi) expressing the V genes of the Fab reactive with the antigen as a complete IgG1 antibody molecule in the mouse myeloma.
[0272] An antibody may be an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques.
[0273] An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the invention. Binding fragments of an antibody can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Exemplary fragments include Fv, Fab, Fab′, single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv). Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety. An antibody other than a “bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical.
[0274] The term “antibodyconstruct” as used herein refers to a polypeptide comprising one or more the antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen-binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art. Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.
[0275] The term “antibody-dependent cellular cytotoxicity” or ADCC, also called antibody-dependent cell-mediated cytotoxicity, is an immune mechanism through which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells expressing tumor- or pathogen-derived antigens on their surface. It is mediated by the recruitment of cytotoxic effector cells, such as natural killer (NK) cells, macrophages, and polymorphonuclear leukocytes (PMNs), that express Fc gamma receptors (FcγRs) on their surface.
[0276] The term “antibody-dependent cellular phagocytosis” or ADCP is a potent mechanism of elimination of antibody-coated foreign particles such microbes or tumor cells. Engagement of FcγRIIa and FcγRI expressed on macrophages triggers a signaling cascade leading to the engulfment of the IgG-opsonized particle.
[0277] The term “antigen” as used herein, is meant to refer to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune-system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. The term includes polypeptides which include modifications, such as deletions, additions and substitutions (generally conservative in nature) as compared to a native sequence, as long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
[0278] The term “antigenic drift” as used herein refers to subtle modification of pathogen antigens through random point mutations; it usually involves surface proteins that would normally be the target of neutralizing antibodies.
[0279] The term “antigenic shift” as used herein refers to dramatic modification of viral antigens due to reassortment of genomic segments of two different strains of a virus that simultaneously infect the same individual to generate progeny virions with new combinations of genome segments and thus new proteins.
[0280] The term “antigen presentation” as used herein, generally refers to the display of antigen on the surface of a cell, e.g., in the form of peptide fragments bound to MHC molecules.
[0281] As used herein, the term “antigen presenting cell (APC)” refers to a class of cells capable of displaying on its surface (“presenting”) one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. Examples of professional APCs are dendritic cells and macrophages, though any cell expressing MHC Class I or II molecules can potentially present peptide antigen. An APC can be an irradiated population of PBMCs. An APC can be an “artificial APC,” meaning a cell that is engineered to present one or more antigens. Before a T cell can recognize a foreign protein, the protein has to be processed inside an antigen presenting cell or target cell so that it can be displayed as peptide-MHC complexes on the cell surface.
[0282] As used herein the term “antigen processing” refers to the intracellular degradation of foreign proteins into peptides that can bind to MHC molecules for presentation to T cells.
[0283] The term “apheresis” as used herein refers to a medical technology in which the blood of a donor or patient is passed through an apparatus that separates out one particular constituent and returns the remainder back to the donor or patient's circulation. Leukapheresis is one type of apheresis where leukocytes (white blood cells) are selectively removed.
[0284] The terms “apoptosis” or “programmed cell death” refer to a highly regulated and active process that contributes to biologic homeostasis comprised of a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane, such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without damaging the organism.
[0285] Apoptotic cell death is induced by many different factors and involves numerous signaling pathways, some dependent on caspase proteases (a class of cysteine proteases) and others that are caspase independent. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial response to stress, and cytotoxic T cells, resulting in activation of apoptotic signaling pathways
[0286] The caspases involved in apoptosis convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that then degrade other cellular targets that lead to cell death. The caspases at the upper end of the cascade include caspase-8 and caspase-9. Caspase-8 is the initial caspase involved in response to receptors with a death domain (DD) like Fas.
[0287] Receptors in the TNF receptor family are associated with the induction of apoptosis, as well as inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. The Fas-FasL interaction plays an important role in the immune system and lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis removes self-reactive lymphocytes. Fas signaling also is involved in immune surveillance to remove transformed cells and virus infected cells. Binding of Fas to oligimerized FasL on another cell activates apoptotic signaling through a cytoplasmic domain termed the death domain (DD) that interacts with signaling adaptors including FAF, FADD and DAX to activate the caspase proteolytic cascade. Caspase-8 and caspase-10 first are activated to then cleave and activate downstream caspases and a variety of cellular substrates that lead to cell death.
[0288] Mitochondria participate in apoptotic signaling pathways through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals, and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac/DIABLO is released from mitochondria and inhibits IAP proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs as family members form complexes that enter the mitochondrial membrane, regulating the release of cytochrome c and other proteins. TNF family receptors that cause apoptosis directly activate the caspase cascade, but can also activate Bid, a Bcl-2 family member, which activates mitochondria-mediated apoptosis. Bax, another Bcl-2 family member, is activated by this pathway to localize to the mitochondrial membrane and increase its permeability, releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation, blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria that is released from mitochondria by apoptotic stimuli. While cytochrome C is linked to caspase-dependent apoptotic signaling, AIF release stimulates caspase-independent apoptosis, moving into the nucleus where it binds DNA. DNA binding by AIF stimulates chromatin condensation, and DNA fragmentation, perhaps through recruitment of nucleases.
[0289] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing self-cleavage and activation of caspase-9. Caspase-3, -6 and -7 are downstream caspases that are activated by the upstream proteases and act themselves to cleave cellular targets.
[0290] Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores, and triggering apoptosis, perhaps through cleavage of caspases, although caspase-independent mechanisms of Granzyme B mediated apoptosis have been suggested.
[0291] Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways to create a nucleosomal ladder is a cellular response characteristic of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD). DFF/CAD is activated through cleavage of its associated inhibitor ICAD by caspases proteases during apoptosis. DFF/CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and perhaps recruit CAD to chromatin. Another apoptosis activated protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is localized to mitochondria in normal cells. EndoG may play a role in the replication of the mitochondrial genome, as well as in apoptosis. Apoptotic signaling causes the release of EndoG from mitochondria. The EndoG and DFF/CAD pathways are independent since the EndoG pathway still occurs in cells lacking DFF.
[0292] Hypoxia, as well as hypoxia followed by reoxygenation can trigger cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or potentiate apoptosis due to many stimuli that activate the mitochondrial cell death pathway. Loberg, R D, et al., J. Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated to induce caspase 3 activation and to activate the proapoptotic tumor suppressor gene p53. It also has been suggested that GSK-3 promotes activation and translocation of the proapoptotic Bcl-2 family member, Bax, which, upon aggregation and mitochondrial localization, induces cytochrome c release. Akt is a critical regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of the antiapoptotic effects of Akt.
[0293] The term “attenuate” as used herein refers to render less virulent, to weaken or reduce in force, intensity, effect or quantity.
[0294] As used herein, the term “autologous” is meant to refer to being derived from the same individual.
[0295] As used herein, the term “autophagy” refers to the digestion and breakdown by a cell of its own organelles and proteins in lysosomes.
[0296] The terms “B lymphocyte” or “B cell” are used interchangeably to refer to a broad class of lymphocytes, which are precursors of antibody-secreting cells, that express clonally diverse cell surface immunoglobulin (Ig) receptors (BCRs) recognizing specific antigenic epitopes. Mammalian B-cell development encompasses a continuum of stages that begin in primary lymphoid tissue (e.g., human fetal liver and fetal/adult marrow), with subsequent functional maturation in secondary lymphoid tissue (e.g., human lymph nodes and spleen). The functional/protective end point is antibody production by terminally differentiated plasma cells. A mature B cell can be activated by an encounter with an antigen that expresses epitopes that are recognized by its cell surface immunoglobulin (Ig). The activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B cell activation) or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T cell (“cognate help process”).[LeBien, T W & T F Tedder, B lymphocytes: how they develop and function. Blood (2008) 112 (5): 1570-80].
[0297] The term “B cell receptor” or “BCR” as used herein refers to the antigen-receptor complex of B lineage cells, which is composed of a membrane bound Ig (mIg) monomer plus the Igα/Igβ complex required for intracellular signaling.
[0298] The term “beta2-microglobulin” as used herein refers to the light chain of the MHC class I proteins, encoded outside the MHC. It binds noncovalently to the heavy or a, chain.
[0299] The term “binding” and its various grammatical forms means a lasting attraction between chemical substances. Binding specificity involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross-interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners.
[0300] The term “binding specificity” as used herein involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross-interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners.
[0301] The term “bioavailable” and its other grammatical forms as used herein refers to the ability of a substance to be absorbed and sued by the body.
[0302] The term “biocompatible” as used herein refers to a material that is generally non-toxic to the recipient and does not possess any significant untoward effects to the subject and, further, that any metabolites or degradation products of the material are non-toxic to the subject. Typically a substance that is “biocompatible” causes no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.
[0303] The term “biodegradable” as used herein refers to a material that will erode to soluble species or that will degrade under physiologic conditions to smaller units or chemical species that are, themselves, non-toxic.
[0304] As used herein, the term “biomarker” (or “biosignature”) refers to a peptide, protein, nucleic acid, antibody, gene, metabolite, or any other substance used as an indicator of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The term “indicator” as used herein refers to any substance, number or ratio derived from a series of observed facts that may reveal relative changes as a function of time; or a signal, sign, mark, note or symptom that is visible or evidence of the existence or presence thereof. Once a proposed biomarker has been validated, it may be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint, such as survival or irreversible morbidity. If a treatment alters the biomarker, and that alteration has a direct connection to improved health, the biomarker may serve as a surrogate endpoint for evaluating clinical benefit. Clinical endpoints are variables that can be used to measure how patients feel, function or survive. Surrogate endpoints are biomarkers that are intended to substitute for a clinical endpoint; these biomarkers are demonstrated to predict a clinical endpoint with a confidence level acceptable to regulators and the clinical community.
[0305] As used herein the term “CD1d” is meant to refer to a family of transmembrane glycoproteins, which are structurally related to the MHC proteins and form heterodimers with beta-2-microglobulins that mediate the presentation of primarily lipid and glycolipid antigens of self or microbial origin to T cells.
[0306] The term “CARD domain” as used herein refers to the family subclass of the caspase recruitment domain. The formation of apoptotic and inflammatory multiprotein complexes together with defined signaling episodes in innate immunity heavily relies on members of the death domain family and particularly on the family subclass of the caspase recruitment domain (CARD). [Palacios-Rodriguez, Y. et al., Polypeptide Modulators of Caspase Recruitment Domain (CARD)-Card-mediated protein-protein interactions. J. Biol. Chem. (2011) 286 (52): 44457-66, citing Varfolomeev, E. et al. Cell (2007) 131: 669-81] The interaction between the CARD of Apaf-1 (apoptotic protease-activating factor) and the CARD of procaspase-9 (PC9) in the mitochondria-mediated apoptotic intrinsic pathway is essential for the recruitment of PC9 into the apoptosome and its subsequent activation [Id., citing Acehan, D., et al. Mol. Cell (2002) 9: 423-32]. On the other hand, proteins like those of the NOD-like receptor (NLR) family (in particular NOD-1, NOD-2, and NLRP-1) act as intracellular scrutiny devices and signaling initiators to face microbial aggressions [Id., citing Proell, M. et al. PLoS One (2008) 3: e2119]. The NLR proteins utilize the CARD for binding to downstream signaling molecules through CARD-CARD interactions in order to ultimately initiate the innate immune and inflammatory responses [Id., citing Inohara N., Nufiez G. Nat. Rev. Immunol. (2003) 3, 371-382; Park, H H, et al. Annu. Rev. Immunol. (2007) 25, 561-586].
[0307] The term “carrier” as used herein describes a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active compound of the composition of the described invention. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the mammal being treated. The carrier can be inert, or it can possess pharmaceutical benefits, cosmetic benefits or both. The terms “excipient”, “carrier”, or “vehicle” are used interchangeably to refer to carrier materials suitable for formulation and administration of pharmaceutically acceptable compositions described herein. Carriers and vehicles useful herein include any such materials know in the art which are nontoxic and do not interact with other components. The carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
[0308] The term “CD40” as used herein refers to a tumor necrosis factor receptor (TNFR) superfamily member expressed on APCs, such as dendritic cells (DC), B cells, and monocytes as well as many non-immune cells and a wide range of tumors. Interaction with its trimeric ligand CD40 Ligand (CD40L) on activated T helper cells results in APC activation, required for the induction of adaptive immunity. CD40 on B cells and CD40 ligand on activated helper T cells are co-stimulatory molecules whose interaction is required for the proliferation and class switching of antigen activated naïve B cells. CD40 is also expressed by dendritic cells; where the CD40-CD40L interaction provides co-stimulatory signals to naïve T cells.
[0309] As used herein, the term “cell growth” is the process by which cells accumulate mass and increase in physical size. There are many different examples in nature of how cells can grow. In some cases, cell size is proportional to DNA content. For instance, continued DNA replication in the absence of cell division (called endoreplication) results in increased cell size. Megakaryoblasts, which mature into granular megakaryocytes, the platelet-producing cells of bone marrow, typically grow this way. By a different strategy, adipocytes can grow to approximately 85 to 120 pm by accumulating intracellular lipids. In contrast to endoreplication or lipid accumulation, some terminally differentiated cells, such as neurons and cardiac muscle cells, cease dividing and grow without increasing their DNA content. These cells proportionately increase their macromolecule content (largely protein) to a point necessary to perform their specialized functions. This involves coordination between extracellular cues from nutrients and growth factors and intracellular signaling networks responsible for controlling cellular energy availability and macromolecular synthesis. Perhaps the most tightly regulated cell growth occurs in dividing cells, where cell growth and cell division are clearly separable processes. Dividing cells generally must increase in size with each passage through the cell division cycle to ensure that a consistent average cell size is maintained. For a typical dividing mammalian cell, growth occurs in the G1 phase of the cell cycle and is tightly coordinated with S phase (DNA synthesis) and M phase (mitosis). The combined influence of growth factors, hormones, and nutrient availability provides the external cues for cells to grow. [Guertin, D. A., Sabatini, D. M., “Cell Growth,” in The Molecular Basis of Cancer (4.sup.th Edn) Mendelsohn, J. et al Eds, Saunders (2015), 179-190].
[0310] As used herein, the term “cell proliferation” is meant to refer to the process that results in an increase of the number of cells, and is defined by the balance between cell divisions and cell loss through cell death or differentiation.
[0311] As used herein, the term “chemokine” is meant to refer to a class of chemotactic cytokines that signal leukocytes to move in a specific direction.
[0312] The term “clade” as used herein refers to related organisms descended from a common ancestor.
[0313] The term “class switching”, “isotype switching” or “class switch recombination” as used herein refers to a somatic gene recombination process in activated B cells that replaces one heavy chain constant region with one of a different isotype, switching the isotype of antibodies from IgM to IgG, IgA or IgE. This affects the antibody effector functions but not their antigen specificity.
[0314] As used herein, the term “cognate help” is meant to refer to a process that occurs most efficiently in the context of an intimate interaction with a helper T cell.
[0315] The term “complement” as used herein refers to a system of over 30 soluble and membrane-bound proteins that act through a tightly regulated cascade of pro-protein cleavage and activation to mediate cell lysis through assembly of the membrane attack complex (MAC) composed of complement components C5b, C6, C7, C8, and C9 in a target cell membrane. Intermediates in the complement cascade play a variety of roles in antigen clearance. The activation of complement can lead to lysis of an antibody-opsonized cell by complement-dependent cytotoxicity (CDC) or complement-dependent cell-mediated cytotoxicity (CDCC) [Meyer, S. et al. MAbs (2014) 6 (5): 1133-44].
[0316] The term “component” as used herein, is meant to refer to a constituent part, element or ingredient.
[0317] The term “composition” as used herein, is meant to refer to a material formed by a mixture of two or more substances.
[0318] As used herein, the term “condition” as used herein, is meant to refer to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder.
[0319] As used herein, the term “contact” and its various grammatical forms is meant to refer to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination may occur by any means of administration known to the skilled artisan.
[0320] The term “costimulation” as used herein refers to the second signal required for completion of lymphocyte activation and prevention of anergy, which is supplied by engagement of CD28 by CD80 and CD86 (T cells) and of CD40 by CD40 Ligand (B cells).
[0321] The term “costimulatory molecule” as used herein refers to molecules that are displayed on the cell surface that have a role in enhancing the activation of a T cell that is already being stimulated through its TCR. For example, HLA proteins, which present foreign antigen to the T cell receptor, require costimulatory proteins which bind to complementary receptors on the T cell's surface to result in enhanced activation of the T cell. The term “co-stimulatory molecules” as used herein refers to highly active immunomodulatory proteins that play a critical role in the development and maintenance of an adaptive immune response (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). The two signal hypothesis of T cell response involves the interaction between an antigen bound to an HLA molecule and with its cognate T cell receptor (TCR), and an interaction of a co-stimulatory molecule and its ligand. Specialized APCs, which are carriers of a co-stimulatory second signal, are able to activate T cell responses following binding of the HLA molecule with TCR. By contrast, somatic tissues do not express the second signal and thereby induce T cell unresponsiveness (Id.). Many of the co-stimulatory molecules involved in the two-signal model can be blocked by co-inhibitory molecules that are expressed by normal tissue (Id.). In fact, many types of interacting immunomodulatory molecules expressed on a wide variety of tissues may exert both stimulatory and inhibitory functions depending on the immunologic context (Id.).As used herein the term “co-stimulatory receptor” is meant to refer to a cell surface receptor on naïve lymphocytes through which they receive signals additional to those received through the antigen receptor, and which are necessary for the full activation of the lymphocyte. Examples are CD30 and CD40 on B cells, and CD27 and CD28 on T cells.
[0322] As used herein, the term “cross-protection” is used to describe immunity against at least two subgroups, subtypes, strains and/or variants of a virus, bacteria, parasite or other pathogen with a single inoculation with one subgroup, subtype, strain and/or variant thereof.
[0323] The term “culture” and its other grammatical forms as used herein, is meant to refer to a process whereby a population of cells is grown and proliferated on a substrate in an artificial medium.
[0324] The term “cytokine” as used herein refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor (“TNF”)-related molecules, including TNFα and lymphotoxin; immunoglobulin super-family members, including interleukin 1 (“IL-1”); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines. Non-limiting examples of cytokines include e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12/IL-23 P40, IL13, IL-15, IL-15/IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-β, IFNγ, GM-CSF, Groα, MCP-1 and TNF-α.
[0325] The term “cytotoxic T lymphocytes” (CTLs) as used herein, is meant to refer to effector CD8+ T cells. Cytotoxic T cells kill by inducing their targets to undergo apoptosis. They induce target cells to undergo programmed cell death via extrinsic and intrinsic pathways.
[0326] The term damage-associated molecular patterns” or “DAMPS” as used herein refers to molecules released by stressed or dying cells that bind to pattern recognition molecules (PRMs) and induce inflammation.
[0327] The term “dendritic cells (DC)” as used herein refers to professional antigen presenting cells, which induce naïve T cell activation and effector differentiation. [Patente, T A, et al., Frontiers Immunol. (2019) doi.org/10.3389/fimmu.2018.03176]. Human DC are identified by their high expression of major histocompatibility complex (MHC) class II molecules (MHC-II) and of CD11c, both of which are found on other cells, like lymphocytes, monocytes and macrophages [Id., citing Carlens J, et al. J Immunol. (2009) 183:5600-7; Drutman S B, et al. J Immunol. (2012) 188:3603-10; Hochweller K, S et al. Eur J Immunol. (2008) 38:2776-83; Huleatt J W, Lefrangois L. J Immunol. (1995) 154:5684-93; Rubtsov A V, et al. Blood (2011) 118:1305-15; Probst H C, et al. Clin Exp Immunol. (2005) 141:398-404; Vermaelen K, Pauwels R. Cytometry (2004) 61A:170-7]. DC express many other molecules which allow their classification into various subtypes. Although some of the DC subtypes were originally described as macrophages, DC and macrophages have distinct characteristics [Id., citing Delamarre L, Science (2005) 307:1630-4; Geissmann F, et al. Science (2010) 327:656-61; van Montfoort N, et al. Proc Natl Acad Sci USA. (2009) 106:6730-5] and ontogeny, so that, currently, little doubt remains that they belong to distinct lineages [Id., citing Haniffa M, et al. (2013) 120:1-49; Hashimoto D, et al. Immunity (2013) 38:792-804; Hettinger J, et al. Nat Immunol. (2013) 14:821-30; McGovern N, et al. Immunity (2014) 41:465-77; Naik S H, et al. Nature (2013) 496:229-32; Schulz C, et al. Science (2012) 336:86-90; Schraml B U, et al. Cell (2013) 154:843-58; Wang J, et al. Mol Med Rep. (2017) 16:6787-93; Yona S, et al. Immunity (2013) 38:79-91]. DC are found in two different functional states, “mature” and “immature”. These are distinguished by many features, but the ability to activate antigen-specific naïve T cells in secondary lymphoid organs is the hallmark of mature DC [Id., citing Hawiger D, Inaba K, et al. J Exp Med. (2001) 194:769-79; Steinman R M, et al. Ann NY Acad Sci. (2003) 987:15-25; Worbs T, et al. Nat Rev Immunol. (2017) 17:30-48]. DC maturation is triggered by tissue homeostasis disturbances, detected by the recognition of pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMPs) [Id., citing Hemmi H, et al. Chem Immunol Aller. (2005) 86:120-135, Cerboni S, et al. Adv Immunol. (2013) 120:211-237]. Maturation turns on metabolic, cellular, and gene transcription programs allowing DC to migrate from peripheral tissues to T-dependent areas in secondary lymphoid organs, where T lymphocyte-activating antigen presentation may occur [Id., citing Alvarez D, et al. Immunity (2008) 29:325-42; Dong H, Bullock T N J. Front Immunol. (2014) 5:24; Friedl P, Gunzer M. Trends Immunol. (2001) 22:187-91; Henderson R A, et al. J Immunol. (1997) 159:635-43; Randolph G J, et al. Nature Rev Immunol. (2005) 5:617-28 Imai Y, et al. Histol Histopathol. (1998) 13:469-510]. During maturation, DC lose adhesive structures, reorganize the cytoskeleton and increase their motility [Id., citing Winzler C, et al. J Exp Med. (1997) 185:317-28). DC maturation also leads to a decrease in their endocytic activity but increased expression of MHC-II and co-stimulatory molecules [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476-83; Steinman R M. Annu Rev Immunol. (2012) 30:1-22; Trombetta E S, Mellman I. Annu Rev Immunol. (2005) 23:975-1028]. Mature DC express higher levels of the chemokine receptor, CCR7 [Id., citing Forster R, et al. Cell (1999) 99:23-33; Ohl L, et al. Immunity (2004) 21:279-88; Sallusto F, et al. Eur J Immunol. (1998) 28:2760-9; Steinman R M. The control of immunity and tolerance by dendritic cell. Pathol Biol. (2003) 51:59-60] and secrete cytokines, essential for T-cell activation [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476-83, Caux C, et al. J Exp Med. (1994) 180:1263-72; Jensen S S, Gad M. J Inflamm (Lond) (2010) 7:37; Tan J K H, O'Neill H C. J Leukocyte Biol. (2005) 78:319-324; Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343-353]. Thus, the interaction between mature DC and antigen-specific T cells is the trigger of antigen-specific immune responses [Id., citing Luft T., Blood (2006) 107:4763-9, Jonuleit H. Arch Dermatol Res. (1996) 289:1-8]. When interacting with CD4+ T cells, DC may induce their differentiation into different T helper (T.sub.H) subsets [Id., citing Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343-353] such as T.sub.H1 [Amsen D, et al. Cell (2004) 117:515-26; Constant S, et al. J Exp Med (1995) 182:1591-6; Hosken N A, et al. J Exp Med. (1995) 182:1579-84; Kadowaki N. Allergol Int. (2007) 56:193-9; Maekawa Y, et al. Immunity (2003) 19:549-59; Pulendran B, et al. Proc Natl Acad Sci USA. (1999) 96:1036-41, Th2 [Id., citing Constant S, et al. J Exp Med (1995) 182:1591-6, Hosken N A, et al. J Exp Med. (1995) 182:1579-84, Jenkins S J, P. et al. J Immunol. (2007) 179:3515-23, Soumelis V, et al. Nat Immunol. (2002) 3:673-6801, T.sub.H17 [Id., citing Bailey S L, Nat Immunol. (2007) 8:172-80; Iezzi G, et al. Proc Natl Acad Sci USA. (2009) 106:876-81; Huang G, et al. Cell Mol Immunol. (2012) 9:287-951, or other CD4+ T cell subtypes [Id., citing Levings M K, et al. Blood (2005) 105:1162-91. T cell differentiation in each subtype is a complex phenomenon, that can be influenced by the cytokines in the DC tissue of origin [Id., citing Rescigno M. Dendritic cell-epithelial cell crosstalk in the gut. Immunol Rev. (2014) 260:118-281, their maturation state [Id., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476-831 and cause of tissue imbalance [Id., citing Vega-Ramos J, et al. Curr Opin Pharmacol. (2014) 17:64-701. DCs present a unique characteristic: the ability to perform cross-presentation [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517-32; Delamarre L, Mellman I. Semin Immunol. (2011) 23:2-11; Jung S, et al. Immunity (2002) 17:211-20; Segura E, Amigorena S. Adv Immunol. (2015) 127:1-31; Segura E, Villadangos J A. Curr Opin Immunol. (2009) 21:105-1101, defined as the presentation, in the context of class I MHC molecules (MHC-I), of antigens captured from the extracellular milieu. This feature allows DC to trigger responses against intracellular antigens from other cell types, thus providing means for the system to deal with threats that avoid professional APC [Id., citing Coulon P-G, et al. J Immunol. (2016) 197:517-32, Bevan M J. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med. (1976) 143:1283-8, Sinchez-Paulete A R, et al. Ann Oncol. (2017) 28:xii74. doi: 10.1093/annonc/mdx727] and, even, to prime CD8+ lymphocytes in the absence of CD4+ T cells [Id., citing McCoy K D, et al. J Exp Med. (1999) 189:1157-62, Young J W, Steinman R M. J Exp Med. (1990) 171:1315-321. Cross-presentation is involved also in the induction of tolerance to intracellular self-antigens that are not expressed by APC and, then, called, cross-tolerance [Kurts C, et al. J Exp Med. (1997) 186:239-45, Rock K L, Shen L. Immunol Rev. (2005) 207:166-831.
[0328] Before receiving maturation stimuli, DC are said to be in an “immature state.” Immature DC are poor inducers of naïve lymphocyte effector responses, since they have low surface expression of co-stimulatory molecules, low expression of chemokine receptors, and do not release immunostimulatory cytokines [Id., citing Trombetta E S, Mellman I. Annu Rev Immunol. (2005) 23:975-1028, Steinman R M, Swanson J. J Exp Med. (1995) 182:283-81. These “immature” cells, though, are very efficient in antigen capture due to their high endocytic capacity, via receptor-mediated endocytosis, including lectin-[Id., citing Geijtenbeek T B, et al. Cell (2000) 100:575-585; Sallusto F, et al. J Exp Med. (1995) 182:389-400; Valladeau J, et al. Cell Immunol. (1994) 159:323-30; Medzhitov R, et al. Nature (1997) 388:394-7; Muzio M, et al. J Immunol. (2000) 164:5998-6004], FC- and complement receptors [Id., citing Muzio M, et al. J Immunol. (2000) 164:5998-6004) and macropinocytosis (Id., citing Sallusto F, et al. J Exp Med. (1995) 182:389-400). Thus, immature DCs act not only as sentinels against invading pathogens [Id., citing Worbs T, et al. Nat Rev Immunol. (2017) 17:30-48, Wilson N S, et al. Blood (2004) 103:2187-95], but also as tissue scavengers, capturing apoptotic and necrotic cells [Id., citing Albert M L, et al. Nature (1998) 392:86-9).
[0329] This latter feature confers to immature DC an essential role in the induction and maintenance of immune tolerance [Id., citing Steinman R M, et al. Ann NY Acad Sci. (2003) 987:15-25, Castellano G, et al. Mol Immunol. (2004) 41:133-40; Deluce-Kakwata-Nkor N, et al. Transfus Clin Biol. (2018) 25:90-5; Liu J, Cao X. J Autoimmun. (2015) 63:1-12; Shiokawa A, et al. Immunology (2017) 152:52-64]. Apoptotic cells that arise in consequence of natural tissue turnover [Id., citing Huang F P, et al. J Exp Med. (2000) 191:435-44, Steinman R M, et al. J Exp Med. (2000) 191:411-416] are internalized by DCs but do not induce their maturation [Id., citing Steinman R M, et al. Ann NY Acad Sci. (2003) 987:15-25, Liu K, et al. J Exp Med. (2002) 196:1091-1097; Stuart L M, et al. J Immunol. (2002) 168:1627-35; Wallet M A, et al. J Exper Med. (2008) 205:219-32]. Thus, their antigens are presented to T cells without the activating co-stimulatory signals that a mature DC would deliver, resulting in T cell apoptosis [Id., citing Kurts C, et al. J Exp Med. (1997) 186:239-45, Hong J, et al. Chin Med J. (2013) 126:2139-44], anergy [Id., citing Manicassamy S, Pulendran B. Immunol Rev. (2011) 241:206-27, Zhu H-C, et al. Cell Immunol. (2012) 274:12-8] or development into Tregs [Id., citing Saito M, et al. J Exper Med. (2011) 208:235-49, Sela U, et al., PLoS ONE (2016) 11:e0146412).
[0330] These “tolerogenic DC” express less co-stimulatory molecules and proinflammatory cytokines, but upregulate the expression of inhibitory molecules (like PD-L1 and CTLA-4), secrete anti-inflammatory cytokines (IL-10, for example) [Id., citing Manicassamy S, Pulendran B. Immunol Rev. (2011) 241:206-27, Grohmann U, et al. Nat Immunol. (2002) 3:1097-101; Morelli A E, Thomson A W. Nature Rev Immunol. (2007) 7:610-21; Sakaguchi S, et al. Nat Rev Immunol. (2010) 10:490-500] and are essential to prevent responses against healthy tissues [Id., citing Hawiger D. J Exp Med. (2001) 194:769-79, Steinman R M, et al. Ann NY Acad Sci. (2003) 987:15-25, Idoyaga J, et al. J Clin Invest. (2013) 123:844-54; Mahnke K, et al. Blood (2003) 101:4862-9; Yates S F, et al. J Immunol (2007) 179:967-76; Yogev N, et al. Immunity (2012) 37:264-75].
[0331] However, in some contexts, immature DC can be harmful to the body. It is known that DC that are unable to induce lymphocyte effector responses may contribute to the immune system's failure to fight infections [Id., citing Campanelli A P, et al. J Infect Dis. (2006) 193:1313-22, Montagnoli C, et al. J Immunol. (2002) 169:6298-308] or tumors [Id., citing Baleeiro R B, et al. Cancer Immunol Immunother (2008) 57:1335-45; Almand B, et al. Clin Cancer Res. (2000) 6:1755-66; Bella S D, et al. Br J Cancer (2003) 89:1463-72; Dunn G P, et al. Immunity (2004) 21:137-48; Johnson D J, Ohashi P S. Anna NY Acad Sci. (2013) 1284:46-51; Vicari A P, et al. Semin Cancer Biol. (2002) 12:33-42]. In these situations, DC, even after recognition of pathogens or other changes in microenvironment, fail to increase the co-stimulatory molecules required to activate T cells, thus allowing the disease to “escape” immune control.
[0332] The term “DAD” as used herein refers to diffuse alveolar damage (DAD), which is manifested by injury to alveolar lining and endothelial cells, pulmonary edema, hyaline membrane formation and later by proliferative changes involving alveolar and bronchiolar lining cells and interstitial cells (Katzenstein, A L et al. Am J Pathol (1976) 85:209).
[0333] The term “derived from” as used herein, is meant to encompasses any method for receiving, obtaining, or modifying something from a source of origin.
[0334] The term “detectable marker” encompasses both selectable markers and assay markers. The term “selectable markers” refers to a variety of gene products to which cells transformed with an expression construct can be selected or screened, including drug-resistance markers, antigenic markers useful in fluorescence-activated cell sorting, adherence markers such as receptors for adherence ligands allowing selective adherence, and the like.
[0335] The term “detectable response” as used herein, is meant to refer to any signal or response that may be detected in an assay, which may be performed with or without a detection reagent. Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray diffraction. Alternatively, a detectable response may be the result of an assay to measure one or more properties of a biologic material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity or elemental composition. A “detection reagent” is any molecule that generates a detectable response indicative of the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences and enzymes. To facilitate detection, a detection reagent may comprise a marker.
[0336] The term “differentiate” and its various grammatical forms as used herein, are meant to refer to the process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied with a more specialized function.
[0337] The terms “disease” or “disorder” as used herein refer to an impairment of health or a condition of abnormal functioning.
[0338] The term “dose” as used herein, is meant to refer to the quantity of a therapeutic substance prescribed to be taken at one time. The term “maximum tolerated dose” as used herein is meant to refer to the highest dose of a drug or treatment that does not cause unacceptable side effects.
[0339] The term “dye” (also referred to as “fluorochrome” or “fluorophore”) as used herein refers to a component of a molecule which causes the molecule to be fluorescent. The component is a functional group in the molecule that absorbs energy of a specific wavelength and re-emits energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the dye and the chemical environment of the dye. Many dyes are known, including, but not limited to, FITC, R-phycoerythrin (PE), PE-Texas Red Tandem, PE-Cy5 Tandem, propidium iodem, EGFP, EYGP, ECF, DsRed, allophycocyanin (APC), PerCp, SYTOX Green, courmarin, Alexa Fluors (350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, chromomycin A3, mithramycin, YOYO-1, SYTOX Orange, ethidium bromide, 7-AAD, acridine orange, TOTO-1, TO-PRO-1, thiazole orange, TOTO-3, TO-PRO-3, thiazole orange, propidium iodide (PI), LDS 751, Indo-1, Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP, AmCyan1, Y77W, S65A, S65C, S65L, S65T, ZsGreen1, ZsYellow1, DsRed2, DsRed monomer, AsRed2, mRFP1, HcRed1, monochlorobimane, calcein, the DyLight Fluors, cyanine, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5 conjugates, PE-Cy7 conjugates, APC-Cy7 conjugates, Red 613, fluorescein, FluorX, BODIDY-FL, TRITC, X¬rhodamine, Lissamine Rhodamine B, Texas Red, TruRed, and derivatives thereof.
[0340] The term “ECOG performance status scale” as used herein refers to a scale used to assess how a patient's disease is progressing, assess how the disease affects the daily living abilities of the patient, and determine appropriate treatment and prognosis.
[0341] The term “effective dose” as used herein, generally refers to that amount of an immunogen comprising an internal conserved protein, or an immunogenic fragment thereof, of an infectious agent or pathogen described herein, or a vaccine comprising the immunogen, sufficient to induce immunity, to control and/or ameliorate an infection or to reduce at least one symptom of an infection and/or to enhance the efficacy of another dose of a immunogen or vaccine comprising the immunogen. An effective dose may refer to the amount of immunogen or vaccine comprising the immunogen sufficient to delay or minimize the onset of an infection. An effective dose may also refer to the amount of immunogen or vaccine comprising the immunogen that provides a therapeutic benefit in the treatment or management of an infection. Further, an effective dose is the amount with respect to an immunogen or vaccine comprising the immunogen of the disclosure alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection. An effective dose may also be the amount sufficient to enhance a subject's (e.g., a human's) own immune response against a subsequent exposure to an infectious agent. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and/or reduces the severity of symptoms.
[0342] The term “effective amount” as used herein, is meant to refer to an amount of immunogen or vaccine comprising the immunogen necessary or sufficient to realize a desired biologic effect. An effective amount of the composition would be the amount that achieves a selected result, and such an amount could be determined as a matter of routine experimentation by a person skilled in the art. For example, an effective amount for preventing, controlling, treating and/or ameliorating an infection could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to immunogens or vaccines comprising the immunogen of the disclosure. The term is also synonymous with “sufficient amount”
[0343] The term “effector cell” as used herein refers to a cell that carries out a final response or function. The main effector cells of the immune system, for example, are activated lymphocytes and phagocytes.
[0344] The term “effector functions” as used herein refers to the actions taken by effector cells and antibodies to eliminate foreign entities, and includes, without limitation, cytokine secretion, cytotoxicity, and antibody-mediated clearance.
[0345] The term “endogenous” as used herein refers to any material from or produced inside an organism, cell, tissue or system.
[0346] The term “enrich” as used herein refers to increasing the proportion of a desired substance, for example, to increase the relative frequency of a subtype of cell compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary to any enrichment scheme. Selection methods include, without limitation, magnetic separation and FACS. Regardless of the specific technology used for enrichment, the specific markers used in the selection process are critical, since developmental stages and activation-specific responses can change a cell's antigenic profile.
[0347] As used herein, the terms “expanding a population of cytokine killer T cells (CKTCs)” or “cytokine killer T cell (CKTC)expansion” are meant to refer to a process wherein a population of cytokine killer T cells undergoes a series of cell divisions and thereby expands in cell number (for example, by in vitro culture). The term “expanded superactivated cytokine killer T cells” relates to superactivated cytokine killer T cells obtained through cell expansion.
[0348] As used herein, the term “expression” is meant to encompass production of an observable phenotype by a gene, usually b directing the synthesis of a protein. It includes the biosynthesis of mRNA, polypeptide biosynthesis, polypeptide activation, e.g., by post-translational modification, or an activation of expression by changing the subcellular location or by recruitment to chromatin.
[0349] As used herein the term “Fas” is meant to refer to a type 2 membrane protein found on lymphocytes that belongs to the TNF superfamily. In cells that express Fas, engagement of the cell death receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation.
[0350] The term “flow cytometry” as used herein, is meant to refer to a tool for interrogating the phenotype and characteristics of cells. It senses cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area. The relative light-scattering and color-discriminated fluorescence of the microscopic particles is measured. Flow analysis and differentiation of the cells is based on size, granularity, and whether the cell is carrying fluorescent molecules in the form of either antibodies or dyes. As the cell passes through the laser beam, light is scattered in all directions, and the light scattered in the forward direction at low angles (0.5-10°) from the axis is proportional to the square of the radius of a sphere and so to the size of the cell or particle. Light may enter the cell; thus, the 90° light (right-angled, side) scatter may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, the differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content may be facilitated. Flow cytometers are multiparameter, recording several measurements on each cell; therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS), which allows isolation of distinct cell populations too similar in physical characteristics to be separated by size or density, uses fluorescent tags to detect surface proteins that are differentially expressed, allowing fine distinctions to be made among physically homogeneous populations of cells.
[0351] The terms “follicular helper T cell” (“T.sub.HF”), and “circulatory follicular helper CD4+ T cells” [“cT.sub.HF” ] as used herein are used interchangeably to refer to a type of effector CD4 T cell that resides in lymphoid follicles and provides help to B cells for antibody production.
[0352] As used herein, the terms “formulation” and “composition” are used interchangeably herein to refer to a product of the present disclosure that comprises all active and inert ingredients. The terms “pharmaceutical formulation” or “pharmaceutical composition” as used herein refer to a formulation or composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease.
[0353] The terms “functional equivalent” and “functionally equivalent” are used interchangeably herein to refer to substances, molecules, polynucleotides, proteins, peptides, or polypeptides having similar or identical effects or use.
[0354] The term “GALTs” as used herein refers to gut-associated lymphoid tissues, which are part of the mucosa-associated lymphoid tissues (MALTs). The histological components of GALTs mainly includes Peyer's patches, crypt patches, isolated lymphoid follicles (ILFs) appendix and mesenteric lymph nodes (mLNs). [Jiao, Y. et al., Crosstalk between gut microbiota and innate immunity and its implication in autoimmune disease. Front. Immunol. (2020) 11: 282; citing Brandtzaeg, P. et al. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol. (2008) 1: 31; Mowat, A M. Anatomical basis of tolerance and immunity to intestinal antigens. Nat. Rev. Immunol. (2003) 3: 331-41] constituent cells of GALTs include microfold (M) cells, which are capable of transferring antigens but not processing or presenting them [Id., citing Mabbott, N A et al. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium, Mucosal Immunol. (2013) 6:666]. Conventional lymphocytes, such as helper T cells (T.sub.H cells) (Id., citing Dunkley, M., Husband, A. Distribution and functional characteristics of antigen-specific helper T cells arising after Peyer's patch immunization. J. Immunol. (1987) 61: 475; Kiyono, H. e al. Murine Peyer's patch T cell clones. Characterization of antigen-specific helper T cells for immunoglobulin A responses. J. Exp. Med. (1982) 156: 1115-30]; Tregs (Id., citing Coombes, J L., et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGFβ-and-retinoid acid-dependent mechanism. J. Exp. Med. (2007) 204: 1757-64; Siddiqui, K., Powrie, F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol. (2008) 1 (Suppl. 1): 534-8), cytotoxic T lymphocytes (Id. citing Nelson, D L et al. Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT). Cell Immunol. (1976) 22: 65-75), IgA producing B cells (Id., citing Mora, J R et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science (2006) 314: 1157-60), phagocytes, including dendritic cells (Id., citing Coombes, J L., et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGFβ-and-retinoid acid-dependent mechanism. J. Exp. Med. (2007) 204: 1757-64; Siddiqui, K., Powrie, F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol. (2008) 1 (Suppl. 1): 534-8), macrophages, and other nonconventional lymphocytes, such as innate lymphoid cells (ILCs) (Id., citing Pearson, C. et al. Lymphoid microenvironments and innate lymphoid cells in the gut. Trends Immunol. (2012) 33: 289-96; Wojno, E D T; Artis, D. Innate lymphoid cells: balancing immunity, inflammation and tissue repair in the intestine. Cell Host Microbe (2012) 12: 445-57). The gut microbiota shapes the structural development of GALTs and primes its immune response to initiate host defense and to maintain tolerance against commensal bacteria via PRR-PAMP recognition and epigenetic modulators like short-chain fatty acids (SCFAs). [Id.]
[0355] The terms “GATA-3” and “GATA binding protein 3” are used interchangeably to refer to a member of the GATA family of conserved zinc-finger transcription factors, several of which are involved in hematopoiesis. GATA-3 is highly expressed in T cells and a wide variety of other tissues, including the CNS and fetal liver. In T cells, Gata3 acts at multiple stages of thymocyte differentiation. It is indispensable for early thymic progenitor differentiation [Hosoya, T. et al., J Exp Med. 2009 206(13):2987-3000] and for thymocytes to pass through beta selection and T cell commitment. Gata3 is also necessary for single-positive CD4 thymocyte development as well as for T.sub.H1-T.sub.H2 lineage commitment [Ting, C N et al., Nature. (1996) 384(6608):474-8; Thang, D H et al., J Biol Chem. (1997) 272(34):21597-603; Zheng W, Flavell R A. Cell. (1997) 89(4):587-96; Zhang, D H et al., J Immunol. (1998) 161(8):3817-21; Pai, S Y et al. Immunity (2003) 19(6):863-753]. As master regulator of Th2 lineage commitment, GATA3 acts either as a transcriptional activator or repressor through direct action at many critical loci encoding cytokines, cytokine receptors, signaling molecules as well as transcription factors that are involved in the regulation of T(h)1 and T(h)2 differentiation [Jenner, R G et al., Proc Natl Acad Sci USA. (2009) 106(42):17876-81]. For example, it regulates the expression of 7b2 lineage specific cytokine gene such as IL5 and represses the T.sub.H1 lineage specific genes IL-12 receptor β2 and STAT4 as well as neutralizing RUNX3 function through protein-protein interaction. Mice lacking Gata3 produce IFN-gamma rather than T.sub.H2 cytokines (IL5 and IL13) in response to infection [Zhu, J et al., Nat Immunol. (2004) 5(11):1157-65]. It acts in mutual opposition to the transcription factor T-bet, as T-bet promotes whereas GATA3 represses Fut7 transcription [Hwang, E S et al., Science. (2005) 21; 307(5708):430-3]. It also acts with Tbx21 to regulate cell lineage-specific expression of lymphocyte homing receptors and cytokine in both T.sub.H1 and T.sub.H2 lymphocyte subsets [Chen, G Y et al., Proc Natl Acad Sci USA. (2006) 103(45):16894-9]. Enforced expression of Gata3 during T cell development induced CD4(+)CD8(+) double-positive (DP) T cell lymphoma [Nawijn, M C et al., J Immunol. (2001) 167(2):724-32a; Nawijn, M C et al., J Immunol. (2001) 167(2):715-23]. Gata3 is essential for the expression of the cytokines IL-4, IL-5 and IL-13 that mediate allergic inflammation. Gata3 overexpression causes enhanced allergen-induced airway inflammation and airway remodeling, including subepithelial fibrosis, and smooth muscle cell hyperplasia [Kiwamoto, T et al., Am J Respir Crit Care Med. (2006) 174(2):142-51]. It additionally has a critical function in regulatory T cells and immune tolerance since deletion of Gata3 specifically in regulatory T cells led to a spontaneous inflammatory disorder in mice [Wang, Y et al., Immunity (2011) 35(3):337-48].
[0356] The term “graft-versus-host disease” or “GvHD”, as used herein refers to a complication of allogeneic transplantation in which donated T cells from a non-identical donor view the recipient's body as foreign and the donated cells attack the tissues of the recipient.
[0357] The term “granulocyte-macrophage colony-stimulating factor (GM-CSF) as used herein refers to a cytokine involved in the growth and differentiation of cells of the myeloid lineage, including dendritic cells, monocytes and tissue macrophages, and granulocytes.
[0358] The term “granulocytes” as used herein refers to myeloid leukocytes that harbor large intracellular granules containing microbe-destroying hydrolytic enzymes, and includes neutrophils, basophils and eosinophils. In synergy with other cytokines such as stem cell factor, IL-3, erythropoietin, and thrombopoietin, it also stimulates erythroid and megakaryocyte progenitor cells (Barreda, D R, et al, Developmental & Comparative Immunol. (2004) 28(50: 509-554). GM-CSF is produced by multiple cell types, including stromal cells, Paneth cells, macrophages, dendritic cells (DCs), endothelial cells, smooth muscle cells, fibroblasts, chondrocytes, and T.sub.H1 and T.sub.H17 T cells (Francisco-Cruz, A. et al, Medical Oncology (2014) 31: 774 et al.).
[0359] The term “helper T cells” or “T.sub.H” cells as used herein refers to effector CD4 T cells that stimulate or “help” B cells to make antibody in response to antigenic challenge. T.sub.H2, T.sub.H1 and the T.sub.HF subsets of effector CD4 T cells can perform this function.
[0360] The term “homeostatic proliferation” as used herein refers to a process of activation and proliferation of leukocytes in a lymphopenic environment. T cell homeostatic proliferation is driven by T cell receptor interactions with self-peptide-MHC complexes and responsiveness to homeostatic cytokines, such as IL7, IL-15, and possibly IL-21. [Gattinoni, L. et al. Natur Revs. Cancer 12: 671-684].
[0361] The term “herd immunity” as used herein refers to protection conferred to unvaccinated individuals in a population produced by vaccination of others and reduction in the natural reservoir for infection.
[0362] The term “heterologous immunity” as used herein refers to an immunity that can develop to one pathogen after a host has had exposure to non-identical pathogens.
[0363] The term “heterosubtypic immunity” (“HSI”) as used herein refers to immunity based on immune recognition of antigens conserved across all viral strains.
[0364] The term “heterotypic” as used herein is used to refer to being of a different or unusual type or form (e.g., different subgroup, subtype, strain and/or variant of a virus, bacteria, parasite or other pathogen).
[0365] The term “homotypic” as used herein is used to refer to being of the same type or form, e.g., same subgroup, subtype, strain and/or variant of a virus, bacteria, parasite or other pathogen.
[0366] The term “IL-4Rα” as used herein refers to the cytokine-binding receptor chain for IL-4.
[0367] The terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject's immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject. The term “immunological response” to an antigen or composition as used herein, is meant to refer to the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. One aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or γδ T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
[0368] The term “immune phenotype” or “immunotype” as used herein refers to the collective frequency of various immune cell populations and their functional responses to stimuli (cell signaling and antibody responses). [See Kaczorowski, K J et al. Proc. Nat. Acad. Sci. USA (2017) doi/10.1073/pnas.1705065114]
[0369] The terms “immune surveillance” or “immunological surveillance” are used interchangeably to refer to a monitoring process by the immune system to detect and destroy virally infected and neoplastically transformed cells in the body.
[0370] The term “immune system” as used herein refers to the body's system of defenses against disease, which comprises the innate immune system and the adaptive immune system. The innate immune system provides a non-specific first line of defense against pathogens. It comprises physical barriers (e.g. the skin) and both cellular (granulocytes, natural killer cells) and humoral (complement system) defense mechanisms. The reaction of the innate immune system is immediate, but unlike the adaptive immune system, it does not provide permanent immunity against pathogens. The adaptive immune response is the response of the vertebrate immune system to a specific antigen that typically generates immunological memory.
[0371] The term “immunocompromised” as used herein refers to having a weakened immune system and a reduced ability to fight infections and other diseases. The term “immunocompromised” as used herein refers to having a weakened immune system and a reduced ability to fight infections and other diseases. Immunocompromised subjects include patients receiving long-term (>3 months) or high-dose (>0.5 mg/kg/day) steroids or other immunosuppressant drugs, organ or bone marrow transplant recipients, patients with a solid tumor requiring chemotherapy in the last 5 years or with a hematological malignancy whatever the time since diagnosis and who received treatments, patients with leukemia or lymphoma, patients with primary immune deficiency; patients with HIV or AIDS; patients with autoimmune conditions, patients with asthma, which causes the immune system to overreact to harmless substances); patients of advanced age; and smokers.
[0372] The term “immunological repertoire” refers to the collection of transmembrane antigen-receptor proteins located on the surface of T and B cells. [Benichou, J. et al. Immunology (2011) 135: 183-191)] The combinatorial mechanism that is responsible for encoding the receptors does so by reshuffling the genetic code, with a potential to generate more than 10.sup.18 different T cell receptors (TCRs) in humans [Id., citing Venturi, Y. et al. Nat. Rev. Immunol. (2008) 8: 231-8] and a much more diverse B-cell repertoire. These sequences, in turn, will be transcribed and then translated into protein to be presented on the cell surface. The recombination process that rearranges the gene segments for the construction of the receptors is key to the development of the immune response, and the correct formation of the rearranged receptors is critical to their future binding affinity to antigen. For example, diversity of the TCR gene is generated by rearrangement of the V and J gene segments during T cell development in the thymus. (Makino, Y., et al (1993) J. Exptl Med. 177: 1399-1408). The TCR V and J gene segments, like Ig genes, possess recombination signals in which heptamer and nonamer sequences, separated by a 12/23 bp spacer, are flanked by germline V and J gene segments. Id.
[0373] The term “immunogen” and its various grammatical forms as used herein is used interchangeably with the term “antigen”.
[0374] The terms “immunomodulatory”, “immune modulator”, “immunomodulatory,” and “immune modulatory” are used interchangeably herein to refer to a substance, agent, or cell that is capable of augmenting or diminishing immune responses directly or indirectly, e.g., by expressing chemokines, cytokines and other mediators of immune responses.
[0375] As used herein, the term “immunostimulatory amount” refers to an amount of an immunogenic composition that stimulates an immune response by a measurable amount, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay.
[0376] As used herein the term “immunosuppressive amount” refers to an amount of an immunosuppressive composition that suppresses an immune response, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay.
[0377] The term “inflammasome” as used herein refers to a pro-inflammatory protein complex that is formed after stimulation of the intracellular NOD-like receptors. Production of an active caspase in the complex processes cytokine proteins into active cytokines.
[0378] The term “inflammation” as used herein refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators. Inflammation is often characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Traditionally, inflammation has been divided into acute and chronic responses.
[0379] The term “acute inflammation” as used herein refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes. Examples of injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents.
[0380] The term “chronic inflammation” as used herein refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity.
[0381] The term “inflammatory mediators” or “inflammatory cytokines” as used herein refers to the molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, and proinflammatory cytokines, including, but not limited to, interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IF-γ), and interleukin-12 (IL-12). Among the pro-inflammatory mediators, IL-1, IL-6, and TNF-α are known to activate hepatocytes in an acute phase response to synthesize acute-phase proteins that activate complement.
[0382] The term “infusion” as used herein refers to the introduction of fluid other than blood into a vein.
[0383] The terms “inhibiting”, “inhibit” or “inhibition” are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof. Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. The term “injury” as used herein refers to damage or harm to a structure or function of the body caused by an outside agent or force, which may be physical or chemical. As used herein, the term “interferon gamma” (IFN-y) is meant to refer to a soluble cytokine that is a member of the type II interferon class, which is secreted by cells of both the innate and adaptive immune systems. The active protein is a homodimer that binds to the interferon gamma receptor, which triggers a cellular response to viral and microbial infections.
[0384] The term “interleukin (IL)” as used herein refers to a cytokine secreted by, and acting on, leukocytes. Interleukins regulate cell growth, differentiation, and motility, and stimulates immune responses, such as inflammation. Examples of interleukins include, without limitation, interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin 37 (IL-37).
[0385] As used herein, the term “interleukin-2” (IL-2) is meant to refer to a type of cytokine made by a type of T-lymphocyte that increases the growth and activity of other T lymphocytes and B lymphocytes and affects the development of the immune system. IL-2 made in the laboratory is called aldesleukin.
[0386] As used herein, the term “interleukin 4” (IL-4) is a pleiotropic cytokine whose actions are generally antagonistic to those of interferon gamma. Because IL-4R is widely expressed, IL-4 influences almost all cell types. In T cells, IL-4 is crucial for the differentiation and growth of the Th2 subset. As such, IL-4 promotes the establishment of the humoral response necessary to combat pathogens that live and reproduce extracellularly. In B cells, IL-4 stimulates growth and differentiation and induces upregulation of MHC class II and FcεRII (CD23). IL-4 also promotes isotype switching in murine B cells to IgG1 and IgE but inhibits switching to IgG2a, IgG2b, and IgG3. IL-4 is a growth factor for mast cells and plays a major regulatory role in allergic responses since these involve IgE-mediated mast cell degranulation. IL-4 is also important for defense against helminth worms because the IgE production promoted by IL-4 allows eosinophils bearing FcεRIIB to carry out efficient ADCC. In macrophages, IL-4 inhibits the secretion of pro-inflammatory chemokines and cytokines such as TNF and IL-1β, impairs the ability of these cells to produce reactive oxygen and nitrogen intermediates, and blocks IFNγ-induced expression of cellular adhesion molecules such as ICAM and E-selectin. However, IL-4 can also induce DCs and macrophages to upregulate their synthesis of IL-12, supplying a negative feedback mechanism to regulate the Th2 response. Mak, T W, Saunders, M E, Chapter 17, “Cytokines and Cytokine Receptors,” in The Immune Response, Basic and Clinical Principles (2006), Academic Press, pp. 463-516).
[0387] As used herein, the terms “interleukin-7” (IL-7) also known as “lymphopoietin-1”, are meant to refer to a type of cytokine made by cells that cover and support organs, glands and other structures in the body that causes the growth of T lymphocytes and B lymphocytes.
[0388] As used herein, the term “interleukin-12” (IL-12) is meant to refer to a type of cytokine made mainly by B lymphocytes and macrophages that causes other immune cells to make cytokines and increase the growth of T lymphocytes. It may also block the growth of new blood vessels.
[0389] As used herein, the term “interleukin-15” (IL-15) is meant to refer to a type of cytokine that acts through its specific receptor, IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 regulates T and natural killer cell activation and proliferation. IL-15 and IL-2 share many biological activities. They are found to bind common hematopoietin receptor subunits, and may compete for the same receptor, and thus negatively regulate each other's activity. The number of CD8+ memory cells is shown to be controlled by a balance between IL-15 and IL2. IL-15 induces the activation of JAK kinases, as well as the phosphorylation and activation of transcription activators STAT3, STAT5, and STAT6. Studies of the mouse counterpart suggested that IL-15 may increase the expression of apoptosis inhibitor BCL2L1/BCL-x(L), possibly through the transcription activation activity of STAT6, and thus prevent apoptosis.
[0390] The term “isolated” is used herein to refer to material, such as, but not limited to, a nucleic acid, peptide, polypeptide, protein, or cell which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. The terms “substantially free” or “essentially free” are used herein to refer to considerably or significantly free of, or more than about 95% free of, or more than about 99% free of such components. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment.
[0391] The term “labeling” as used herein refers to a process of distinguishing a compound, structure, protein, peptide, antibody, cell or cell component by introducing a traceable constituent. Common traceable constituents include, but are not limited to, a fluorescent antibody, a fluorophore, a dye or a fluorescent dye, a stain or a fluorescent stain, a marker, a fluorescent marker, a chemical stain, a differential stain, a differential label, and a radioisotope.
[0392] The term “lectin” as used herein refers to a carbohydrate-binding protein.
[0393] The term “lymphocyte” refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte's surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions. Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells),
[0394] The term “lymphocyte activation” refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells. For example, a mature B cell can be activated by an encounter with an antigen that expresses epitopes that are recognized by its cell surface immunoglobulin Ig). The activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B cell activation) or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T cell (“cognate help process”). T-cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adapter proteins that link the TCR to the ras pathway, phospholipase Cγ1, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the antigen presenting cell (APC). The soluble product of an activated B lymphocyte is immunoglobulins (antibodies). The soluble product of an activated T lymphocyte is lymphokines (meaning cytokines produced by lymphocytes).
[0395] The term “macrophage” as used herein refers to a mononuclear, actively phagocytic cell arising from monocyte stem cells in the bone marrow. These cells are widely distributed in the body and vary in morphology and motility. Phagocytic activity is typically mediated by serum recognition factors, including certain immunoglobulins and components of the complement system, but also may be nonspecific. Macrophages also are involved in both the production of antibodies and in cell-mediated immune responses, particularly in presenting antigens to lymphocytes. They secrete a variety of immunoregulatory molecules.
[0396] The terms “Major Histocompatibility Complex (MHC), MHC-like molecule” and “HLA” are used interchangeably herein to refer to cell-surface molecules that display a molecular fraction known as an epitope or an antigen and mediate interactions of leukocytes with other leukocyte or body cells. MHCs are encoded by a large gene group and can be organized into three subgroups—class I, class II, and class III. In humans, the MHC gene complex is called HLA (“Human leukocyte antigen”); in mice, it is called H-2 (for “histocompatibility”). Both species have three main MHC class I genes, which are called HLA-A, HLA-B, and HLA-C in humans, and H2-K, H2-D and H2-L in the mouse. These encode the α chain of the respective MHC class I proteins. The other subunit of an MHC class I molecule is β2-microglobulin. The class II region includes the genes for the α and β chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ in humans. Also in the MHC class II region are the genes for the TAP:TAP2 peptide transporter, the PSMB (or LMP) genes that encode proteasome subunits, the genes encoding the DMα and BMO chains (DMA and DMB), the genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). The class II genes encode various other proteins with functions in immunity. The DMA and DMB genes encoding the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule. [Janeways Immunobiology. 9th ed., G S, Garland Science, Taylor & Francis Group, 2017. pps. 232-233]. In humans, there are three MHC class II isotypes: HLA-DR, HLA-DP, and HLA-DQ, encoded by α and β chain genes within the Human Leukocyte Antigen (HLA) locus on chromosome 6 [Wosen, J E et al. Front. Immunol. (2018) doi.10.3389/fimmu.2018.02144].
[0397] The term “MHC restriction” as used herein refers to the requirement that APC or target cells express MHC molecules that a T cell recognizes as self in order for T cell to respond to the antigen presented by that APC or target cell (T cells will only recognize antigens presented by their own MHC molecules). For example, CD8 T cells bind class I MHC which are expressed on most cells in the body, and CD4 T cells bind class II MHC which are only expressed on specialized APCs.
[0398] MHC-like molecules, while not encoded by the same gene group as true MHCs, have the same folding and overall structure of MHCs, specifically MHC class I molecules, and thus possesses similar biological functions, such as antigen presentation.
[0399] MR1, a nonclassical histocompatibility molecule which activates MAIT cells, and the CD1 family of molecules are examples of MHC-like molecules.
[0400] CD1 consists of two groups based on amino acid homology: group 1, which includes CD1a, b, and c; and group 2, which consists of CD1d. Exemplary CD1 antigenic ligands include, without limitation, dideoxymycobactin (Cd1a); glycose monomycolate (Cd1b), sulfatide (CD1a-d)mycolic acid, (CD1b), mannosyl-β1-phosphodolicol (Cd1c) alpha-galactosylceramide (alpha-GalCer, CD1d); phenylpentamethyl dihydrobenzofuransulphonate (CD1d), isoglobotriheoxylceramide (CD1d); palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (CD1d); and α-galacturonosyl ceramide (CD1d). Group 1 CD1 molecules can present antigens to a wide variety of T cells, whereas CD1d presents antigens mostly to NKT cells. (Brutkiewicz. “CD1d Ligands: The Good, the Bad, and the Ugly.” The Journal of Immunology (2006) 177 (2) 769-775). While CD1d structurally resembles MHC Class I molecules, it traffics through the endosome of the exogenous antigen presentation pathway. The binding groove of the CD1d molecule tethers the lipid tail of a glycolipid antigen, while the carbohydrate head group of the antigen projects out of the groove for recognition by the TCR of the NKT cell. (Wah, MakTak, et al. “Chapter 11: NK, γδ T and NKT Cells.” Primer to the Immune Response. Elsevier, 2014).
[0401] CD1 molecules are glycosylated heterodimers composed of a heavy chain polypeptide noncovalently associated with β2-microglobulin (β2m). Group I and II CD1 proteins are mainly expressed on cortical thymocytes, B-cells (CD1c) and antigen presenting cells (APC), such as dendritic cells (DC). The group II isoform, CD1d, is additionally expressed on macrophages, epithelial cells and hepatocytes [Id., citing Brigl, M.; Brenner, M B. Annu. Rev. Immunol. (2004) 22 (1): 817-90].
[0402] CD1 mediates T-cell responses through the presentation of self and foreign lipids, glycolipids, lipopeptides, or amphipathic small molecules to TCRs [Wu, D. et al. “Glycolipids as immunostimulating agents.” Bioorg. Med. Chem. (2008) 16 (3): 1073-83, citing Brigl, M., Brenner, M B. Annu Rev. Immunol. (2004) 22 (1): 817-90; Porcelli, S A, Modlin, R L. Annu. Rev. Immunol. (1999) 17 (1): 297-329; Savage, P B et al. Chemm. Socy Rev. (2007) 35 (9): 771-9].CD1d presents lipid antigens, and requires the presence of particular mechanisms to induce uptake of these molecules by APCs and subsequent loading onto CD1d molecules. Lipid transfer protein such as apolipoprotein E and fatty acid amide hydrolase (FAAH) have been shown to enhance the presentation of certain antigens by CD1d. Loading efficiency can be enhanced by specific proteins, such as saposins and microsomal triglyceride transfer protein, present in the endosomal and lysosomal compartments of cells by promoting lipid antigen exchange. Similar to MHC antigens, lipid antigens can also be processed by lysosomal enzymes to yield active compounds, as demonstrated in the case of CD1d for synthetic antigens, microbial antigens, and self-antigens. [Giradi and Zajonc (2012). “Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells.” Immunol Rev. 250(1): 167-179.]
[0403] MHC tetramers are used for the detection of antigen-specific T cell populations. CD1d tetramer is a reagent prepared by tetramerization of complexes of CD1d and β2m by PE- or APC-labeled streptavidin. Binding this reagent to α-GalCer enables highly sensitive detection of CD1d-restricted NKT cells. PBMCs are incubated at room temperature for 5 minutes with 40 μl of Clear Back (Human Fc receptor blocking reagent, MBL code no. MTG-001). CD3-FITC and human CD1d tetramer-PE (with or without binding of α-GalCer) are added and incubated for 30 minutes at 4° C. protected from light. Cells are analyzed by flow cytometry.
[0404] As used herein, the terms “marker” or “cell surface marker” are used interchangeably herein to refer to an antigenic determinant or epitope found on the surface of a specific type of cell. Cell surface markers can facilitate the characterization of a cell type, its identification, and eventually its isolation. Cell sorting techniques are based on cellular biomarkers where a cell surface marker(s) may be used for either positive selection or negative selection, i.e., for inclusion or exclusion, from a cell population.
[0405] The term “mediated” and its various grammatical forms as used herein refers to depending on, acting by or connected through some intervening agency.
[0406] The term “memory cells” as used herein refers to B and T lymphocytes generated during a primary immune response that remain in a quiescent state until fully activated by a subsequent exposure to specific antigen (secondary immune response). Memory cells generally are more sensitive than naïve lymphocytes to antigen and respond rapidly on reexposure to the antigen that originally induced them. During an immune response, naïve T cells (T.sub.N) are primed by antigen-presenting cells (APCs). Depending on the strength and quality of stimulatory signals, proliferating T cells progress along a differentiation pathway that culminates in the generation of terminally differentiated short-lived effector T (T.sub.EFF) cells. When antigenic and inflammatory stimuli cease, primed T cells become quiescent and enter into the memory stem cell (T.sub.SCM), central memory (T.sub.CM) cell or effector memory (T.sub.EM) cell pools, depending on the signal strength received. T.sub.SCM cells possess stem cell-like attributes to a greater extent than any other memory lymphocyte population. Although both T.sub.CM and T.sub.EM cells can also undergo self-renewal, the capacity to form diverse progeny is progressively restricted, so that only T.sub.SCM cells are capable of generating all three memory subsets and Tu cells; T.sub.CM cells can give rise to T.sub.CM, T.sub.EM and T.sub.EFF cells, and T.sub.EM cells can only produce themselves and T.sub.EFF cells. [Gattinoni, L. et al. Nature Revs. Cancer 12 (2012) 671-84].
[0407] The term “mitogen” as used herein refers to a substance that stimulates mitosis.
[0408] The term “mobilize” and its various grammatical forms as used herein refers to putting into motion or use; becoming ready; being capable of being moved quickly and with relative ease.
[0409] The term “modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.
[0410] The term “mucosa-associated lymphoid tissue” or MALT, as used herein is a generic term for all organized lymphoid tissue found at mucosal surfaces in which an adaptive immune response can be initiated. It comprises GALT, NALT and BALT (when present). The term “mucosa-associated invariant T cells (MAIT)” as used herein refers primarily to γδT cells with limited diversity present in the mucosal immune system that respond to bacterially derived folate derivatives presented by the nonclassical MHC class 1b molecule MR1.
[0411] The term “mucosal epithelia” as used herein refers to mucus-coated epithelia lining the body's internal cavities that connect with the outside (e.g., the gut, airways, and vaginal tract).
[0412] The term “mucosal mast cells” as used herein refers to specialized mast cells present in mucosa. They produce little histamine but large amounts of prostaglandins and leukotrienes.
[0413] As used herein, the term “mutation” refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).
[0414] The term “myeloid” as used herein means of or pertaining to bone marrow. Granulocytes and monocytes, collectively called myeloid cells, are differentiated descendants from common progenitors derived from hematopoietic stem cells in the bone marrow. Commitment to either lineage of myeloid cells is controlled by distinct transcription factors followed by terminal differentiation in response to specific colony-stimulating factors and release into the circulation. Upon pathogen invasion, myeloid cells are rapidly recruited into local tissues via various chemokine receptors, where they are activated for phagocytosis as well as secretion of inflammatory cytokines, thereby playing major roles in innate immunity. [Kawamoto, H., Minato, N. Intl J. Biochem. Cell Biol. (2004) 36 (8): 1374-9].
[0415] The abbreviation “NFκB” as used herein refers to a proinflammatory transcription factor that switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and inhibitors of apoptosis, resulting in amplification of the inflammatory response [Barnes, P J, (2016) Pharmacol. Rev. 68: 788-815]. The molecular pathways involved in NF-κB activation include several kinases. The classic (canonical) pathway for inflammatory stimuli and infections to activate NF-κB signaling involve the IKK (inhibitor of κB kinase) complex, which is composed of two catalytic subunits, IKK-α and IKK-β, and a regulatory subunit IKK-γ (or NFκB essential modulator [Id., citing Hayden, M S and Ghosh, S (2012) Genes Dev. 26: 203-234]. The IKK complex phosphorylates Nf-κB-bound IκBs, targeting them for degradation by the proteasome and thereby releasing NF-κB dimers that are composed of p65 and p50 subunits, which translocate to the nucleus where they bind to κB recognition sites in the promoter regions of inflammatory and immune genes, resulting in their transcriptional activation. This response depends mainly on the catalytic subunit IKK-β (also known as IKK2), which carries out IκB phosphorylation. The noncanonical (alternative) pathway involves the upstream kinase NF-κB-inducing kinase (NIK) that phosphorylates IKK-a homodimers and releases RelB and processes p100 to p52 in response to certain members of the TNF family, such as lymphotoxin-β [Id., citing Sun, S C. (2012) Immunol. Rev. 246: 125-140]. his pathway switches on different gene sets and may mediate different immune functions from the canonical pathway. Dominant-negative IKK-β inhibits most of the proinflammatory functions of NF-κB, whereas inhibiting IKK-a has a role only in response to limited stimuli and in certain cells, such as B-lymphocytes. The noncanonical pathway is involved in development of the immune system and in adaptive immune responses. The coactivator molecule CD40, which is expressed on antigen-presenting cells, such as dendritic cells and macrophages, activates the noncanonical pathway when it interacts with CD40L expressed on lymphocytes [Id., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol. 151: 179-89].
[0416] The term “natural killer (NK) cells” as used herein is meant to refer to lymphocytes in the same family as T and B cells, classified as group I innate lymphocytes. They have an ability to kill tumor cells without any priming or prior activation, in contrast to cytotoxic T cells, which need priming by antigen presenting cells. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response. Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by a balance of several germ-line encoded inhibitory and activating receptors such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs). The presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells. Engagement of KIR receptors blocks NK activation and, paradoxically, preserves their ability to respond to successive encounters by triggering inactivating signals. Therefore, if a KIR is able to sufficiently bind to MHC Class I, this engagement may override the signal for killing and allows the target cell to live. In contrast, if the NK cell is unable to sufficiently bind to MHC Class I on the target cell, killing of the target cell may proceed. Consequently, those tumors which express low MHC Class I and which are thought to be capable of evading a T-cell-mediated attack may be susceptible to an NK cell-mediated immune response instead.
[0417] The term “natural killer T cell” or “NKT” as used herein, is meant to refer to invariant natural killer T (iNKT) cells, also known as type-I NKT cells, as well as all subsets of non-invariant (Vα24− and Vα24+)natural killer T cells, which express CD3 and an αβ T cell receptor (TCR) (herein termed “natural killer αβ T cells”) or γδ TCR (herein termed “natural killer γδ T cells”), all of which have demonstrated capacity to respond to non-protein antigens presented by CD1 antigens. The non-invariant NKT cells share in common with type-I NKT cells the expression of surface receptors commonly attributed to natural killer (NK) cells, as well as a TCR of either αβ or γδ TCR gene locus rearrangement/recombination. Accordingly, as used herein, the term “NKT cells” refers to a population of cells that includes CD3+Vα24+ NKT cells, CD3+Vα24− NKT cells, CD3+Vα24− CD56+ NKT cells, CD3+Vα24−CD161+ NKT cells, CD3+γδ− TCR+ T cells, and mixtures thereof.
[0418] The term “invariant natural killer T cell” as used herein, is meant to be used interchangeably with the term “iNKT,” and is meant to refer to a subset of T-cell receptor (TCR)c-expressing cells that express a restricted TCR repertoire that, in humans, is composed of a Vα24-Jα18 TCRα chain, which is, for example, coupled with a Vβ11 TCRβ chain. iNKT is meant to encompass all subsets of CD3+Vα24+ type-I NKT cells (CD3+CD4+CD8−Vα24+, CD3+CD4−CD8+Vα24−+, and CD3+CD4−CD8−Vα24+) as well as those cells, which can be confirmed to be type-I NKT cells by gene expression or other immune profiling, but have down-regulated surface expression of Vα24 (CD3+Vα24−). This includes cells which either do or do not express the regulatory transcription factor FOXP3. Unlike conventional T cells, which mostly recognize peptide antigens presented by MHC molecules, iNKT cells recognize glycolipid antigens presented by the non-polymorphic MHC class 1-like CD1d.
[0419] The term “nebulizer” as used herein refers to a device used to administer liquid medication in the form of a mist inhaled into the lungs.
[0420] The term “neutrophils” or “polymorphonuclear neutrophils (PMNs)” as used herein refers to the most abundant type of white blood cells in mammals, which form an essential part of the innate immune system. They form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils. Neutrophils are normally found in the blood stream. During the beginning (acute) phase of inflammation, particularly as a result of bacterial infection and some cancers, neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as interleukin-8 (IL-8) and C5a in a process called chemotaxis, meaning the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent.
[0421] The term “non-expanded” as used herein, is meant to refer to a cell population that has not been grown in culture (in vitro) to increase the number of cells in the cell population.
[0422] The term “non-replicating” or “replication-impaired” virus refers to a virus that is not capable of replication to any significant extent in the majority of normal mammalian cells or normal primary human cells.
[0423] The term “normal healthy subject” as used herein refers to a subject having no symptoms or other evidence of a viral infection.
[0424] The term “Nucleotide-binding Oligomerization Domain (NOD)-like receptors (NLRs)” as used herein refers to innate sensors that detect microbial products or cellular damage in the cytoplasm or activate signaling pathways, and are expressed in cells that are routinely exposed to bacteria, such as epithelial cells, macrophages and dendritic cells. Some NLRs activate NFκB to initiate the same inflammatory responses as the TLRs, while others trigger a distinct pathway that induces cell death and the production of pro-inflammatory cytokines. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96].
[0425] Subfamilies of NLRs can be distinguished based on the other protein domains they contain. For example the NOD subfamily has an amino-terminal caspase recruitment domain (CARD), which is structurally related to the T1R death domain in MyD88, and can dimerize with CARD domains on other proteins to induce signaling. NOD proteins recognize fragments of bacterial cell wall peptidoglycans, although it is not known if they do so through direct binding or through accessory proteins. Id. At 96. NOD1 senses γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycans of Gram negative and some Gram positive bacteria, whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycans of most bacteria. Id. Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their function is less well understood. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96-98]
[0426] When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine-threonine kinase RIP2 (also known as RICK and RIPK2), which associates with the E3 ligases cIAP1, CIAP2, and XIAP, whose activity generates a polyubiquitin scaffold, which recruits TAK1 and IKK and results in activation of NFκB. NFκB then induces the expression of genes for inflammatory cytokines and for enzymes involved in the production of NO. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 97].
[0427] Macrophages and dendritic cells express both TLRs and NOD1 and NOD2, and are activated by both pathways. In epithelial cells, NOD1 may also function as a systemic activator of innate immunity. NOD2 is strongly expressed in the Paneth cells of the gut where it regulates the expression of potent anti-microbial peptides such as the α- and β-defensins. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 97].
[0428] Other members of the NOD family, including NLRX1 and NLRC5, have been identified, but their function is less well understood. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 96-98]
[0429] The NLRP family, another subfamily of NLR proteins, has a pyrin domain in place of the CARD domain at their amino termini. Humans have 14 NLR proteins containing pyrin domains, of which NLRP3 (also known as NAPL3 or cryopyrin) is the best characterized. NLRP3 resides in an inactive form in the cytoplasm, where its leucine rich repeat (LRR) domains are thought to bind the head-shock chaperone protein HSP90 and the co-chaperone SGT1. NRLP3 signaling is induced by reduced intracellular potassium, the generation of reactive oxygen species, or the disruption of lysosomes by particulate or crystalline matter. For example, death of nearby cells can release ATP into the extracellular space, which would activate the purinergic receptor P2X7, which is a potassium channel, and allow potassium ion efflux. A model proposed for ROS-induced NLRP3 activation involves intermediate oxidation of sensor proteins collectively called thioredoxin (TRX). Normally TRX proteins are bound to thioredoxin-interacting protein (TXNIP). Oxidation of TRX by ROS causes dissociation of TXNIP from TRX. The free TXNIP may then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both cases, NLRP3 activation involves aggregation of multiple monomers via their leucine-rich repeat (LRR) and NOD domains to induce signaling. Phagocytosis of particulate matter (e.g. the adjuvant alum), may lead to the rupture of lysosomes and release of the active protease cathepsin B, which can activate NLRP3. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 98-99].
[0430] NLR signaling, as exemplified by NLRP3, leads to the generation of pro-inflammatory cytokines and to cell death through formation of an inflammasome, a multiprotein complex. Activation of the inflammasome proceeds in several stages. Aggregation of NLRP molecules triggers autocleavage of procaspase I, which releases active caspase 1—Aggregation of LRR domains of several NLRP3 molecules, or other NLRP molecules by a specific trigger or recognition event, which induces the pyrin domains of NLRP3 to interact with pyrin domains of ASC (also called PYCARD), an adaptor protein composed of an amino terminal pyrin domain and a carboxy terminal CARD domain, which further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and the CARD domains facing outward; the CARD domains then interact with CARD domains of the inactive protease pro-caspase 1, initiating its CARD-dependent polymerization into discrete caspase 1 filaments. Active caspase 1 then carries out ATP-dependent proteolytic processing of proinflammatory cytokines, particularly 1L-1β and IL-18, into their active forms, and induces a form of cell death (pyroptosis) associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 99-1001.
[0431] A priming step, which can result from TLR signaling, must first occur in which cells inducer and translate the mRNAs that encode the pro-forms of IL-1, IL-18 or other cytokines for inflammasome activation to produce inflammatory cytokines. For example, the TLR-3 agonist poly I:C can be used experimentally to prime cells for triggering of the inflammasome. Janeways Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 1001.
[0432] Inflammasome activation also can involve proteins of the PYHIN family, which have an H inversion (HIN) domain in place of an LRR domain. There are four PYIN proteins in humans. Id. At 100. A noncanonical inflammasome (caspase I-independent) pathway uses the protease caspase 11, which therefore is both a sensor and an effector molecule, to detect intracellular LPS. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 1011.
[0433] Besides activating effector functions and cytokine production, another outcome of the activation of innate sensing pathways is the induction of co-stimulatory molecules on tissue dendritic cells and macrophages. B7.1 (CD80) and B7.2 (CD86), for example, which are induced on macrophages and tissue dendritic cells by innate sensors such as TLRs in response to pathogenic recognition, are recognized by specific co-stimulatory receptors expressed by cells of the adaptive immune response, particularly CD4 T cells, and their activation by B7 is an important step in activating adaptive immune responses. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017, at 105].
[0434] The term “overall survival” (OS) as used herein, is meant to refer to the length of time from either the date of diagnosis or the start of treatment for a disease that patients diagnosed with the disease are still alive.
[0435] The term “oxygen saturation” (SpO.sub.2) as used herein refers to a measurement of how much oxygen the blood is carrying as a percentage of the maximum it could carry. For a healthy individual, the normal SpO.sub.2 should be between 96% to 99%.
[0436] The term “parenteral” as used herein refers to introduction into the body by means other than through the digestive tract, for example, without limitation, by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle), intravenously (i.e., an injection into a vein), or infusion techniques.
[0437] The term “pathogenesis” as used herein refers to the development of a disease and the chain of events leading to that disease and its sequelae.
[0438] The term “pathological” as used herein refers to indicative of or caused by disease.
[0439] The term “pathophysiology” and its various grammatical forms as used herein refers to derangement of function in an individual or organ due to a disease.
[0440] The term “pattern recognition receptors” or “PRRs” as used herein, is meant to refer to receptors that are present at the cell surface to recognize extracellular pathogens; in the endosomes where they sense intracellular invaders, and finally in the cytoplasm. They recognize conserved molecular structures of pathogens, called pathogen associated molecular patterns (PAMPs) specific to the microorganism and essential for its viability. PRRs are divided into four families: toll-like receptors (TLR); nucleotide oligomerization receptors (NLR); C-type leptin receptors (CLR), and RIG-1 like receptors (RLR).
[0441] The term “peptide” is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. Peptides are typically 9 amino acids in length, but can be as short as 8 amino acids in length, and as long as 14 amino acids in length. A series of amino acids are considered an “oligopeptide” when the amino acid length is greater than about 14 amino acids in length, typically up to about 30 to 40 residues in length. When the amino acid residue length exceeds 40 amino acid residues, the series of amino acid residues is termed a “polypeptide”.
[0442] As used herein, the term “perforin” is meant to refer to a molecule that can insert into the membrane of target cells and promote lysis of those target cells. Perforin-mediated lysis is enhanced by enzymes called granzymes.
[0443] The terms “peripheral blood mononuclear cells” or “PBMCs” are used interchangeably herein to refer to blood cells having a single round nucleus such as, for example, a lymphocyte or a monocyte. When a Ficoll fractionation of peripheral blood method is used, PBMCs remain at the less dense, upper interface of the Ficoll layer, often referred to as the buffy coat, and are the cells collected. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, and only a small percentage of dendritic cells.
[0444] The term “pharmaceutical composition” is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease.
[0445] The term “pharmaceutically acceptable carrier” as used herein is meant to refer to any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated polypeptide of the present disclosure will remain stable and bioavailable. The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
[0446] The term “pharmaceutically acceptable salt” as used herein is meant to refer to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts may be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. By “pharmaceutically acceptable salt” is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe pharmaceutically acceptable salts in detail in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH, Zurich, Switzerland: 2002). The salts may be prepared in situ during the final isolation and purification of the compounds described within the present disclosure or separately by reacting a free base function with a suitable organic acid. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts may be prepared in situ during the final isolation and purification of compounds described within the disclosure by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like. Pharmaceutically acceptable salts also may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium or magnesium) salts of carboxylic acids may also be made.
[0447] The term “plasma cell” as used herein refers to terminally differentiated B cells that secrete antibody. They may be short-lived, with no isotype switching or somatic hypermutation, or long lived, meaning they undergo isotype switching and somatic hypermutation.
[0448] The term “plasmablasts” as used herein refer to proliferating progeny of an activated B cell. Plasmablasts become plasma cells. Antigen binding to the BCR triggers activation of Src family kinases such as Lyn and Fyn leading to phosphorylation of Igα (CD79a) and Igβ (CD79b), recruitment of Syk kinase and subsequent recruitment and phosphorylation of BLNK, Btk and PLCγ [Luo, W. et al. J. Immunol. (2014) 193(2): 909-20, citing Packard, T A & Cambier, J C. F1000 prime reports (2013) 5: 40]. These events activate the Ras pathway, PKC pathway and calcium flux, eventually triggering the activation of NF-κB, Erk and JNK. These positive signals are normally counterbalanced by negative signals that limit B cell activation and prevent spontaneous B cell proliferation and differentiation to plasma cells [Id., citing Nitschke, L. Curr. Opin. Immunol. (2005) 17: 2990-97]. Negative signals are generated by a series of membrane receptors (CD22, CD72, FcγRIIb, PIR-B, Siglec-G, etc.) that are phosphorylated by Lyn. This allows them to recruit phosphatases such as SHP1 and SHIP1 that reverse phosphorylation of signaling molecules in the BCR pathway and dampen BCR signaling [Id., citing Poe, J C & Tedder, T F, Trends Immunol. (2012) 33: 413-20; Tsubata, T. Infectious disorders drug targets (2012) 12: 181-90; Vang, T. et al. Annu. Rev. Immunol. (2008) 26: 29-55].
[0449] The term “Plasmalyte A” as used herein refers to a sterile, nonpyrogenic isotonic solution in a single dose container for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C.sub.6H.sub.11NaO.sub.7); 368 mg of Sodium Acetate Trihydrate, USP (C.sub.2H.sub.3NaO.sub.2.3H.sub.2O); 37 mg of Potassium Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCl.sub.2.6H.sub.2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
[0450] The term “potentiate” and its other grammatical forms as used herein means to increase the power, effect, or potency, of; to enhance, to augment the activity of.
[0451] The term “prevention” as used herein, is meant to refer to a process of prophylaxis in which an animal (e.g., a mammal, and most especially a human) is exposed to an immunogen of the present disclosure prior to the induction or onset of the disease process. This could be done where an individual is at high risk for any viral infection based on the living or travel to the virus pandemic areas. Alternatively, the immunogen could be administered to the general population as is frequently done for any infectious diseases. Alternatively, the term “suppression” is often used to describe a condition wherein the disease process has already begun but obvious symptoms of said condition have yet to be realized. Thus, the cells of an individual may have been infected but no outside signs of the disease have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression.
[0452] The term “priming” as used herein refers to the process whereby T cells and B cell precursors encounter the antigen for which they are specific. The term “unprimed cells” (also referred to as virgin, naïve, or inexperienced cells) as used herein refers to T cells and B cells that have generated an antigen receptor (TCR for T cells, BCR for B cells) of a particular specificity, but have never encountered the antigen. For example, before helper T cells and B cells can interact to produce specific antibody, the antigen-specific T cell precursors must be primed.
[0453] Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation. [Janeway, C A, Jr., “The priming of helper T cells, Semin. Immunol. (1989) 1(1): 13-20]. Helper T cells express CD4, but not all CD4 T cells are helper cells. Id. The signals required for clonal expansion of helper T cells differ from those required by other CD4 T cells. The critical antigen-presenting cell for helper T cell priming appears to be a macrophage; and the critical second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1). Id. If the primed T cells and/or B cells receive a second, co-stimulatory signal, they become activated T cells or B cells.
[0454] The term “progression” as used herein refers to the course of a disease as it becomes worse or spreads in the body.
[0455] The term “proliferate” and its various grammatical forms as used herein is meant to refer to the process that results in an increase of the number of cells, and is defined by the balance between cell division and cell loss through cell death or differentiation.
[0456] The term “protect” or “protection of” a subject from developing a disease or from becoming susceptible to an infection as referred herein means to partially or fully protect a subject. As used herein, to “fully protect” means that a treated subject does not develop a disease or infection caused by an agent such as a virus, bacterium, fungus, protozoa, helminth, and parasites, or caused by a cancer cell. To “partially protect” as used herein means that a certain subset of subjects may be fully protected from developing a disease or infection after treatment, or that the subject does not develop a disease or infection with the same severity as an untreated subject.
[0457] The term “protective immune response” or “protective response” as used herein, is meant to refer to an immune response mediated by antibodies against an infectious agent, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof. Vaccines of the present disclosure can stimulate the production of antibodies that, for example, neutralize infectious agents, block infectious agents from entering cells, block replication of said infectious agents, and/or otherwise protect host cells from infection and destruction. The term can also refer to an immune response that is mediated by T-lymphocytes and/or other white blood cells against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates a viral infection or reduces at least one symptom thereof.
[0458] As used herein, the term “purify” is meant to refer to freeing from extraneous or undesirable elements.
[0459] The term “pyroptosis” as used herein refers to a form of programmed cell death that is associated with abundant pro-inflammatory cytokines, such as IL-1β and IL-18 produced through inflammasome activation.
[0460] The term “reduce” and its various grammatical forms as used herein refers to a diminution, a decrease, an attenuation or abatement of a degree, intensity, extent, size, amount, density or number.
[0461] The Renin-Angiotensin-aldosterone System (RAAS) or renin-angiotensin system (RAS) is a critical regulator of blood volume and systemic vascular resistance. It is composed of three major compounds: renin, angiotensin II, and aldosterone, which act to elevate arterial pressure in response to decreased renal blood pressure, decreased salt delivery to the distal convoluted tubule, and/or beta agonism.
[0462] Angiotensin II (Ang II), the primary physiological product of the RAAS system, is a potent vasoconstrictor. Angiotensin converting enzyme (ACE) catalyzes the transformation of angiotensin I (Ang I) to Ang II. Ang II elicits its effects by activating two receptors: type 1 angiotensin II (AT1) receptor and type 2 angiotensin II (AT2) receptor [Ingraham, N E, et al. Eur. Respir. J. (2020); DOI: 10.1183/13993003.00912-2020, citing Balakumar, P. & Jagadeesh, G. Cell Signal (2014) 26: 2147-60]. Ang II action through AT1 receptor causes a cascade with resultant inflammation, vasoconstriction, and atherogenesis [Id., citing Strawn, W B & Ferrario, C M., Curr Opin. Lipido. (2002) 13: 505-12]. These effects also promote insulin resistance and thrombosis [Id., citing Dandona, P. et al. J. Hum. Hypertens. (2007) 21: 20-27]. In contrast, AT2 receptor stimulation causes vasodilation, decreased platelet aggregation, and the promotion of insulin action. However, the expression of AT2 receptor is low in healthy adults [Id., citing Dandona, P. et al. J. Hum. Hypertens. (2007) 21: 20-27]. As such, Ang II's effects in adults are modulated and balanced indirectly by angiotensin II converting enzyme (ACE2), which converts Ang II into lung-protective Angiotensin-(1-7) (Ang-[1-7]), similar to effects seen from AT2 receptor stimulation [Id., citing Ghazi, L. & Grawz, P. F1000Research 2017; 6: F1000, Faculty Rev-1297. doi:10.12688/f1000research.9692.1; Warner, F J et al. Cell Mol. Life Sci. (2004) 61: 2704-13].
[0463] The term “restore” and its various grammatical forms as used herein refers to bringing back to a former or normal condition, to recover or renew.
[0464] The term “retinoic acid receptor (RAR) and “retinoid X receptor” as used herein refer to nuclear hormone receptors that mediate both the organismal and cellular effects of intracellular retinoic acids and their synthetic analogs. The terms RORγt and RORα as used herein refer to transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in T.sub.H17 cells and have been suggested to play a role in T.sub.H17 differentiation.
[0465] The term “secondary lymphoid tissues” as used herein refers to sites where lymphocytes interact with each other and nonlymphoid cells to generate immune responses to antigens. These include the spleen, lymph nodes, and mucosa-associated lymphoid tissues (MALT).
[0466] As used herein, the term “secretion” and its various grammatical forms is meant to refer to production by a cell of a physiologically active substance and its movement out of the cell in which it is formed.
[0467] The term “senescence” as used herein refers to a biological process by which cells undergo growth arrest after extensive replication.
[0468] The term “sequelae” and its various grammatical forms as used herein means a pathological condition resulting from a prior disease, injury or attack.
[0469] The term “shock” as used herein refers to a critical condition brought on by a sudden drop in blood flow through the body, where the circulatory system fails to maintain adequate blood flow, sharply curtailing the delivery of oxygen and nutrients to vital organs.
[0470] The term “sign” as used herein refers to a healthcare provider's evidence of disease.
[0471] The term “specification” as used herein refers to a list of tests, references to analytical procedures, and appropriate acceptance criteria that are numerical limits, ranges or other criteria for the test described that establishes the set of criteria to which material should conform to be considered acceptable for its intended use. The term “conformance to specification” means that the material, when tested according to the listed analytical procedures, will meet the listed acceptance criteria.
[0472] As used herein, the term “stimulate” in any of its grammatical forms as used herein is meant to refer to inducing activation or increasing activity.
[0473] The term “stimulate an immune cell” or “stimulating an immune cell” as used herein is meant to refer to a process (e.g., involving a signaling event or stimulus) causing or resulting in a cellular response, such as activation and/or expansion, of an immune cell, e.g. a CD8+ T cell.
[0474] The term “subject” as used herein is meant to refer to any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The present disclosure above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.
[0475] As used herein, the phrase “subject in need thereof” is meant to refer to a patient that (i) will be administered an immunogenic composition (e.g. a population of SCKTCs) according to the described invention, (ii) is receiving an immunogenic composition (e.g. a population of SCKTCs) according to the described invention; or (iii) has received an immunogenic composition (e.g. a population of SCKTCs) according to the described invention, unless the context and usage of the phrase indicates otherwise.
[0476] As used herein, the term “sufficient to stimulate cytokine killer T cell (CKTC) cell expansion” refers to an amount or level of a signaling event or stimulus, e.g. an amount of alpha-galactosylceramide (αGalCer), or an analog or functional equivalent thereof, that promotes preferential expansion of a CKT cell.
[0477] As used herein, the term “sufficient to stimulate CKT cell activation” refers to an amount or level of a signaling event or stimulus, e.g. an amount of IL-2, IL-7, IL-15 and IL-12, that promotes cytokine secretion or cell-killing activity of a CKT cell.
[0478] As used herein, the term “superactivated cytokine killer T cells” (or SCKTCs) refers to cells derived from cytokine killer T cells (CKTCs) by contacting CKTCs in vitro with cytokines IL-2, IL-7, IL-15 and IL-12 in a predetermined order and time of addition.
[0479] The term “susceptible subject” as used herein refers to an individual vulnerable to developing infection when their body is invaded by an infectious agent. Examples of individuals vulnerable to developing a serious lung infection include, without limitation, the very young, the elderly, those who are ill; those who are receiving immunosuppressants; those with long term health conditions; those that are obese; and those who are physically weak, e.g., due to malnutrition or dehydration.
[0480] The term “symptom” as used herein refers to a patient's subjective evidence of disease.
[0481] The term “Tbet” as used herein refers to a T.sub.H1 cell transcription factor. Differential expression of the T.sub.H1 cell transcription factor T bet and a closely related T-box family transcription, factor particularly in CD8+ T cells, Eomesodermin (Eomes) facilitates the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection. [Paley, M A et a., Science (2012) 338: 1220-125]. During chronic infections, T-bet is reduced in virus-specific CD8+ T cells; this reduction correlates with T cell dysfunction. In contrast, Eomes mRNA expression is up-regulated in exhausted CD8+ T cells during chronic infection. [Id.]
[0482] The terms “T cell” or “T lymphocyte” or are used interchangeably to refer to cells that mediate a wide range of immunologic functions, including the capacity to help B cells develop into antibody-producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. T cells recognize antigens on the surface of antigen presenting cells (APCs) and mediate their functions by interacting with, and altering, the behavior of these APCs. T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells. T-cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adapter proteins that link the TCR to the ras pathway, phospholipase Cγ1, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the antigen presenting cell (APC).
[0483] Although the lineage relationship between T cell subsets remains controversial, T cells cluster in populations that can be arranged as a progressive continuum on the basis of phenotypic, functional and transcriptional attributes. T lymphocytes transition through progressive stages of differentiation that are characterized by a stepwise loss of functional and therapeutic potential in the order from naive T (T.sub.N) cells to T memory stem cells (T.sub.SCM) (the most immature antigen experienced T cells), to T central memory (T.sub.CM) cells, which patrol central lymphoid organs, to Teffector memory (T.sub.EM) cells, which patrol peripheral tissues. In contrast to T.sub.N cells, memory T cells are capable of rapidly releasing cytokines on restimulation. T.sub.CM cells more efficiently secrete IL-2 and T.sub.EM have an increased capacity for IFNγ release and cytotoxicity. All antigen-experienced T cells upregulate the common IL-2 and IL-15β receptor (IL-2RD) conferring the ability to undergo homeostatic proliferation in response to IL-15, and also display high amounts of CD95 (also known as FAS), a receptor that provides either costimulatory or pro-apoptotic signals depending on the efficiency of CD95 signaling complex formation and on which particular intracellular signaling proteins are part of the complex. [Gattinoni, L. et al. Natur Revs. Cancer 12: 671-684].
[0484] The term “T cell antigen” as used herein is meant to refer to a protein or fragment thereof which can be processed into a peptide that can bind to either Class I MHC, Class II MHC, non-classical MHC, or CD1 family molecules (collectively antigen presenting molecules), and in this combination can engage a T cell receptor on a T cell.
[0485] The term “T cell epitope” as used herein is meant to refer to a short peptide molecule that binds to a class I or II MHC molecule and that is subsequently recognized by a T cell. T cell epitopes that bind to class I MHC molecules are typically 8-14 amino acids in length, and most typically 9 amino acids in length. T cell epitopes that bind to class II MHC molecules are typically 12-20 amino acids in length. In the case of epitopes that bind to class II MHC molecules, the same T cell epitope may share a common core segment, but differ in the length of the carboxy- and amino-terminal flanking sequences due to the fact that ends of the peptide molecule are not buried in the structure of the class II MHC molecule peptide-binding cleft as they are in the class I MHC molecule peptide-binding cleft.
[0486] The term “T cell exhaustion” as used herein refers to a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Modulating pathways overexpressed in exhaustion—for example, by targeting programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4)—can reverse this dysfunctional state and reinvigorate immune responses [Wherry E J and Kurachi, M. Nature (2015) 15: 486-99, citing Wherry E J. Nat. Immunol. (2011) 131:492-499; Schietinger A, Greenberg P D. Trends Immunol. (2014) 35:51-60; Barber D L, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. (2006) 439:682-687; Nguyen L T, Ohashi P S. Nat. Rev. Immunol. (2014) 15:45-56]. The level and duration of chronic antigen stimulation and infection seem to be key factors that lead to T cell exhaustion and correlate with the severity of dysfunction during chronic infection. Examples of inhibitory receptors include the inhibitory pathways mediated by PD1 in response to binding of PD1 ligand 1 (PDL1) and/or PDL2. [Id., citing Okazaki T, et al., Nature Immunol. (2013) 14:1212-1218, Odorizzi P M, Wherry E J. J. Immunol. (2012) 188:2957-2965, Araki K, et al. Cold Spring Harb. Symp. Quant. Biol. (2013) 78:239-247]. Exhausted T cells can co-express PD1 together with lymphocyte activation gene 3 protein (LAG3), 2B4 (also known as CD244), CD160, T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM3; also known as HAVCR2), CTLA4 and many other inhibitory receptors [Id., citing Blackburn S D, et al. Nat. Immunol. (2009) 10:29-37]. Typically, the higher the number of inhibitory receptors co-expressed by exhausted T cells, the more severe the exhaustion. It has been suggested that inhibitory receptors such as PD1 might regulate T cell function in several ways [Id., citing Schietinger A, Greenberg P D. Trends Immunol. (2014) 35:51-60; Odorizzi P M, Wherry E J. J. Immunol. (2012) 188:2957-29651, e.g., by ectodomain competition, which refers to inhibitory receptors sequestering target receptors or ligands and/or preventing the optimal formation of microclusters and lipid rafts (for example, CTLA4); second, through modulation of intracellular mediators, which can cause local and transient intracellular attenuation of positive signals from activating receptors such as the TCR and co-stimulatory receptors [Id., citing Parry R V, et al. Molec. Cell. Biol. (2005) 25:9543-9553; Yokosuka T, et al. J. Exp. Med. (2012) 209:1201-1217; Clayton K L, et al. J. Immunol. (2014) 192:782-7911; and third, through the induction of inhibitory genes [Id., citing Quigley M, et al. Nat. Med. (2010) 16:1147-1151]. Co-stimulatory receptors also are involved in T cell exhaustion [Id., citing Odorizzi P M, Wherry E J. J. Immunol. (2012) 188:2957-29651. For example, desensitization of co-stimulatory pathway signaling through the loss of adaptor molecules can serve as a mechanism of T cell dysfunction during chronic infection. The signaling adaptor tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) is downregulated in dysfunctional T cells in HIV progressors, as well as in chronic LCMV infection [Id., citing Wang C, et al. J. Exp. Med. (2012) 209:77-911. Adoptive transfer of CD8+ T cells expressing TRAF1 enhanced control of chronic LCMV infection compared with transfer of TRAF1-deficient CD8+ T cells, which indicates a crucial role for TRAF1-dependent co-stimulatory pathways in this setting [Id., citing Wang C, et al. J. Exp. Med. (2012) 209:77-911. It has also been possible to exploit the potential beneficial role of co-stimulation to reverse exhaustion by combining agonistic antibodies to positive co-stimulatory pathways with blockade of inhibitory pathways. 4-1BB (also known as CD137 and TNFRSF9) is a TNFR family member and positive co-stimulatory molecule that is expressed on activated T cells. Combining PD1 blockade and treatment with an agonistic antibody to 4-1BB dramatically improved exhausted T cell function and viral control [Id, citing Vezys V, et al. J. Immunol. (2011) 187:1634-16421. Soluble molecules are a second class of signals that regulate T cell exhaustion; these include immunosuppressive cytokines such as IL-10 and transforming growth factor-β (TGFβ) and inflammatory cytokines, such as type I interferons (IFNs) and IL-6. [Id.]
[0487] The term “T cell mediated immune response” as used herein is meant to refer to a response that occurs as a result of recognition of a T cell antigen bound to an antigen presenting molecule on the cell surface of an APC, coupled with other interactions between costimulatory molecules on the T cell and APC. This response serves to induce T cell proliferation, migration, and production of effector molecules, including cytokines and other factors that can injure cells.
[0488] The term “T cell receptor” (TCR) as used herein, is meant to refer to a complex of integral membrane proteins that participate in the activation of T cells in response to an antigen. The TCR expressed by the majority of T cells consisting of α and β chains. A small group of T cells express receptors made of γ and δ chains. Among the α/β T cells are two sublineages: those that express the coreceptor molecule CD4 (CD4+ cells), and those that express CD8 (CD8+ cells). These cells differ in how they recognize antigen and in their effector and regulatory functions. CD4+ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms. CD8+ T cells, can develop into cytotoxic T-lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs.
[0489] Naive conventional CD4 T cells can differentiate into four distinct T cell populations, a process that is determined by the pattern of signals they receive during their initial interaction with antigen. These 4 T cell populations are T.sub.H1, T.sub.H2, T.sub.H17, and induced regulatory T (iTreg) cells. Th1 cells, which are effective inducers of cellular immune responses, mediate immune responses against intracellular pathogens, and are responsible for the induction of some autoimmune diseases. Their principal cytokine products are IFNγ (which enhances several mechanisms important in activating macrophages to increase their microbiocidal activity), lymphotoxin α (LTα), and IL-2, which is important for CD4 T cell memory. Th2 cells, which are effective in helping B cells develop into antibody producing cells, mediate host defense against extracellular parasites, are important in the induction and persistence of asthma and other allergic disease, and produce IL-4, IL-5, IL-9, IL-10 (which suppresses T.sub.H1 cell proliferation and can suppress dendritic cell function), IL-13, IL-25 (signaling through IL-17RB, enhances the production of IL-4, IL-5, and IL-13 by a c-kit-FcεRI-nonlymphocyte population, serves as an initiation factor as well as an amplification factor for T.sub.H2 responses) and amphiregulin. IL-4 and IL-10 produced by T.sub.H2 cells block IFNγ production by T.sub.H1 cells. T.sub.H17 cells produce IL-17a, IL-17f, IL-21, and IL-22. IL-17a can induce many inflammatory cytokines, IL6 as well as chemokines such as IL-8 and plays an important role in inducing inflammatory responses. Treg cells play a critical role in maintaining self-tolerance and in regulating immune responses. They exert their suppressive function through several mechanisms, some of which require cell-cell contact. The molecular basis of suppression in some cases is through their production of cytokines, including TGFβ, IL-10, and IL-35. TGFβ produced by T reg cells may also result in the induction if iTreg cells from naïve CD4 T cells. CD4+ T-cells bear receptors on their surface specific for the B-cell's class II/peptide complex. B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B-cell activation. Zhu, J. and Paul, W E, Blood (2008) 112: 1557-69). Resting naïve CD8+ T cells, when primed by antigen presenting cells that have acquired antigens from the infected macrophages through direct infection or cross-presentation in secondary lymphoid organs, such as lymph nodes and spleen, react to pathogens by massive expansion and differentiation into cytotoxic T lymphocyte effector cells that migrate to all corners of the body to clear the infection. In the majority of viral infections, however, CD8 T cell activation requires CD4 effector T cell help to activate dendritic cells for them to become able to stimulate a complete CD8 T cell response. CD4 T cells that recognize related antigens presented by the APC can amplify the activation of naïve CD8 T cells by further activating the APC. B7 expressed by the dendritic cell first activates the CD4 T cells to express IL-2 and CD40 ligand. CD40 ligand binds CD40 on the dendritic cell, delivering an additional signal that increases the expression of B7 and 4-1BBL by the dendritic cell, which in turn provides additional co-stimulation to the naïve CD8 T cell. The IL-2 produced by activated CD4 T cells also acts to promote effector CD T cell differentiation.
[0490] The CD3 (TCR complex) is a protein complex composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains, which associate with the T cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. Together, the TCR, the ζ-chain and CD3 molecules comprise the TCR complex. The intracellular tails of CD3 molecules contain a conserved motif known as the immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling capacity of the TCR. Upon phosphorylation of the ITAM, the CD3 chain can bind ZAP70 (zeta associated protein), a kinase involved in the signaling cascade of the T cell.
[0491] The term “T follicular helper (T.sub.FH) cells” as used herein refers to a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They develop within secondary lymphoid organs (SLO) and can be identified based on their unique surface phenotype, cytokine secretion profile, and signature transcription factor. They support B cells to produce high-affinity antibodies toward antigens, in order to develop a robust humoral immune response and are crucial for the generation of B cell memory. They are essential for infectious disease control and optimal antibody responses after vaccination. Stringent control of their production and function is critically important, both for the induction of an optimal humoral response against thymus-dependent antigens but also for the prevention of self-reactivity. [Gensous, N. et al. Front. Immunol. (2018) doi.org/10.3389/finmmu.2018.01637).
[0492] The term “T.sub.H1 cells” as used herein refers to a lineage of CD4+ effector T cells that promotes cell-mediated immune responses and is required for host defense against intracellular viral and bacterial pathogens. They are mainly involved in activating macrophages but can also help stimulate B cells to produce antibody. T.sub.H1 cells secrete IFN-gamma, IL-2, IL-10, and TNF-alpha/beta. IL-12 and IFN-γ make naive CD4+ T cells highly express T-bet and STAT4 and differentiate to T.sub.H1 cells. (Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15-44)/
[0493] The term “T.sub.H2 cells” as used herein refers to a lineage of CD4+ effector T cells that secrete IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25. These cells are required for humoral or antibody-mediated immunity and play an important role in coordinating the immune response to large extracellular pathogens. IL-4 makes naive CD4+ T cells highly express STAT6 and GATA3 and differentiate to T.sub.H2 cells. (Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15-44)/
[0494] The term “T.sub.H17 cells” as used herein refers to a CD4+ T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues, resulting in production of inflammatory cytokines and recruitment of leukocytes, especially neutrophils, thus creating a link between innate and adaptive immunity. [Tesmer, L A, et al., Immunol. Rev. (2008) 223: 87-113]. The key transcription factor in T.sub.H17 cell development is RORγt.
[0495] The term “Treg” or “regulatory T cells” as used herein refers to effector CD4 T cells that inhibit T cell responses and are involved in controlling immune reactions and preventing autoimmunity. The natural regulatory T cell lineage that is produced in the thymus is one subset. The induced regulatory T cells that differentiate from naïve CD4 T cells in the periphery in certain cytokine environments is another subset. Tregs are most commonly identified as CD3+CD4+CD25+FoxP3+ cells in both mice and humans. Additional cell surface markers include CD39, 5′ Nucleotidase/CD73, CTLA-4, GITR, LAG-3, LRRC32, and Neuropilin-1. Tregs can also be identified based on the secretion of immunosuppressive cytokines including TGF-beta, IL-10, and IL-35. Cell surface molecules CTLA-4, LAG-3, and neuropilin-1 (Nrp1) impair dendritic cell (DC)-mediated Tconv activation: CTLA-4 and LAG-3 outcompete CD28 and T cell receptor expressed on conventional T cells for binding to CD80/86 and MHC class II on DCs, and Nrp1 stabilizes DC-Treg contact, thereby preventing antigen presentation to conventional T cells [Ikebuchi, R. et al. Front. Immunol. (2019) doi.org/10.3389/finmmu.2019.01098].
[0496] The terms “therapeutic amount”, “effective amount”, an “amount effective”, or “pharmaceutically effective amount” of an active agent are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “therapeutic amount”, “effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described disclosure. In prophylactic or preventative applications of the described disclosure, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.
[0497] The term “therapeutic effect” as used herein is meant to refer to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.
[0498] For any therapeutic agent described herein the therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.
[0499] General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.
[0500] Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug's plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.
[0501] Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the rates and extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.
[0502] The term “therapeutic window” as used herein is meant to refer to a concentration range that provides therapeutic efficacy without unacceptable toxicity. Following administration of a dose of a drug, its effects usually show a characteristic temporal pattern. A lag period is present before the drug concentration exceeds the minimum effective concentration (“MEC”) for the desired effect. Following onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This reaches a peak, after which drug elimination results in a decline in the effect's intensity that disappears when the drug concentration falls back below the MEC. Accordingly, the duration of a drug's action is determined by the time period over which concentrations exceed the MEC. The therapeutic goal is to obtain and maintain concentrations within the therapeutic window for the desired response with a minimum of toxicity. Drug response below the MEC for the desired effect will be subtherapeutic, whereas for an adverse effect, the probability of toxicity will increase above the MEC. Increasing or decreasing drug dosage shifts the response curve up or down the intensity scale and is used to modulate the drug's effect. Increasing the dose also prolongs a drug's duration of action but at the risk of increasing the likelihood of adverse effects. Accordingly, unless the drug is nontoxic, increasing the dose is not a useful strategy for extending a drug's duration of action.
[0503] The term “thrombosis” as used herein refers to the formation of a blood clot (thrombus) within a blood vessel, which prevents blood from flowing normally through the circulatory system. For example, endothelial infection with influenza virus has been shown to increase the adhesion of human platelets to primary human lung microvascular endothelial cells via fibronectin, contributing to mortality from acute lung injury. [Sugiyama, M G et al. J. Virol. (2016) 90 (4): 1812-21] A blood clot that forms in the veins (a venous thromboembolism) can cause deep vein thrombosis and pulmonary embolisms. Deep vein thrombosis (DVT) occurs when a blood clot forms in a major vein, usually in the leg, which stops blood from flowing easily through the vein, which can lead to swelling, discoloration and pain. Patients with DVT are at risk for developing post-thrombotic syndrome (PTS), which can involve chronic leg swelling, calf pain calf heaviness/fatigue, skin discoloration and/or venous ulcers. A pulmonary embolism (PE) is a blood clot that has traveled to the lungs. It often starts as a DVT where a piece of the clot breaks off and is carried to the lungs. PE can block the flow of blood to the lungs, causing serious damage to the lungs and affecting a person's ability to breath, which can lead to serious injury and death A blood clot that forms in the arteries (atherothrombosis) can lead to heart attack and stroke.
[0504] The term “tissue-resident memory T cell” or “T.sub.RM” as used herein refers to memory lymphocytes that do not migrate after taking up residence in barrier tissues, where they are retained long term. They appear to be specialized for rapid effector function after restimulation with antigen or cytokines at sites of pathogen entry.
[0505] The term “toll-like receptor (TLR)” as used herein refers to innate receptors on macrophages, dendritic cells, and some other cells, that recognize pathogens and their products, such as bacterial lipopolysaccharide (LPS). Recognition stimulates the receptor-bearing cells to produce cytokines that help initiate immune responses. For example, TLR-1 is a cell surface toll-like receptor that acts in a heterodimer with TLR-2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-2 is a cell surface toll-like receptor that acts in a heterodimer with either TLR-1 or TLR-6 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-4 is a cell surface toll-like receptor that, in conjunction with accessory proteins MD-2 and CD14, recognizes bacterial lipopolysaccharide and lipoteichoic acid. TLR5 is a cell surface toll-like receptor that recognizes the flagellin protein of bacterial flagella. TLR 6 is a cell surface toll-like receptor that acts in a heterodimer with TLR2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR3 is an endosomal toll-like receptor that recognizes double-stranded viral RNA. TLR-7 is an endosomal toll-like receptor that recognizes single-stranded viral RNA. TLR-8 is an endosomal toll-like receptor that recognizes single-stranded viral RNA. TLR-9 is an endosomal toll-like receptor that recognizes DNA containing unmethylated CpG.
[0506] The term “TRAIL” as used herein refers to tumor necrosis factor-related apoptosis-inducing ligand, a member of the TNF cytokine family expressed on the cell surface of some cells, e.g., NK cells, that induces cell death in target cells by ligation of the “death” receptors DR4 and DR5.
[0507] The term “treat” or “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).
[0508] The term “treatment” as used herein is meant to refer to one or more of (i) the prevention of infection or reinfection, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
[0509] The term “TRIM21” as used herein refers to tripartite motif-containing 21, a cytosolic Fc receptor and E3 ligase that is activated by IgG and can ubiquitinate viral proteins after an antibody coated virus enters the cytoplasm.
[0510] The term “TRIM25” as used herein refers to an E3 ubiquitin ligase involved in signaling by RIG-1 and MDA-5 for the activation of MAVs.
[0511] The terms “variants”, “mutants”, and “derivatives” are used herein to refer to nucleotide or polypeptide sequences with substantial identity to a reference nucleotide or polypeptide sequence. The differences in the sequences may be the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” or “derivatives” of the original sequence. A skilled artisan likewise can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements, but biologically equivalent to the wild type sequence. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, for example, an epitope for an antibody. The techniques for obtaining such variants, including, but not limited to, genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the skilled artisan.
[0512] The term “vascular permeability” as used herein means the net amount of a solute, typically a macromolecule, that has crossed a vascular bed and accumulated in the interstitium in response to a vascular permeabilizing agent or at a site of pathological angiogenesis. [Nagy, J A, et al. Angiogenesis (2008) 11(2): 1009-119].
[0513] The term “virus immune escape” or “virus escape” as used herein refers to mechanisms by which viruses evade the immune system of the host.
[0514] The terms “viral load” or “viral burden” as used herein refer to a measurement of the amount of a virus in an organism, typically in the bloodstream, usually stated in virus particles per milliliter.
[0515] The term “wild-type” as used herein refers to the most common phenotype of an organism, strain, gene, protein, nucleic acid, or characteristic as it occurs in nature. The terms “wild-type” and “naturally occurring” are used interchangeably.
EMBODIMENTS
1. Method of Preparing a Cell Product
[0516] According to one aspect, the present disclosure describes a method for preparing a pharmaceutical composition comprising an enriched population of superactivated cytokine killer T cells (SCKTCs) comprising, in order
[0517] (a) isolating a population of mononuclear cells (MCs) comprising a population of cytokine killer T cells (CKTCs);
[0518] (b) transporting the preparation of (a) to a processing facility under sterile conditions;
[0519] (c) on Day 0, placing the population of MCs in a suspension culture system in a serum free culture medium;
[0520] (d) on Day 6, contacting the culture system of step (c) with the serum free culture medium containing IL-2 and IL-7,wherein the contacting stimulates CKTC activation;
[0521] (e) on Day 7, pulsing the CKTCs of step (d) with an enriched population of CD1d− expressing antigen presenting cells (APCs) derived from the MCs in (a) loaded with a-GalCer;
[0522] (f) on Day 8-13, replenishing the culture medium every 1-3 days from day 7 to day 14 with fresh serum-free culture medium;
[0523] (g) on Day 14, adding CD1d expressing APCs loaded with α-GalCer;
[0524] (h) on Day 14+1 to Day 14+6, replenishing the culture medium of the cells with fresh serum-free culture medium every 1-3 days;
[0525] (i) on Day 14+7 replenishing the culture medium of the culture with fresh serum-free culture medium and pulsing with CD1d expressing APCs loaded with α-GalCer;
[0526] (j) on Day 14+8 to Day 14+13, replenishing the culture medium of the culture with fresh serum-free culture medium;
[0527] on Day 14+14, replenishing the culture medium of the culture with fresh serum-free culture medium and pulsing with CD1d-expressing APCs loaded with α-GalCer;
[0528] (k) on Day 14+15 to Day 14+20, replenishing the culture medium of the culture with fresh serum-free culture medium;
[0529] on day 14+21 replenishing the culture medium of the culture with fresh serum-free culture medium and adding IL-12;
[0530] (l) on Day 14+22 harvesting the amplified enriched superactivated population of SCKTCs from the culture system to form a SCKTC cell product; and
[0531] (m) filling and finishing aliquots of the SCKTC cell product comprising 2×10.sup.8-1×10.sup.9 SCKTCs into a container;
[0532] (n) optionally cryopreserving the SCKTC cell product in the vapor phase of a liquid nitrogen freezer in a serum-free cryo freezing medium.
[0533] According to some embodiments, the method further comprises transporting the SCKTC cell product from the processing facility to a treatment facility. According to some embodiments, the transporting step is initiated within at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours of the addition of IL-12.
[0534] According to some embodiments, in step (a) the frequency of the population of CKTCs from the donor represents <0.5% of the total MNC population.
[0535] According to some embodiments, activation and expansion steps of the method are performed in tissue culture flasks. According to some embodiments, activation and expansion steps of the method are performed in gas permeable cell culture bags. According to some embodiments, activation and expansion steps of the method are performed in a closed system. According to some embodiments, the closed system is fully automated.
[0536] According to some embodiments, the population of PBMCs comprises subpopulations of T lymphocytes, NK cells, B lymphocytes, and monocytes. According to some embodiments, the subpopulation of T lymphocytes comprises NKT cells, CD4+ T cells, and CD8+ T cells.
[0537] According to some embodiments, the SCKTC cell product in step (l) is proliferation competent.
[0538] According to some embodiments, a source of the mononuclear cells (MCs) is blood. According to some such embodiments, the blood is peripheral blood and the MCs are peripheral blood MCs (PBMCs). According to some embodiments, the PBMCs are derived from a human subject. According to some embodiments, the donor of the MCs is autologous to the recipient. According to some embodiments, the donor of the MCs is allogeneic to the recipient.
[0539] According to some embodiments, leukapheresis is performed at a blood collection center. PBMCs then are isolated using an apparatus containing a spinning chamber (e.g., a Sepax c-Pro System (Cytiva)). The blood separates into its components (plasma, platelet-rich plasma, leukocytes and red blood cells) by gravity along the wall of the chamber. Mononuclear cells are sorted out and collected.
[0540] According to some embodiments, the MCs can be isolated from whole peripheral blood at room temperature as follows. 2 ml of defibrinated or anti-coagulin-treated blood and an equal volume of balanced salt solution is added to a 10 ml centrifuge tube. The blood and buffer are mixed. Ficoll-Paque media (3 ml—Cytiva) is added to the centrifuge tube. The diluted blood sample (4 ml) is layered onto the Ficoll-Paque media solution and centrifuged at 400 g for 30-40 min with the brake off. The upper layer containing plasma and platelets is drawn off, leaving the mononuclear cell layer undisturbed at the interface. The layer of mononuclear cells is transferred to a sterile centrifuge tube using a sterile pipette and washed with centrifugation.
[0541] According to some embodiments, the population of MCs comprising a population of CKTCs can be derived from stem cells. The term “stem cells” as used herein refers to undifferentiated cells having high proliferative potential with the ability to self-renew that can generate daughter cells that can undergo terminal differentiation into more than one distinct cell phenotype. Stem cells are distinguished from other cell types by two characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions.
[0542] Embryonic stem cells (EmSC) are stem cells derived from an embryo that are pluripotent, i.e., they are able to differentiate in vitro into endodermal, mesodermal and ectodermal cell types. Induced pluripotent stem cells (iPSCs) offer an extensive capacity for self-renewal without the ethical concerns faced by EmSCs. iPSCs can be induced and redifferentiated to cells in the immune system, specifically to HSCs and fully differentiated immune cells, including NIT cells [Jiang, Z. et al. Cellular & Molec. Immunol. (2014) 11: 17-24].
[0543] Adult (somatic) stem cells are undifferentiated cells found among differentiated cells in a tissue or organ. Their primary role in vivo is to maintain and repair the tissue in which they are found. Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscles, skin, teeth, gastrointestinal tract, liver, ovarian epithelium, and testis. Adult stem cells are thought to reside in a specific area of each tissue, known as a stem cell niche, where they may remain quiescent (non-dividing) for long periods of time until they are activated by a normal need for more cells to maintain tissue, or by disease or tissue injury.
[0544] According to some embodiments, the stem cells comprise hematopoietic stem cells. Hematopoietic stem cells (also known as the colony-forming unit of the myeloid and lymphoid cells (CFU-M,L), HSCs, or CD34+ cells) are rare pluripotential cells within the blood-forming organs that are responsible for the continued production of blood cells during life. While there is no single cell surface marker exclusively expressed by hematopoietic stem cells, it generally has been accepted that human HSCs have the following antigenic profile: CD34+, CD59+, Thy1+(CD90), CD38low/−, C-kit−/low and, lin−. HSCs can generate a variety of cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, and the T and B lymphocytes.
[0545] According to some embodiments, the HSCs can be derived from adult bone marrow, umbilical cord, umbilical cord blood, placental tissue, or fetal liver. According to some embodiments, the HSCs can be purified by positive or negative selection cell separation methods. Positive selection cell separation methods involve directly labeling desired cells for selection with an antibody or a ligand that targets a specific cell surface protein. In immunomagnetic separation methods, the antibody or ligand is linked to a magnetic particle, allowing the labeled cells to be retained in the final isolated fraction after incubation of the same in a magnetic field. Negative selection cell separation methods involve laveling unwanted cell types for removal with antibodies or ligands targeting specific cell surface proteins. In immunomagnetic separation methods, the antibodies or ligands are linked to magnetic particles, allowing the labeled, unwanted cells to be depleted from the final isolated fraction by incubating the sample in a magnetic field. Since the desired cells are not specifically targeted by antibodies or ligands, they remain unbound by particles. According to some embodiments, magnetic bead activated cell sorting, a positive selection technique, can be used for purifying the CD34+ cell population from the mononuclear cells. According to some embodiments, negative selection protocols can be employed to reduce the risk of decreasing the quantity and activity of the desired cells such protocols. According to some embodiments a pure SCKTC population is achieved from HSCs by the method without positive or negative cell separation methods.
Antigen Presenting Cells
[0546] An antigen presenting cell is a class of cell capable of displaying on its surface one or more antigens in the form of a peptide-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. Examples of professional APCs are dendritic cells and macrophages, although any cell expressing MHC Class I molecules or MHC Class II molecules can potentially present peptide antigen. According to some embodiments, an APC can be a cell or population of cells that is engineered to present one or more antigens (i.e. an artificial APC (aAPC). According to some embodiments, an APC can be irradiated population of PBMCs. According to some embodiments, the irradiated population of pBMCs comprises a subpopulation of cells expressing CD1d.
[0547] According to some embodiments, the antigen is a non-peptide antigen. According to some embodiments, the antigen is a lipid antigen. According to some embodiments, the antigen is alpha-GalCer. According to some embodiments, the population of APCs loaded with alpha-GalCer is a population of dendritic cells. According to some embodiments, a population of dendritic cells loaded with αGalCer is prepared by a method comprising (a) isolating a population of CD14+ mononuclear cells (MCs); (b) culturing the population of CD14+ MCs in a culture system; thereby inducing differentiation of the CD14+ MCs into dendritic cells; (c) contacting the culture system with αGalCer, wherein the contacting is sufficient to load the dendritic cells with αGalCer.
(a) Isolating a Population of CD14+ MCs
[0548] Monocytes are circulating blood leukocytes with a fundamental capacity to differentiate into macrophages. In the right environment, monocytes can also differentiate into specialized antigen-presenting dendritic cells (moDCs). [Qu, C. et al. Intl J. Infectious Disease (2014) 19: 1-5]. The major subset of monocytes consists of CD14.sup.high CD16.sup.negative (CD14.sup.++CD16.sup.−). The CD16 expressing monocytes are usually divided into CD14.sup.high CD16.sup.low (CD14.sup.++CD16.sup.+) and CD14.sup.low CD16.sup.high(CD.sup.14+CD16.sup.++) subsets [Id., citing Ziegler-Heitbrock, L. et al. Blood (2010) 116: e74-e80]; both subsets of monocytes can differentiate into dendritic cells (DCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 when cultured in vitro. They internalize soluble and particulate antigens similarly, and both are able to stimulate T cell proliferation in autologous and allogeneic cultures [Id., citing Sanchez-Torres, C. et al., Int'l Immunol. (2001) 13: 1571-81; Sallusto, F. and Lanzavecchia, A. J. Exp. Med. (1994) 179: 1109-18; Romani, N. et al. J. Exp. Med. (1994) 180: 83-93]. However, CD16.sup.+ moDCs express higher levels of CD86, CD11a, and CD11c, and show lower expression of CD1a and CD32 compared to CD16.sup.− moDCs. LPS-stimulated CD16.sup.− moDCs express increased levels of IL-12 p40 mRNA and secrete greater amounts of IL-12 p70 than CD16.sup.+ moDCs, whereas levels of transforming growth factor beta 1 (TGF-β1) mRNA are higher in CD16.sup.+ moDCs. Moreover, CD4+ T cells stimulated with CD16.sup.+ moDCs secrete increased amounts of IL-4 compared to those stimulated by CD16.sup.− moDCs [Id., citing Sanchez-Torres, C. et al. Intl. Immunol. (2001) 13: 1571-81]. Using an in vitro transendothelial migration model, monocytes were demonstrated to migrate across an endothelial barrier in vitro and differentiate into DCs, which reverse-migrate back across the endothelial layer, or into macrophages, which remain in the subendothelial matrix [Id., citing Randolph, G J et al. Blood (1998) 92: 4167-77]. In this model, the CD14+CD16+ monocytes were found to be more likely to become DCs than the CD14.sup.+CD16.sup.− monocytes [Id., citing Randolph, G J et al. J. Exp. Med. (2002) 196: 517-27], indicating that the CD14.sup.+CD16.sup.+ monocytes might be precursors of DCs. The classical CD14+ monocytes develop the non-classical CD14.sup.+CD16.sup.+ monocytes; CD14.sup.+CD16.sup.+ monocytes may represent a more mature version. Id., citing Randolph, G J J. Exp. Med. (2002) 196: 517-27].
[0549] According to some embodiments, CD14+ monocytes are sorted out of a population of PBMCs using CD14+ microbeads (e.g., Miltenyi, Dynabeads™). For MACS separation, cells are magnetically labeled with CD14 microbeads and separated on a column which is placed in the magnetic field of a MACS separator. The magnetically labeled CD14+ cells are retained in the column while the unlabeled CD14− cells, which are depleted of CD14+ cells run through. After removal of the column from the magnetic field, the magnetically retained CD14+ cells can be eluted as a positively selected cell fraction. According to some embodiments, the eluted CD14+ monocytes are viable.
(b) Culturing the CD14+ MCs to Induce Differentiation of DCs
[0550] According to some embodiments, a viable enriched population of DCs is prepared from about 5×10.sup.8 to 5×10.sup.9 MCs, i.e., about 5.0×10.sup.8, about 5.1×10.sup.8, about 5.2×10.sup.8, about 5.3×10.sup.8, about 5.4×10.sup.8, about 5.5×10.sup.8, about 5.6×10.sup.8, about 5.7×10.sup.8, about 5.8×10.sup.8, about 5.9×10.sup.8, about 6.0×10.sup.8, about 6.1×10.sup.8, about 6.2×10.sup.8, about 6.3×10.sup.8, about 6.4×10.sup.8, about 6.5×10.sup.8, about 6.6×10.sup.8, about 6.7×10.sup.8, about 6.8×10.sup.8, about 6.9×10.sup.8, about 7.0×10.sup.8, about 7.1×10.sup.8, about 7.2×10.sup.8, about 7.3×10.sup.8, about 7.4×10.sup.8, about 7.5×10.sup.8, about 7.6×10.sup.8, about 7.7×10.sup.8, about 7.8×10.sup.8, about 7.9×10.sup.8, about 8.0×10.sup.8, about 8.1×10.sup.8, about 8.2×10.sup.8, about 8.3×10.sup.8, about 8.4×10.sup.8, about 8.5×10.sup.8, about 8.6×10.sup.8, about 8.7×10.sup.8, about 8.8×10.sup.8, about 8.9×10.sup.8, about 9.0×10.sup.8, about 9.1×10.sup.8, about 9.2×10.sup.8, about 9.3×10.sup.8, about 9.4×10.sup.8, about 9.×10.sup.8, about 9.6×10.sup.8, about 9.7×10.sup.8, about 9.8×10.sup.8, about 9.9×10.sup.8, about 1×10.sup.9, about 1.1×10.sup.9, about 1.2×10.sup.9, about 1.3×10.sup.9, about 1.4×10.sup.9, about 1.5×10.sup.9, about 1.6×10.sup.9, about 1.7×10.sup.9, about 1.8×10.sup.9, about 1.9×10.sup.9, about 2.0×10.sup.9, about 2.1×10.sup.9, about 2.2×10.sup.9, about 2.3×10.sup.9, about 2.4×10.sup.9, about 2.5×10.sup.9, about 2.6×10.sup.9, about 2.7×10.sup.9, about 2.8×10.sup.9, about 2.9×10.sup.9, about 3.0×10.sup.9, about 3.1×10.sup.9, about 3.2×10.sup.9, about 3.2×10.sup.9, about 3.3×10.sup.9, about 3.4×10.sup.9, about 3.5×10.sup.9, about 3.6×10.sup.9, about 3.7×10.sup.9, about 3.8×10.sup.9, about 3.9×10.sup.9, about 4.0×10.sup.9, about 4.1×10.sup.9, about 4.2×10.sup.9, about 4.3×10.sup.9, about 4.4×10.sup.9, about 4.5×10.sup.9, about 4.6×10.sup.9, about 4.7×10.sup.9, about 4.8×10.sup.9, about 4.9×10.sup.9, about 5.0×10.sup.9 MCs.
[0551] According to some embodiments, the culturing of CD14+ monocytes induces differentiation of the monocytes to DCs.
[0552] According to some embodiments, at least 30% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 35% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 40% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 45% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 50% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 55% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 60% of the dendritic cell population constitutively expresses CD1d. According to some embodiments, at least 65% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 70% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 75% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 80% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 85% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 90% of the monocyte derived population of DCs constitutively expresses CD1d. According to some embodiments, at least 95% of the monocyte derived population of DCs constitutively expresses CD1d.
(c) Loading αGalCer
[0553] According to some embodiments, the enriched population of DCs is contacted and loaded with α-GalCer or a derivative or analog thereof. According to some embodiments the enriched population of DCs is contacted and loaded with α-GalCer up to 2 hrs before pulsing the CKTCs. According to some embodiments, the DC population loaded with α-GalCer is a mixture of adherent and suspension cells.
[0554] According to some embodiments, the concentration of αGalCer, or an analog or functional equivalent thereof, ranges from about 50 ng/ml to about 500 ng/ml, from about 100 ng/ml to about 500 ng/ml, from about 150 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 250 ng/ml to about 500 ng/ml, from about 300 ng/ml to about 500 ng/ml, from about 350 ng/ml to about 500 ng/ml, from about 400 ng/ml to about 500 ng/ml, or from about 450 ng/ml to about 500 ng/ml. According to some embodiments, the concentration of αGalCer, or an analog or functional equivalent thereof, is maintained at a concentration of about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 110 ng/ml, about 120 ng/ml, about 130 ng/ml, about 140 ng/ml, about 150 ng/ml, about 160 ng/ml, about 170 ng/ml, about 180 ng/ml, about 190 ng/ml, about 200 ng/ml, about 210 ng/ml, about 220 ng/ml, about 230 ng/ml, about 240 ng/ml, about 250 ng/ml, about 260 ng/ml, about 270 ng/ml, about 280 ng/ml, about 290 ng/ml, about 300 ng/ml, about 310 ng/ml, about 320 ng/ml, about 330 ng/ml, about 340 ng/ml, about 350 ng/ml, about 360 ng/ml, about 370 ng/ml, about 380 ng/ml, about 390 ng/ml, about 400 ng/ml, about 410 ng/ml, about 420 ng/ml, about 430 ng/ml, about 440 ng/ml, about 450 ng/ml, about 460 ng/ml, about 470 ng/ml, about 480 ng/ml, about 490 ng/ml, or about 500 ng/ml. According to some embodiments, the αGalCer, or an analog or functional equivalent thereof is maintained at a constant concentration. According to some embodiments, the concentration of α-Gal Ser is about 200 ng/ml.
[0555] α-GalCer, also known as KRN7000, is a simplified glycolipid analogue of agelasphin, which was originally isolated from a marine sponge Agelas mauritianus (Kobayahi et al., Oncol Res. α-GalCer is composed of an a-linked galactose, a phytosphingosine and an acyl chain. Alpha-GalCer is composed of a galactose head group that is linked through the a-hydroxyl to the sphingosine chain (18 carbons). The sphingosine chain is further linked to the fatty acyl chain (26 carbons). The structural chemical formula (A) and ball and stick formula (B) for alpha-galactosyl ceramide (α-GalCer, also known as N-[(2S,3S,4R)-3,4-dihydroxy-1-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoctadecan-2-yl]hexacosanamide; Krn7000; KRN7000, alpha-GalCer, PubChem CID 2826713, molecular formula C.sub.50H.sub.99N0.sub.9, molecular weight 858.3 g/mol) are shown below.
A.
[0556] ##STR00001##
[0557] Recognition of the α-GalCer-CD1d complex by the type-I NKT cell TCR results in the secretion of a range of cytokines, and the initiation of a powerful immune response.
[0558] Several analogs of α-GalCer have been prepared and described, e.g.,
##STR00002## ##STR00003##
[0559] Of these, OCH, an alpha-GalCer analogue with a shorter phytosphingosine chain, stimulates type-I NKT cells to secrete higher amounts of IL-4 than IFN-γ, triggering the immune response toward T.sub.H2 [Hung, J T et al. Journal of Biomedical Science 2017, 24:221, while alpha-C-GalSer is a T.sub.H1-biasing CD1d agonist. [Wojno, J. et al. ACS Biology (2012) 7: 847-55]. Other synthetic glycolipids or alpha-GalCer analogs chemically modified to induce more precise and predictable cytokine profile than alpha-GalCer also have been synthesized and tested. [See, e.g., Hung, J-T et al. Journal of Biomedical Science (2017) 24:22]. U.S. Pat. Nos. 9,365,496, and 10,765,648, each of which is incorporated by reference in its entirety herein, also describes various alpha-GalCer analogs with the general structural formula:
##STR00004##
where in some embodiments, n is 1, 2, or 3.
[0560] Beta-ManCer, another class of type-I NKT cell agonist, also has been described [O'Konek, J J et al., J Clin Invest. 2011 February; 121(2):683-94].
##STR00005##
[0561] This compound has an identical ceramide structure to that of alpha-GalCer, which contributes to the binding with CD1d, with a beta-linked mannose instead of alpha-linked galactose. Because it had been believed in the field that the alpha-linked sugar moiety was a critical feature of alpha-GalCer to elicit tumor immunity, the discovery of the relatively strong anti-tumor activity of beta-ManCer was unexpected. While the protection induced by beta-ManCer was type-I NKT cell-dependent, the protection was independent of IFN-γ but dependent on TNF-α and nitric oxide synthase (NOS). Furthermore, consistent with their distinct mechanisms of protection, alpha-GalCer and beta-ManCer synergize to induce tumor immunity when suboptimal doses were used. In addition, beta-ManCer has much weaker ability to induce long-term anergy in type-I NKT cells than alpha-GalCer [O'Konek, J J et al, Clin Cancer Res. 2013 Aug. 15; 19(16):4404-11]. Similar to alpha-GalCer, beta-ManCer can enhance the effect of a tumor vaccine [Mattarollo, S R et al., Blood. (2012) Oct. 11; 120(15):3019-29].
Activation of the CKTC Population
[0562] According to some embodiments, a fresh population of DCs is added to the IL-2 and IL-7 stimulated CKTC culture. According to some embodiments, the population of DCs is cryopreserved, thawed and then added to the CKTC culture. According to some embodiments the population of DCs derived from PBMCs that is added to the 1-1.5×10.sup.6 CKTCs ranges from about 1×10.sup.6 to about 1×10.sup.7 DCs, i.e., about 1.0×10.sup.6, about 1.1×10.sup.6, about 1.2×10.sup.6, about 1.3×10.sup.6, about 1.4×10.sup.6, about 1.5×10.sup.6, about 1.6×10.sup.6, about 1.7×10.sup.6, about 1.8×10.sup.6, about 1.9×10.sup.6, about 2.0×10.sup.6, about 2.1×10.sup.6, about 2.2×10.sup.6, about 2.3×10.sup.6, about 2.4×10.sup.6, about 2.5×10.sup.6, about 2.6×10.sup.6, about 2.7×10.sup.6, about 2.8×10.sup.6, about 2.9×10.sup.6, about 3.0×10.sup.6, about 3.1×10.sup.6, about 3.2×10.sup.6, about 3.3×10.sup.6, about 3.4×10.sup.6, about 3.5×10.sup.6, about 3.6×10.sup.6, about 3.7×10.sup.6, about 3.8×10.sup.6, about 3.9×10.sup.6, about 4.0×10.sup.6, about 4.1×10.sup.6, about 4.2×10.sup.6, about 4.3×10.sup.6, about 4.4×10.sup.6, about 4.5×10.sup.6, about 4.6×10.sup.6, about 4.7×10.sup.6, about 4.8×10.sup.6, about 4.9×10.sup.6, about 5.0×10.sup.6, about 5.1×10.sup.6, about 5.2×10.sup.6, about 5.3×10.sup.6, about 5.4×10.sup.6, about 5.5×10.sup.6, about 5.6×10.sup.6, about 5.7×10.sup.6, about 5.8×10.sup.6, about 5.9×10.sup.6, about 6.0×10.sup.6, about 6.1×10.sup.6, about 6.2×10.sup.6, about 6.3×10.sup.6, about 6.4×10.sup.6, about 6.5×10.sup.6, about 6.6×10.sup.6, about 6.7×10.sup.6, about 6.8×10.sup.6, about 6.9×10.sup.6, about 7.0×10.sup.6, about 7.1×10.sup.6, about 7.2×10.sup.6, about 7.3×10.sup.6, about 7.4×10.sup.6, about 7.5×10.sup.6, about 7.6×10.sup.6, about 7.7×10.sup.6, about 7.8×10.sup.6, about 7.9×10.sup.6, about 8.0×10.sup.6, about 9×10.sup.6, or about 1×10.sup.7 DCs.
[0563] According to some embodiments of the methods describe herein, the concentration of IL-2 (Recombinant Human IL-2 GMP Protein (R&D Systems, cat #202-GMP) is between about 10 U/ml to about 100 U/ml, for example between about 10 U/ml to about 100 U/ml, about 15 U/ml to about 100 U/ml, about 20 U/ml to about 100 U/ml, about 25 U/ml to about 100 U/ml, about 30 U/ml to about 100 U/ml, about 35 U/ml to about 100 U/ml, about 40 U/ml to about 100 U/ml, about 45 U/ml to about 100 U/ml, about 50 U/ml to about 100 U/ml, about 55 U/ml to about 100 U/ml, about 60 U/ml to about 100 U/ml, about 65 U/ml to about 100 U/ml, about 70 U/ml to about 100 U/ml, about 75 U/ml to about 100 U/ml, about 80 U/ml to about 100 U/ml, about 85 U/ml to about 100 U/ml, about 90 U/ml to about 100 U/ml, or about 95 U/ml to about 100 U/ml. According to some embodiments, the concentration of IL-2 is about 10 U/ml, about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, about 50 U/ml, about 55 U/ml, about 60 U/ml, about 65 U/ml, about 70 U/ml, about 75 U/ml, about 80 U/ml, about 85 U/ml, about 90 U/ml, about 95 U/ml, or about 100 U/ml.
[0564] According to some embodiments of the methods describe herein, the concentration of IL-7 (Recombinant Human IL-7 GMP Protein (R&D Systems, cat #207-GMP) is between about 10 ng/ml to about 200 ng/ml, for example between about 10 ng/ml to about 200 ng/ml, about 20 ng/ml to about 200 ng/ml, about 30 ng/ml to about 200 ng/ml, about 40 ng/ml to about 200 ng/ml, about 50 ng/ml to about 200 ng/ml, about 60 ng/ml to about 200 ng/ml, about 70 ng/ml to about 200 ng/ml, about 80 ng/ml to about 200 ng/ml, about 90 ng/ml to about 200 ng/ml, about 100 ng/ml to about 200 ng/ml, about 110 ng/ml to about 200 ng/ml, about 120 ng/ml to about 200 ng/ml, about 130 ng/ml to about 200 ng/ml, about 140 ng/ml to about 200 ng/ml, about 150 ng/ml to about 200 ng/ml, about 160 ng/ml to about 200 ng/ml, about 170 ng/ml to about 200 ng/ml, about 180 ng/ml to about 200 ng/ml, or about 190 ng/ml to about 200 ng/ml. According to some embodiments, the concentration of IL-7 is about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 110 ng/ml, about 15 ng/ml, about 120 ng/ml, about 125 ng/ml, about 130 ng/ml, about 135 ng/ml, about 140 ng/ml, about 145 ng/ml, about 150 ng/ml, about 155 ng/ml, about 160 ng/ml, about 165 ng/ml, about 170 ng/ml, about 175 ng/ml, about 180 ng/ml, about 185 ng/ml, about 190 ng/ml, about 195 ng/ml, or about 200 ng/ml.
[0565] According to some embodiments, IL-15 is added between about day 13 and day 15 of culture. According to some embodiments, IL-15 is added at about day 13 of culture. According to some embodiments, IL-15 is added at about day 14 of culture. According to some embodiments, IL-15 is added at about day 15 of culture.
[0566] According to some embodiments, the concentration of IL-15 (Recombinant Human IL-15 GMP Protein (R&D Systems, cat #247-GMP) is between about 10 ng/ml to about 100 ng/ml, for example between about 10 ng/ml to about 100 ng/ml, about 15 ng/ml to about 100 ng/ml, about 20 ng/ml to about 100 ng/ml, about 25 ng/ml to about 100 ng/ml, about 30 ng/ml to about 100 ng/ml, about 35 ng/ml to about 100 ng/ml, about 40 ng/ml to about 100 ng/ml, about 45 ng/ml to about 100 ng/ml, about 50 ng/ml to about 100 ng/ml, about 55 ng/ml to about 100 ng/ml, about 60 ng/ml to about 100 ng/ml, about 65 ng/ml to about 100 ng/ml, about 70 ng/ml to about 100 ng/ml, about 75 ng/ml to about 100 ng/ml, about 80 ng/ml to about 100 ng/ml, about 85 ng/ml to about 100 ng/ml, about 90 ng/ml to about 100 ng/ml, or about 95 ng/ml to about 100 ng/ml. According to some embodiments, the concentration of IL-15 is about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, or about 100 ng/ml.
[0567] According to some embodiments, IL-12 (Recombinant Human IL-12 GMP Protein (R&D Systems, cat #219-GMP) is added about one day before cell harvest. According to some embodiments, the concentration of IL-12 is between about 10 ng/ml to about 100 ng/ml, for example between about 10 ng/ml to about 100 ng/ml, about 15 ng/ml to about 100 ng/ml, about 20 ng/ml to about 100 ng/ml, about 25 ng/ml to about 100 ng/ml, about 30 ng/ml to about 100 ng/ml, about 35 ng/ml to about 100 ng/ml, about 40 ng/ml to about 100 ng/ml, about 45 ng/ml to about 100 ng/ml, about 50 ng/ml to about 100 ng/ml, about 55 ng/ml to about 100 ng/ml, about 60 ng/ml to about 100 ng/ml, about 65 ng/ml to about 100 ng/ml, about 70 ng/ml to about 100 ng/ml, about 75 ng/ml to about 100 ng/ml, about 80 ng/ml to about 100 ng/ml, about 85 ng/ml to about 100 ng/ml, about 90 ng/ml to about 100 ng/ml, or about 95 ng/ml to about 100 ng/ml. According to some embodiments, the concentration of IL-12 is about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, or about 100 ng/ml.
[0568] According to some embodiments, the SCKTCs can be purified by positive or negative selection cell separation methods. Positive selection cell separation methods involve directly labeling desired cells for selection with an antibody or a ligand that targets a specific cell surface protein. In immunomagnetic separation methods, the antibody or ligand is linked to a magnetic particle, allowing the labeled cells to be retained in the final isolated fraction after incubation of the same in a magnetic field. Negative selection cell separation methods involve laveling unwanted cell types for removal with antibodies or ligands targeting specific cell surface proteins. In immunomagnetic separation methods, the antibodies or ligands are linked to magnetic particles, allowing the labeled, unwanted cells to be depleted from the final isolated fraction by incubating the sample in a magnetic field. Since the desired cells are not specifically targeted by antibodies or ligands, they remain unbound by particles. According to some embodiments, magnetic bead activated cell sorting, a positive selection technique, can be used for purifying a specific cell population from the mononuclear cells. According to some embodiments, negative selection protocols can be employed to reduce the risk of decreasing the quantity and activity of the desired cells such protocols.
[0569] According to some embodiments a pure SCKTC population is achieved without positive or negative cell separation methods. According to some embodiments, the pulsing with DCs loaded with alpha-GalCer enables the increased purity of the SCKTC population without positive or negative selection cell separation methods. According to some embodiments, the SCKTCs prepared by the process are at least 80% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 81% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 82% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 83% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 84% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 85% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 86% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 87% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 88% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 89% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 90% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 91% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 92% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 93% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 94% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 95% pure SCKTCs. According to some embodiments, the SCKTCs prepared by the process are at least 96% pure SCKTCs, According to some embodiments, the SCKTCs prepared by the process are at least at least 97% pure SCKTCs.
[0570] According to some embodiments, the method comprises replenishing the culture medium in the culture system with fresh serum-free culture medium every 1 to 3 days, i.e., at least every 3 days, at least every 2 days, or every day. According to some embodiments cells are counted to about 0.8-1.5×10.sup.6 cells/ml and then fed with the fresh serum-free culture medium based on the cell count. According to some embodiments, the replenishing step includes adding to the culture system a pulse comprising an enriched population of DCs derived from PBMCs that are loaded with αGalCer or an analog or functional equivalent thereof. According to some embodiments, the number of pulses of DCs loaded with α-GalCer added to the SCKTC culture is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 pulses.
[0571] According to some embodiments, the serum-free culture medium comprises X-VIVO-15 serum-free medium.
CKTC Activation
[0572] According to some embodiments, the population of CKTCs of the described invention comprises a subpopulation of CD3+ T cells. According to some embodiments, the population of CKTCs comprises a subpopulation of NKT cells. According to one embodiment, the subpopulation of NKT cells comprises CD3+Vα24+ cells. According to one embodiment, the subpopulation of NKT cells comprises CD3+Vα24− cells. According to one embodiment, the subpopulation of NKT cells comprises CD3+CD56+ cells. According to some embodiments, the subpopulation of NKT cells comprise a subpopulation of type 1 NKT cells. According to some embodiments, the T cell receptor of the subpopulation of NKT cells comprises a Vα24-Jα18 TCRα chain. According to some embodiments, the T cell receptor of the subpopulation of NKT cells comprises a Vα24-Jα18 TCRα chain and a Vβ11 β chain. According to some embodiments, the subpopulation of NKT cells recognize glycolipid antigens presented by CD1d. According to some embodiments, the glycolipid antigen is αGalCer or an analog or functional equivalent thereof.
[0573] In nature, when type-I NKT cells are stimulated with α-GalCer, they produce IFN-γ. Simultaneously, they activate antigen-presenting cells (APCs) through CD40-CD40L interaction, especially inducing DCs to mature and up-regulate co-stimulatory receptors such as CD80 and CD86. DCs also produce IL-12 upon their interaction with type-I NKT cells. IL-12 induces more IFN-γ production by other T cells and plays a critical role together with IFN-γ in the activation of downstream effectors such as NK cells, CD8+ T cells and γδ T cells (Paget et al., J Immunol. 2012 Apr. 15; 188(8):3928-39). The interaction of type-I NKT cells with APCs offers activation signals to (i.e., licenses) APCs to render them able to cross-prime to CD8+ T cells through the induction of CD70 and CCL17 (Taraban et al., J Immunol. 2008 Apr. 1; 180(7):4615-20; Fujii et al., Immunol Rev. 2007 December; 220( ):183-98).
[0574] According to some embodiments, the activating of the population of CKTCs can comprise one or more of inducing secretion of a cytokine by the population of CKTCs, stimulating proliferation of the population of CKTCs, or modulating expression of one or more markers on the cell surface of the CKTCs. According to some embodiments, the cytokine whose expression is modulated is one or more cytokine selected from the group consisting of IFNγ, IL-4, IL-5, IL-6, or IL-10.
[0575] Activation of the population of CKTCs can be measured by various assays as described herein. Exemplary activities that may be measured include the induction of proliferation, the induction of expression of activation markers in the population of CKTCs, the induction of cytokine secretion by the population of CKTCs, the induction of signaling in the population of CKTCs, and an increase in the cytotoxic activity of the population of CKTCs.
Cytokine Secretion
[0576] The activation of CKTCs to form SCKTCs may be assessed or measured by determining secretion of cytokines, including one or more of gamma interferon (IFNγ), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6) or interleukin-10 (IL-10). According to some embodiments, an ELISA is used to determine cytokine secretion, for example secretion of gamma interferon (IFNγ), IL-4, IL-5, IL-6 or IL-10. According to some embodiments, the ELISPOT (enzyme-linked immunospot) technique may be used to detect CKTCs and SCKTCs that secrete a given cytokine (e.g., gamma interferon (IFNγ)) in response to the methods described herein. For example, a culture system can be set up whereby a population of CKTCs or SCKTCs produced by the methods described herein are cultured within wells that have been coated with anti-IFNγ antibodies. The secreted IFNγ is captured by the coated antibody and then revealed with a second antibody coupled to a chromogenic substrate. Locally secreted cytokine molecules form spots, with each spot corresponding to one IFNγ-secreting cell. The number of spots allows one to determine the frequency of IFNγ-secreting cells in the analyzed sample. The ELISPOT assay has also been described for the detection of tumor necrosis factor alpha (TNFα), IL-4, IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granzyme B-secreting lymphocytes (Klinman D, Nutman T. Current protocols in immunology. New York, N.Y: John Wiley & Sons, Inc.; 1994. pp. 6.19.1-6.19.8, incorporated by reference in its entirety herein).
[0577] According to some embodiments, cytokine secretion is quantified by cytokine bead assay. Bead populations with distinct fluorescence intensities are coated with capture antibodies specific for IFN-γ and IL4 and mixed together to form a bead array that is resolved in a flow cytometer. During the assay procedure, the inflammatory cytokine capture beads are mixed with recombinant standards or SCKTCs and incubated with PE-detection antibodies. The intensity of PE fluorescence of each complex reveals the concentration of that cytokine.
[0578] Flow cytometric analyses of intracellular cytokines may be used to measure the cytokine content in culture supernatants, but provide no information on the number of NKT cells that actually secrete the cytokine. When lymphocytes are treated with inhibitors of secretion, such as monensin or brefeldin A, they accumulate cytokines within their cytoplasm upon activation. After fixation and permeabilization, intracellular cytokines can be quantified by cytometry. This technique allows the determination of the cytokines produced, the type of cells that produce these cytokines, and the quantity of cytokine produced per cell.
[0579] According to some embodiments, cytokine production by the enriched population of SCKTCs is characterized as IL-4 low, IL-5 low, IL-6 low, IL-10 low, IFNγ high.
[0580] According to one embodiment, the amount of IFN-γ produced by the population of cells into the culture supernatant is at least about 500 pg/ml; 1000 pg/ml; 1500 pg/ml; 2000 pg/ml, at least about 2500 pg/ml, at least about 3000 pg/ml, at least about 3500 pg/ml, at least about 4000 pg/ml, at least about 4500 pg/ml, at least about 5000 pg/ml, at least about 5500 pg/ml, at least about 6000 pg/ml, at least about 6500 pg/ml, at least about 7000 pg/ml, at least about 7500 pg/ml, at least about 8000 pg/ml, at least about 8500 pg/ml, at least about 9000 pg/ml, at least about 9500 pg/ml, at least about 10,000 pg/ml, at least about 10,500 pg/ml, at least about 11,000 pg/ml, at least about 11,500 pg/ml, at least about 12,000 pg/ml, at least about 12,500 pg/ml, at least about 13,000 pg/ml, at least bout 13,500 pg/ml, or at least about 14,0000 pg/ml.
[0581] According to some embodiments, the amount of IL-4 produced by the population of cells and secreted into the culture supernatant is less than 1000 pg/ml; less than 900 pg/ml; less than 800 pg/ml, less than 700 pg/ml, less than 600 pg/ml; less than 500 pg/ml; less than 400 pg/ml; less than 300 pg/ml; less than 200 pg/ml; less than 100 pg/ml; less than 90 pg/ml; less than 80 pg/ml; less than 70 pg/ml; less than 60 pg/ml; less than 50 pg/ml; less than 40 pg/ml; less than 30 pg/ml; less than 20 pg/ml; less than 10 pg/ml; less than 9 pg/ml; less than 8 pg/ml; less than 7 pg/ml; less than 6 pg/ml; less than 5 pg'ml; or 4 pg/ml, 3 pg/ml; 2 pg/ml or 1 pg/ml. According to some embodiments, the amount of IL-4 produced by the population of SCKTC cells and secreted into the culture supernatant ranges from 1-5 pg/ml; 5-6 pg/ml; 6-7 pg/ml; 7-8-pg/ml; 8-9 pg/ml; 9-10 pg/ml, 10-15 pg/ml; 10-20 pg/ml; 20-30 pg/ml; 30-40 pg/ml; 40-50 pg/ml; 50-60 pg/ml; 60-70 pg/ml; 70-80 pg/ml; 80-90 pg/ml; or 90-100 pg/ml, inclusive.
[0582] According to some embodiments, the ratio of IFNγ to IL-4 is an indicator of one or more T cell effector functions (such as cell killing and cell activation), of the CKTCs and SCKTCs.
[0583] According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 500. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 600. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 700. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 800. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 900. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1000. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1100. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1200. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1300. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1400. According to one embodiment, the ratio in culture supernatants of IFN-γ:IL-4 is at least 1500. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1550. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1600. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1650. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1700. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1750. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1800. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1850. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1900. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 1950. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2000. According to one embodiment, the ratio of IFN-γ:IL-4 is at least 2050. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2100. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2150. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2200. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2250. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2300. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2350. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2400. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2450. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2500. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2550. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2600. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2650. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2700. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2750. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2800. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2850. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2900. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 2950. According to one embodiment, the ratio of IFN-γ:IL-4 in culture supernatants is at least 3000.
Cytotoxicity
[0584] The activation of CKTCs to form SCKTCs may be assessed by assaying cytotoxic activity of the CKTCs at each step of the described method.
[0585] The cytotoxic activity may be assessed by any suitable technique known to those of skill in the art. For example, a sample comprising a population of CKTCs or SCKTCs produced by the methods described herein can be assayed for cytotoxic activity after an appropriate period of time, in a standard cytotoxicity assay. Such assays may include, but are not limited to, the chromium release CTL assay and the ALAMAR BLUE fluorescence assay known in the art. According to some embodiments, cytotoxicity can be assayed in a lactate dehydrogenase (LDH) assay. LDH, a well-established and reliable indicator of cellular toxicity, is a cytosolic enzyme that is released into the cell culture medium upon damage to the plasma membrane. The extracellular LDH is then quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH, which then reduces a tetrazolium salt to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium.
[0586] According to some embodiments, a population of SCKTC cells is collected by centrifugation and their cytotoxicity against A549 cells (human lung epithelial cell line) assessed. According to some embodiments, cytotoxicity is assessed against a genetically modified cell line that expresses increased amounts of CD1d, e.g., a genetically modified A549 cells or Panc-1 (pancreatic carcinoma) cells. According to some embodiments, a population of cells is collected by centrifugation and cytotoxicity against K562 cells (highly undifferentiated and of the granulocytic series, derived from a patient with chronic myeloid leukemia) is assessed. The K562 cell line, derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 bcr-abl hybrid gene, is known to be particularly resistant to apoptotic death. (Luchetti, F. et al, Haematologica (1998) 83: 974-980). According to some embodiments, K562 target cells and SCKTCs are allocated into wells at one or more effector: target ratios, e.g. 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1. After incubation, absorbance is detected by an enzyme-linked immunosorbent assay reader, and the killing rate can be calculated. According to some embodiments, the same assay can be carried out, where cytotoxicity against Jurkat cells (acute T leukemia) is assessed (Somanchi et al., PLoS ONE 10(10): e0141074. https://doi.org/10.1371/journal.pone.0141074).
[0587] According to some embodiments, killing rate can be represented by the following formula:
[0588] According to some embodiments, the killing rate of the CKTC population comprising SCKTCs against a target cell ranges from about 20% to about 85%, inclusive. According to some embodiments, the killing rate of the CKTC population comprising SCKTCs ranges from about 50% to about 75%, inclusive. According to some embodiments, the killing rate of the CKTC population comprising SCKTCs is at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%,at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%.
Proliferation/Expansion
[0589] The ability of the described methods of the invention to induce expansion of the SCKTCs can be evaluated by staining using the fluorescent cell staining dye carboxyfluorescein syccinimidyl ester (CFSE). To compare the initial rate of cell expansion, the cells are stained with CFSE to determine how well the various steps of the described method (i.e. steps (b)-(e)) induced the proliferation of the SCKTCs. CFSE staining provides a quantitative endpoint and allows simultaneous phenotyping of the expanded cells. Every day after stimulation, an aliquot of cells is removed from each culture and analyzed by flow cytometry. CFSE staining makes cells highly fluorescent. Upon cell division, the fluorescence is halved and thus the more times a cell divides the less fluorescent it becomes. The ability of the described method to induce proliferation of the SCKTCs is quantitated by measuring the number of cells that divided once, twice, three times and so on.
[0590] To determine how well the described method promotes long-term growth of the SCKTCs, cell growth curves can be generated. These experiments are set up as are the foregoing CFSE experiments, but no CFSE is used. Every 2-3 days of culture, cells are removed from the respective cultures and counted using a Coulter counter, which measures how many cells are present and the mean volume of the cells. The mean cell volume is the best predictor of when to restimulate the cells. In addition, the phenotypes of the cells that are expanded can be characterized to determine whether a particular subset is preferentially expanded.
[0591] Prior to each restimulation, a phenotypic analysis of the expanding cell populations is performed to determine the presence of particular markers that define the SCKTC population. According to some embodiments, prior to each restimulation, an aliquot of cells is removed from each culture and analyzed by flow cytometry, using Forward Scatter (FS) vs 90° Light Scatter to bitmap the intact lymphocyte population. Gating (rectangular) on this bitmap, CD56 vs CD3 was measured. Gating on the double positives, Vα24 vs. Vβ11 was measured. Perforin and Granzyme B intracellular staining can be used to perform a gross measure to estimate cytolytic potential.
[0592] According to some embodiments, the population of SCKTCs is expanded from about 100- to about 1,000,000-fold, or from about 1,000- to about 1,000,000-fold, e.g., from about 1,000-fold to about 100,000-fold based on the population of starting CKTC cells, i.e., at least about 100-, at least about 200-, at least about 300-, at least about 400-, at least about 500-, at least about 600-, at least about 700-, at least about 800-, at least about 900-, at least about 1000-, at least about 2000-, at least about 3000-, at least about 4000-, at least about 5000-, at least about 6000-, at least about 7000-, at least about 8000-, at least about 9000-, at least about 10,000-, at least about 11,000-, at least about 12,000-, at least about 13,000-, at least about 14,000-, at least about 15,000-, at least about 16,000-, at least about 17,000-, at least about 18,000-, at least about 19,000-, at least about 20,000-, at least about 21,000-, at least about 22,000-, at least about 23,000-, at least about 24,000-, at least about 25,000-, at least about 26,000-, at least about 27,000-, at least about 28,000-, at least about 29,000-, at least about 30,000-, at least about 31,000-, at least about 32,000-, at least about 33,000-, at least about 34,000-, at least about 35,000-, at least about 36,000-, at least about 37,000, at least about 38,000-, at least about 39,000-, at least about 40,000-, at least about 41,000-, at least about 42,000-, at least about 43,000-, at least about 44,000-, at least about 44,000-, at least about 45,000-, at least about 46,000-, at least about 47,000-, at least about 48,000-, at least about 49,000-, at least about 50,000-, at least about 51,000-, at least about 52,000-, at least about 53,000-, at least about 54,000-, at least about 55,000-, at least about 56,000-, at least about 57,000-, at least about 58,000-, at least about 59,000-, at least about 60,000-, at least about 61,000-, at least about 62,000-, at least about 63,000-, at least about 64,000-, at least about 65,000-, at least about 66,000-, at least about 67,000-, at least about 68,000-, at least about 69,000-, at least about 70,000, at least about 71,000-, at least about 72,000-, at least about 73,000-, at least about 74,000-, at least about 75,000-, at least about 76,000-, at least about 77,000-, at least about 78,000-, at least about 79,000-, at least about 80,000-, at least about 81,000-, at least about 82,000-, at least about 83,000-, at least about 84,000-, at least about 85,000-, at least about 86,000-, at least about 87,000-, at least about 88,000-, at least about 89,000-, at least about 90,000-, at least about 91,000-, at least about 92,000-, at least about 93,000-, at least about 94,000-, at least about 95,000-, at least about 96,000-, at least about 97,000-, at least about 98,000-, at least about 99,000-, at least about 100,000-, at least about 200,000-, at least about 300,000-, at least about 400,000-, at least about 500,000-, at least about 600,000-, at least about 700,000-, at least about 800,000-, at least about 900,000-, or at least about 1,000,000-fold.
2. Pharmaceutical Composition Comprising the Cell Product Comprising SCKTCs
[0593] According to some embodiments, the cell product prepared by the method comprises at least about 5×10.sup.8 to about 5×10.sup.10 SCKTCs, inclusive, i.e., at least 5×10.sup.8, 5.1×10.sup.8, 5.2×10.sup.8, 5.3×10.sup.8, 5.4×10.sup.8, 5.5×10.sup.8, 5.6×10.sup.8, 5.7×10.sup.8, 5.8×10.sup.8, 5.9×10.sup.8, 6.0×10.sup.8, 6.1×10.sup.8, 6.2×10.sup.8, 6.3×10.sup.8, 6.4×10.sup.8, 6.5×10.sup.8, 6.6×10.sup.8, 6.7×10.sup.8, 6.8×10.sup.8, 6.9×10.sup.8, 7.0×10.sup.8, 7.1×10.sup.8, 7.2×10.sup.8, 7.3×10.sup.8, 7.4×10.sup.8, 7.5×10.sup.8, 7.6×10.sup.8, 7.7×10.sup.8, 7.8×10.sup.8, 7.9×10.sup.8, 8.0×10.sup.8, 9.0×10.sup.8, 9.1×10.sup.8, 9.2×10.sup.8, 9.3×10.sup.8, 9.4×10.sup.8, 9.5×10.sup.8, 9.6×10.sup.8, 9.7×10.sup.8, 9.8×10.sup.8, 9.9×10.sup.8, 1×10.sup.9, 1.1×10.sup.9, 1.2×10.sup.9, 1.3×10.sup.9, 1.4×10.sup.9, 1.5×10.sup.9, 1.6×10.sup.9, 1.7×10.sup.9, 1.8×10.sup.9, 1.9×10.sup.9, 2.0×10.sup.9, 2.1×10.sup.9, 2.2×10.sup.9, 2.3×10.sup.9, 2.4×10.sup.9, 2.5×10.sup.9, 2.6×10.sup.9, 2.7×10.sup.9, 2.8×10.sup.9, 2.9×10.sup.9, 3.0×10.sup.9, 3.1×10.sup.9, 3.2×10.sup.9, 3.3×10.sup.9, 3.4×10.sup.9, 3.5×10.sup.9, 3.6×10.sup.9, 3.7×10.sup.9, 3.8×10.sup.9, 4.9×10.sup.9, 5.0×10.sup.9, 5.1×10.sup.9, 5.2×10.sup.9, 5.3×10.sup.9, 5.4×10.sup.9, 5.5×10.sup.9, 5.6×10.sup.9, 5.7×10.sup.9, 5.8×10.sup.9, 5.9×10.sup.9, 6.0×10.sup.9, 6.1×10.sup.9, 6.2×10.sup.9, 6.3×10.sup.9, 6.4×10.sup.9, 6.5×10.sup.9, 6.6×10.sup.9, 6.7×10.sup.9, 6.8×10.sup.9, 6.9×10.sup.9, 7.0×10.sup.9, 6.1×10.sup.9, 6.2×10.sup.9, 6.3×10.sup.9, 6.4×10.sup.9, 6.5×10.sup.9, 6.6×10.sup.9, 6.7×10.sup.9, 6.8×10.sup.9, 6.9×10.sup.9, 7.0×10.sup.9, 7.1×10.sup.9, 7.2×10.sup.9, 7.3×10.sup.9, 7.4×10.sup.9, 7.5×10.sup.9, 7.6×10.sup.9, 7.7×10.sup.9, 7.8×10.sup.9, 7.9×10.sup.9, 8.0×10.sup.9, 8.1×10.sup.9, 8.2×10.sup.9, 8.3×10.sup.9, 8.4×10.sup.9, 8.5×10.sup.9, 8.6×10.sup.9, 8.7×10.sup.9, 8.8×10.sup.9, 8.9×10.sup.9, 9.0×10.sup.9, 9.1×10.sup.9, 9.2×10.sup.9, 9.3×10.sup.9, 9.4×10.sup.9, 9.5×10.sup.9, 9.6×10.sup.9, 9.7×10.sup.9, 9.8×10.sup.9, 9.9×10.sup.9, 1.0×10.sup.10, 1.1×10.sup.10, 1.2×10.sup.10, 1.3×10.sup.10, 1.4×10.sup.10, 1.5×10.sup.10, 1.6×10.sup.10, 1.7×10.sup.10, 1.8×10.sup.10, 1.9×10.sup.10, 2.0×10.sup.10, 2.2×10.sup.10, 2.3×10.sup.10, 2.4×10.sup.10, 2.5×10.sup.10, 2.6×10.sup.10, 2.7×10.sup.10, 2.8×10.sup.10, 2.9×10.sup.10, 3.0×10.sup.10, 3.1×10.sup.10, 3.2×10.sup.10, 3.3×10.sup.10, 3.4×10.sup.10, 3.5×10.sup.10, 3.6×10.sup.10, 3.7×10.sup.10, 3.8×10.sup.10, 3.9×10.sup.10, 4.0×10.sup.10, 4.1×10.sup.10, 4.2×10.sup.10, 4.3×10.sup.10, 4.4×10.sup.10, 4.5×10.sup.10, 4.6×10.sup.10, 4.7×10.sup.10, 4.8×10.sup.10, 4.9×10.sup.10, or about 5.0×10.sup.10 SCKTCs. According to some embodiments the SCKTC cell product further contains about 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or about 1.5% DCs.
[0594] According to some embodiments, the SCKTC cell product prepared by the method is formulated with a pharmaceutically acceptable carrier. According to some embodiments the pharmaceutically acceptable carrier can contain one or more of Human Serum Albumin (HSA), Plasmalyte injection ((Multiple Electrolytes Injection), glucose/dextrose, or dextran 40. According to some embodiments, the SCKTCs can be cryopreserved in a freezing medium comprising 10% DM (e.g., cryoStor CS 10) and stored in the vapor phase of a liquid nitrogen freezer (−130° C. or lower). According to some embodiments, the freezing medium may comprise 31.25% (v/v) of Plasmia-Lyte A, 31.25% (v/v) of 5% Dextrose/0.45% sodiumchloride, 10% Dextran 40 (LMD)/5% Dextrose, 20% (v/v) of 25% Human Serum Albumin (HSA), and 7.5% (v/v) Cryoserv® dimethylsulfoxide (DMSO).
[0595] Target quality attributes of the amplified enriched population of SCKTCs of (g) prepared by the described method are shown in Table 4 below.
TABLE-US-00004 TABLE 4 Preliminary minimum acceptable target quality attributes SCKTCs (preliminary minimum Target attribute acceptable specifications) % Va24 + Vβ11 expression 80% after 3 weeks (2.sup.nd DC pulse (range)
1.5 × 10.sup.9 total yield IFN-γ secretion: (range) At least 2500 pg/ml w/ IL-12 stim; 200-750 pg/mL w/o IL-12 stim. IL-4 secretion: (range) 4-5 pg/mL w/ IL-12 stim. IFN-γ: IL-4 ratio: (range)
At least 500 w/ IL-12 stim.
20-200 w/o IL-12 stim. Cytotoxicity against A549 At an Effector Target Ratio of 20: cells (range): 1, ≥50% cytotoxicity (A549) Viability after freeze thaw At least 80% (range) Sterile, endotoxin negative Negative mycoplasma Bacterial and fungus Negative Therapeutic dose (range) at least 0.2 × 10.sup.9 SCKTCs per treatment cycle (30 days)
[0596] According to some embodiments, the properties of the SCKTC cell product are stable and reproducible from batch to batch. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by at least 80% SCKTC viability and stability for at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, or at least 20 hours at room temperature. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by identity of the SCKTCs, as confirmed by expression of cell surface markers by flow cytometry. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by a purity of at least 80% SCKTCs. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by secretion into the culture medium of at least 2500 pg/ml IFN-γ. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by secretion of about 4-5 pg/mL IL-4 into the culture medium. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by an IFN γ:IL4 ratio of at least 500 with IL-12 stimulation. According to some embodiments, the fresh activated and expanded SCKTC cell product prepared by the process is characterized by at least 50% cytotoxicity on A549 target cells at an effector:target ratio of 20:1.
[0597] According to some embodiments, the properties of the cryofrozen and thawed SCKTC cell product are stable and reproducible from batch to batch. According to some embodiments, the cryofrozen and thawed activated and expanded SCKTC cell product prepared by the process after thawing is pulsed with at least 1×10.sup.6 DCs loaded with α-GalCer. According to some embodiments, the cryofrozen, thawed and pulsed SCKTC product is characterized by at least 70% SCKTC viability and stability for at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 18 hours, or at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23, hours, or at least 24 hours at room temperature. According to some embodiments, the cryofrozen and thawed activated and expanded SCKTC cell product prepared by the process is characterized by IFN-γ secretion into the culture medium of at least 2500 pg/mL with IL-12 stimulation. According to some embodiments, the cryofrozen and thawed activated and expanded SCKTC cell product prepared by the process is characterized by an IFN γ:IL4 ratio of at least 500 with IL-12 stimulation. According to some embodiments, the cryofrozen and thawed activated and expanded SCKTC cell product prepared by the process is characterized by at least 50% cytotoxicity on A549 target cells at an effector:target ratio of 20:1.
Markers
[0598] According to some embodiments of the present disclosure, expansion of the SCKTCs using the methods as described herein can be determined by assessing the presence of markers that characterize the SCKTCs, and thereby determining the percent of the SCKTCs in the cell population. According to some embodiments, flow cytometry can be used to determine the presence of a subpopulation of SCKTCs expressing NKT cell markers using Forward Scatter (FS) vs 90° Light Scatter bitmap of the lymphocyte intact lymphocyte population. According to some embodiments, gating (rectangular) on this bitmap, CD56 vs CD3 is measured. According to some embodiments, gating on the double positives, Vα24 vs. Vβ11 is measured. According to some embodiments, a sub population of NKT cells can be determined by the presence of CD3 and CD56 markers (CD3+CD56+ NKT cells). According to some embodiments, binding of an anti-CD3 antibody labeled with a first fluorescent label (e.g. a commercially available fluorescently labeled anti-CD3 antibody, such as anti-CD3-pacific blue (PB) (BD Pharmingen, clone #SP34-2) and an anti-CD56 antibody labeled with a second fluorescent label (e.g. a commercially available fluorescently labeled anti-CD56 antibody, such as anti-CD56-Phycoerythrin (PE)-Cy7 (BD Pharmingen, clone #NCAM16.2)) can be used to determine expression of CD3 and CD56 in the cell population, where binding of the antibody is measured by flow cytometry for, e.g., PB fluorescence or PE fluorescence, and a gate is set based on CD3+CD56+ cells.
[0599] According to some embodiments, a subpopulation of type-I NKT cells can be determined by the presence of TCR Vα and TCR Vβ markers. According to one embodiment, binding of an anti-TCR Vα24 antibody labelled with a first fluorescent label (e.g. a commercially available fluorescently labeled anti-TCR Vα24 antibody, such as anti-TCR Vα24-PE (Beckman Coulter, clone # C15)) and an anti-TCR Vβ11 antibody labeled with a second fluorescent label (e.g. a commercially available fluorescently labeled anti-TCR Vβ11 antibody, such as anti-TCR Vβ-Fluorescein isothiocyanate (FITC) (Beckman Coulter, clone #C21)) can be used to determine expression of Vα24 and Vβ11 in the cell population, where binding of the antibody is measured by flow cytometry for, e.g., PE fluorescence or FITC fluorescence, and a gate is set based on Vα24+Vβ+11 cells.
[0600] According to some embodiments, a subpopulation of NKT cells can be characterized by expression of the markers CD3+Vα24+. According to some embodiments, a subpopulation of NKT cells is characterized by expression of the markers CD3+Vα24−. According to some embodiments, the subpopulation of type-I NKT cells includes cells characterized by the markers CD3+CD56+. According to some embodiments, the subpopulation of type-I NKT cells includes cells characterized by expression of the markers CD3+Vα24+, CD3+Vα24−, CD3+CD56+ and mixtures thereof.
Additional Compatible Actives
[0601] According to some embodiments, the pharmaceutical composition of the described invention can further include one or more compatible active ingredients to provide the composition with another pharmaceutical effect in addition to that provided by the cell product of the described invention. “Compatible” as used herein means that the active ingredients of such a composition are capable of being combined with each other in such a manner so that there is no interaction that would substantially reduce the efficacy of each active ingredient or the composition under ordinary use conditions.
[0602] According to some embodiments, the pharmaceutical composition comprising the SCKTC cell product further comprises an enriched population of NK cells. According to some embodiments, the population of NK cells can be acquired by apheresis of peripheral blood from PBMCs. According to some embodiments, stem cell mobilization, a process whereby CD34+ hematopoietic stem cells are stimulated out of the bone marrow into the blood stream, may be used to harvest the PBMCs. According to some embodiments, Plerixafor in combination with G-CSF may be used to mobilize the CD34+ stem cells into the blood before collection. According to some embodiments, the PBMCs are depleted of CD3+ T cells and/or CD19 B cells with magnetic beads. According to some embodiments, CD3−CD56+NK cells are positively selected with magnetic beads. According to some embodiments, the selected CD3−CD56+ cells or T cell and/or B cell depleted cells are differentiated to NK cells by culturing in NK cell medium containing high IL-2 (2813 U/mL) for 14 days. According to some embodiments, the population of enriched NK cells can be expanded using static cell culture bags or an automated bioreactor. [e.g., see Saito, S. et al. Human Gene Therapy Methods (2013) 24 (4): 241-52; Spanholtz, J. et al. PLoS One (2011) 6 (6): e20740]. According to some embodiments, the NK cells are characterized by flow cytometry for identity/activation markers, for IFN-γ expression and secretion; and for their cytolytic potential against an MHC class I null cell line, e.g., K562.
[0603] According to some embodiments, a pharmaceutical composition comprising a-GalCer may be administered intranasally to activate the infused SCKTCs in situ [See, e.g., Artiaga, Bl et al. Sci Reports (2016) 6: 37999]. For example, excipients that offer mucosal bioadhhesion, in situ gelling tendency, ability to control the rate of drug clearance from the nasal cavity as well as protect the drug from enzymatic degradation are well suited for intranasal delivery. [Remington. The Science and Practice of Pharmacy, 23rd Ed. Adejare, A. Ed. In Chief, Academic (Cambridge, Mass. (2021) at p. 640, citing Upadhyay, S. et al. J. App. Pharm. Sci. (2011) 01 (03): 34-44; Ghori, M U et al. Am. J. Pharmacol. Sci. (2015) 3 (5): 110-19; Alnasser, S. Asian J. Pharm. Clin. Res. (2019) 12 (1): 40-45). Suspending agents act by retarding the agglomeration of particles by minimizing interparticle interaction and/or increasing viscosity of continuous medium (acting as thickeners or viscosity modifiers), thereby decreasing the settling rate of particles. These include inorganic materials, synthetic compounds of polysaccharides. Exemplary suspending agents and thickeners for intranasal administration include colloidal microcrystalline cellulose (MCC) (MCC and sodium carboxymethylcellulose (Na CMC), mesoporous methylcellulose (MPMC), methylcellulse (MC), Na CMC, pectin and polyethylene glycols (PEGs). Preservatives are added to pharmaceuticals to inhibit or prevent microbialgrowth and consequently ensure stability during shelf life. Exemplary preservatives for intranasal administration include methyl paraben, propyl paraben, and benzalkonium chloride. Penetration enhancers promote the transport of the drug across the nasal membrane, thereby improving nasal absorption of the drug. Examples include polysorbates, poloxamers, PEGs, propylene glycol and EDTA. Tonicity agents are used to adjust the osmolality of parenteral, ophthalmic and nasal solutions that directly come in contact with biological fluids. Exemplary tonicity agents include dextrose, glycerin, mannitol, potassium/sodium chloride and sorbitol/sorbitol solution. Buffering agents are weak acids or weak bases that are used to adjust, maintain or prevent rapid changes in the pH of a solution. Commonly used buffers include acetate, citrate, tartarate, phosphate and triethanolamide (TRIS) buffers.
[0604] According to some embodiments, the pharmaceutical composition comprising the SCKTC cell product containing the population of SCKTCs may be administered with a supportive therapy or an additional therapeutic agent, e.g., one or more of an immunomodulatory agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
[0605] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral load.
[0606] According to some embodiments, the additional agent regulates immune cell activation. According to some embodiments, the additional agent modulates T cell exhaustion pathways.
Immunomodulatory Agents
[0607] According to some embodiments, the immunomodulatory agent may comprise methotrexate; a glucocorticoid, cyclosporine, tacrolimus and sirolimus; a recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNFα inhibitor/antagonist; an IL-1β inhibitor; a chimeric IL-1Ra; an IL-6 inhibitor; an IL-12/IL-23 inhibitor selected from ustekinumab, briakinumab; an IL-23 inhibitor selected from guselkumab, tildrakizumab; a compound that targets TLR4 signaling; a p38 MAPK inhibitor, a compound that targets Janus kinase signaling; a compound that targets cell adhesion molecules to reduce leukocyte recruitment; a checkpoint inhibitor or a recombinant anti-inflammatory cytokine.
[0608] According to some embodiments, a physiologic or supraphysiological dose of the recombinant interferon comprising IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, and IFN-ω or a PEGylated form thereof boosts immune defenses of the subject.
[0609] According to some embodiments, the glucocorticoid comprises prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil, or combinations thereof; or the recombinant IL-2 inhibitor comprises denileukin diftitox; or the PDE4 inhibitor comprises cilomilast; or the TNFα inhibitor/antagonist comprises etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or the IL-1β inhibitor comprises rilonacept; canakinumab; or Anakinra; or the IL-6 inhibitor comprises tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab; or the compound that targets TLR4 signaling comprises (ethyl 4-(4′-chlorophenyl) amino-6 methyl-2-oxocyclohex-3-en-1-aote (enamionone E121), JODI 18b; JODI 19, resatorvid, TLR-C34; or C35; or the p38 MAPK inhibitor comprises 4-(4′-fluorophenyl)-2-(4′-methylsulfinylphenyl)-5-(4′-pyridyl)-imidazole (SB203580), trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol (SB239063), ord 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ 67657); or the compound that targets Janus kinase signaling comprises tofacitinub, baricitinib, or upadacitinib; or the compound that targets a cell adhesion molecule to reduce leukocyte recruitment comprises an a4 integrin inhibitor comprising vedolizumab or natalizumab; or the recombinant anti-inflammatory cytokine comprises IL-4, IL-10, or IL-11; or the interferon is in a PEGylated form.
[0610] The term “immune checkpoint molecules” as used herein refers to ligand-receptor pairs that exert inhibitory or stimulatory effects on immune responses. Examples include programmed cell death 1 receptor (PD-1, also known as CD279), thought to regulate T cell proliferation later in the immune response, and its ligand programmed cell death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG3), which suppresses T cells activation and cytokine secretion, thereby ensuring immune homeostasis and shows synergy with PD-1 to inhibit immune responses (Long, L. et al. Genes Cancer (2018) 9 (5-6): 176-89. and cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152), a negative regulator of T cell immune function thought to regulate T cell proliferation early in an immune response [Buchbinder, E I, and Desai, A. Am. J. Clin. Oncol. (39) (10: 98-106). In addition, glucocorticoid-induced TNFR family related gene (GITR), a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes activation; GITR activation by its ligand (GITRL) influences the activity of effector and regulatory T cells, thus participating in the development of immune response against tumors and infectious agents, as well as in autoimmune and inflammatory diseases. [Nocentini, G. et al. Br. J. Pharmacol. (2012) 165 (7): 2089-99] T-cell immunoglobulin and mucin domain 3 (Tim-3) is a checkpoint receptor expressed by a wide variety of immune cells as well as leukemic stem cells. [Acharya, N. et al. J. Immunother. Cancer (2020) 8(10: e000911). T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is an immune checkpoint receptor that can suppress T-cell activation and promote T-cell exhaustion. Inhibition of TIGIT may increase cytotoxic T-cell proliferation and function. Inducible T cell costimulator (ICOS, cluster of differentiation (CD278)) is an activating costimulatory immune checkpoint expressed on activated T cells. Its ligand, ICOSL is expressed on antigen-presenting cells and somatic cells, including tumour cells in the tumour microenvironment. [Solinas, C. et al. ESMO Open. (2020) 5(1): e000544].
[0611] According to some embodiments, the immunomodulator is an immune checkpoint inhibitor. The term “immune checkpoint inhibitor” as used herein refers to a molecule that can block immune checkpoint molecules. Specific immune checkpoint inhibitors, including antibodies against CTLA-4, PD-1 receptor or its ligand PD-L1 include YERVOY™ (Ipilimumab; CTLA-4 antagonist), OPDIVO™ (Nivolumab; PD-1 antagonist) and KEYTRUDA™ (Pembrolizumab; PD-1 antagonist) in multiple tumor indications, with ongoing registration trials in many more.
[0612] According to some embodiments, the immunomodulatory agent comprises recombinant IL-37. According to some embodiments, the immunomodulatory agent comprises recombinant CD24. According to some embodiments, the immunomodulatory agent comprises rIL-37 and rCD24.
[0613] IL-37 is a member of the IL-1 family, which includes IL-1α, IL-1β, IL-18, IL33, IL36α, IL-36β, IL-36γ, IL-37 and IL-38. [Mantovani, A. et al. Immunity (2019) doi.10.1016/j.immuni.2019.03.012]. The IL-1 family of cytokines is divided into three subgroups on the basis of the Il-1 consensus sequence and the signaling receptor chain. These include secreted molecules with agonistic activity [IL-1α, IL-1β, IL-18, IL33, IL36α, IL-36β, IL-36γ], receptor antagonists [IL-1Ra, IL36Ra, and IL-38] and an anti-inflammatory cytokine (IL-37) [Id., citing Dinarello, C A Immunol. Rev. (2018) 281: 8-27]. There is no murine counterpart to human IL-37. [Id.].
[0614] In humans, production of IL-37 is activated by pro-inflammatory stimuli, including cytokines, as a protective mechanism to prevent runaway inflammation and excessive damage. Five transcripts for the human IL-37 gene have been identified (IL-37 a-e). IL-37b is the most complete of these isoforms, is the most abundant and studied, and includes 5 of the 6 exons of the IL-37 gene (all but exon 3). Exons 4,5, and 6 encode for the sequence required for the beta-fold barrel structure and account for the extracellular activity of recombinant IL-37. Conversely, exons 1 2, and 3 may be cleaved in the extracellular environment by unknown proteases. IL-37 isoforms a, b and d share exons 4, 5, and 6 and encode functional proteins. The IL-37 isoforms c and e lack one or more of these exons and likely encode non-functional proteins. [Cavalli, G. and Dinarello, C A. Immunological Revs. (2018) 281 (1): 179-90].
[0615] Low concentrations of recombinant IL-37 most effectively suppress cytokine production in vitro. In nature, IL-37 is a dual function cytokine exerting potent anti-inflammatory effects via two distinct mechanisms, either extracellular (receptor-mediated) or intracellular (nuclear function). As shown in
[0616] Intracelluar/endogenous activity of IL-37. The IL-37 precursor is synthesized in human blood monocytes following stimulation by IL-1 or TLR agonists. in human blood monocytes, Pro-inflammatory stimuli induce an increase in intracellular IL-37 precursor while also triggering the activation of caspase-1, which cleaves the carboxyldomain of the IL-37 precursor. Mature Il-37 then associates with phosphorylated Smad-3, which enables nuclear translocation and regulation of gene transcription. Both the mature and precursor forms of IL-37 are released into the extracellulular space upon cell death or secreted by an unknown mechanism.
[0617] Extracellular/exogenous activity of IL-37. Both the mature and precursor forms of IL-37 are released into the extracellular space upon cell each or secreted by an unknown mechanism. Extracellular proteases process IL-37 precursor into the mature form. IL-37 binds to the IL18Rα and recruits IL1R8. IL-1R8 has a mutated TIR domain, which functions as a sink for MyD88; as a result, there is a weak or no transduction of pro-inflammatory signals, while anti-inflammatory pathways are activated. [Cavalli, G. and Dinearello, C A. Immunological Revs. (2018) 281(1): 179-90].
[0618] Li, et al. (accepted manuscript) examined early response of IL-37 in 254 SARS-CoV-2 infected patients prior to any clinical intervention and determined that higher early IL-37 plasma responses correlated with earlier viral RNA negative conversion, chest CT image improvement and cough relief, resulting in earlier hospital discharge. Higher IL-37 was associated with lower IL-6 and IL-8 and higher IFN-α in these patients. In contrast, low early IL-37 plasma responses predicted severe clinical prognosis in combination with IL-8 and C-reactive protein (CRP), a blood test for inflammation. They reported that Il-37 administration attenuated lung inflammation and alleviated respiratory tissue damage in human angiotensin-converting enzyme 2 (hACE2)-transgenic mice infected with SARS-CoV2.
[0619] CD24, also known as Heat Stable Antigen (HSA) or Small Cell Lung Carcinoma Cluster 4 Antigen, is a heavily glycosylated glycophosphatidylinositol (GPI)-anchored surface protein [Barkal, A. A. et al. Nature (2019) 572 (7769): 392-96, citing Pirruccello S J, LeBien T W The human B cell-associated antigen CD24 is a single chain sialoglycoprotein. J. Immunol. 136, 3779-3784 (1986), Chen G Y, Brown N K, Zheng P, Liu Y Siglec-G/10 in self-nonself discrimination of innate and adaptive immunity. Glycobiology 9, 800-806 (2014)]. Several signal transduction proteins are associated with CD24 activity, including the Src-family protein tyrosine kinases Lyn, Fyn, Fgr, Lck snf Hck, but how these are activated is unknown. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146]. Many ligands have been identified for CD24, including P-, L- and E-Selectin, High Mobility Group Box 1 (HMGB1), Li cell adhesion molecule (L1CAM), Neural cell adhesion molecule (NCAM1) and Siglec-G. [Id., citing Aigner, et al. 1995; Myung, et al. 2011; Tan et al 2016]. To explain the contradictory nature of the processes regulated by CD24, its apparent lack of intrinsic signaling capability or its diverse collection of reported ligands, It has been proposed that CD24 functions as a rheostat to modulate responses transduced by partnered cell surface receptor(s), and that these partners define the biological outcomes observed [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146]. Mechanistically, the variable nature of CD24-mediated effects can be explained by its in cis association with unique cell-type specific signaling partners through direct physical interaction mediated by its modifiable glycosylations. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146].
[0620] It has been hypothesized that the DAMPS released during cell death in viral infection may cause a self-propagating inflammatory response with lasting lung damage. [Tian, R-R et al. Cellular & Molec. Immunol. (2020) 17: 887-888]. Therefore, it has been proposed that the CD24-mediated Siglec10/G interaction is an immune checkpoint that regulates inflammation caused by DAMPS. [Id., citing Chen, G. et al. Science (2009) 323: 1722-25; Liu, Y. et al. Trends Immunol. (2009) 30: 557-61; Fang, X. et al. Cell Mol. Immunol. (2010) 7: 100-103].
[0621] CD24 is known to interact with Sialic Acid Binding Ig Like Lectin 10 (Siglec-10) on innate immune cells in order to dampen damaging inflammatory responses to infection [Id., citing Chen W et al. Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation. (2013) Cell 152(3), 467-478], sepsis [(Id, citing Chen G Y et al. Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. Nature Biotechnology (2011) 29, 428-435), liver damage [Id., citing Chen G Y et al. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science (2009) 323 (5922), 1722-1725], and chronic graft v. host disease [Id., citing Toubai T et al. Siglec-G-CD24 axis controls the severity of graft-versus-host disease in mice. Blood (2014) 123(22), 3512-3513]. The binding of CD24 to Siglec-10 elicits an inhibitory signaling cascade mediated by SHP-1 and/or SHP-2 phosphatases associated with the two immunoreceptor tyrosine-based inhibition motifs (ITIMS) in the cytoplasmic tail of Siglec-10, thereby blocking TLR-mediated inflammation and the cytoskeletal rearrangement required for cellular engulfment by macrophages [Id., citing Crocker P R, Paulson J C, Varki A Siglecs and their roles in the immune system. Nature Reviews Immunology (2007) 7, 255-266; Abram C L, Lowell C A Shp1 function in myeloid cells. J. Leukoc. Biol (2017) 102(3), 657-675 Dietrich J, Cella M, Colonna M Ig-Like Transcript 2 (ILT2)/Leukocyte Ig-Like Receptor 1 (LIR1) Inhibits TCR Signalling and Actin Cytoskeleton Reorganization. J. Immunol. (2001) 166(4), 2514-2521].
[0622] It has been reported that a recombinant fusion protein CD24-Fc (an agonist of Sioglecs, which can fortify the CD24-Siglec innate immune checkpoint) had a therapeutic effect on SIV-induced lung inflammatory lesions. [Tian, R-R et al., Cellular & Molec. Immunol. (2020) 17: 887-88]. Chinese rhesus macaques were infected with simian immunodeficiency virus SIVmac239 via intravenous infusion received either three injections of a recombinant fusion protein CD24-Fc or normal saline on day 56 of infection. Five months later, another cycle of treatment was given to the surviving animals, which were terminated one week after the last dosing. Previous studies had shown that lung lesions developed within 2-4 weeks in SIV-infected rhesus monkeys. By 8 weeks, essentially all monkeys developed lung pathology. The data showed that CD24Fc not only reduced the incidence of viral pneumonia but also qualitatively altered the nature of the pathology in the lung.
[0623] It has also been reported that CD24 is important in regulating T cell survival. T cells must regulate their proliferation to support a long-lived cell population, but can expand their numbers during immune activation. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146, citing Boyman, O. et al Eur. J. Immunol. (2009) 39: 2088-94]. In the absence of CD24, homeostatic proliferation of T cells is markedly reduced, however immune-driven proliferation is less affected [Id., citing Li. O. et al. J. Exp. Med. (2004) 200: 1083-89], likely because it depends on TCR co-receptors [Id., citing Chen, L. and Flies, D B Nat. Rev. Immunol. (2013) 13: 227-42]. When CD24+ T cells are transferred to CD24-knockout mice, excessive and destructive homeostatic T cell proliferation occurs, but CD24 expressed on dendritic cells is sufficient to ameliorate this effect [Id., citing Li, O. et al J. Exp. Med. (2006) 203: 1713-20]. This suggested that CD24 can act in cis on the T cell to regulate TCR signaling, or in trans, where DC-expressed Cd24 can bind and modulate its partner(s) on the T cell.
Other Compatible Actives
[0624] According to some embodiments, the anti-inflammatory agent comprises aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, infliximab, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, or combinations thereof.
[0625] According to some embodiments, the anti-infective agent comprises amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem.
[0626] According to some embodiments, the anti-malarial agent comprises quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; or atovaquone.
[0627] According to some embodiments, the anti-viral agent comprises acyclovir, gancidovir, foscamet; ribavirin; amantadine, azidodeoxythymidine/zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, or delavirdine. According to some embodiments, the anti-viral agent is an agent that inhibits viral entry and decreases viral load.
[0628] According to some embodiments, the anti-fibrotic agent comprises nintedanib, pirfenidone, ord combinations thereof.
Formulations
[0629] Formulations of the pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Exemplary carrier solutions also can contain buffers, diluents and other suitable additives. The term “buffer” as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH. Examples of buffers envisioned by the described invention include, without limitation, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl). In some embodiments, the infusion solution is isotonic to subject tissues.
[0630] Exemplary pharmaceutical compositions of the described invention may comprise a suspension or dispersion of cells in a nontoxic parenterally acceptable diluent or solvent. A solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent. A dispersion is a two-phase system, in which one phase (e.g., particles) is distributed in a second or continuous phase. A suspension is a dispersion in which a finely-divided species is combined with another species, with the former being so finely divided and mixed that it does not rapidly settle out. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride (saline) solution.
[0631] Additional compositions of the present disclosure can be readily prepared using technology which is known in the art such as described in Remington's Pharmaceutical Sciences, 18th or 19th editions, published by the Mack Publishing Company of Easton, Pa., which is incorporated herein by reference.
[0632] Formulations of the pharmaceutical composition may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
[0633] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
[0634] Pharmaceutical compositions that are useful in the methods of the disclosure may be prepared/formulated, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
[0635] According to some embodiments, the pharmaceutical compositions of the described invention may be administered initially, and thereafter maintained by further administrations. For example, according to some embodiments, the pharmaceutical compositions of the described invention may be administered by one method of injection, and thereafter further administered by the same or by different method.
[0636] According to some embodiments, a protein stabilizing agent can be added to the cell product comprising the expended and enriched population of SCKTCs after manufacturing, for example albumin, which may act as a stabilizing agent. According to some embodiments, the albumin is human albumin. According to some embodiments, the albumin is recombinant human albumin. According to some embodiments, the minimum amounts of albumin employed in the formulation may be about 0.5% to about 25% w/w, i.e., about 0.5%, about 1.0%, about 2.0, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25% w/w, including intermediate values, such as about 12.5% w/w.
[0637] According to some embodiments, the pharmaceutical composition may comprise a stabilizing amount of serum. The term “stabilizing amount” as used herein refers to the amount of serum that, when included in the formulation of the pharmaceutical composition of the described invention comprising enriched SCKTCs, enables these cells to retain their T cell effector activity. According to some embodiments, the serum is human serum autologous to a human patient. According to some embodiments, the serum is synthetic serum. According to some embodiments the stabilizing amount of serum is at least about 1.0% (v/v).
[0638] According to some embodiments, the methods of the present disclosure comprise the further step of preparing the pharmaceutical composition by adding a pharmaceutically acceptable excipient, in particular an excipient as described herein, for example a diluent, stabilizer and/or preservative.
[0639] The term “excipient” as employed herein is a generic term to cover all ingredients added to the SCKTC population that do not have a biological or physiological function, which are nontoxic and do not interact with other components.
[0640] Once the final formulation of the pharmaceutical composition has been prepared it will be filled into a suitable container, for example an infusion bag or cryovial.
[0641] According to some embodiments, the methods according to the present disclosure comprises the further step of filling the pharmaceutical composition comprising the cell product containing the expanded and enriched population of SCKTCs or a pharmaceutical formulation thereof into a suitable container, such as an infusion bag and sealing the same to form the cell product.
[0642] According to some embodiments, the product comprising the container filled with the pharmaceutical composition comprising the cell product comprising the expanded and enriched population of SCKTCs of the present disclosure is frozen for storage and transport, for example at about −135° C., for example in the vapor phase of liquid nitrogen. According to some such embodiments, the formulation may also contain a cryopreservative, such as DMSO. The quantity of DMSO generally is from about 5% to about 10%, inclusive, i.e., at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or 10% v/v.
[0643] According to some embodiments, the process of the present disclosure comprises the further step of freezing the pharmaceutical composition, or the cell product comprising the expanded and enriched population of SCKTCs of the present disclosure. According to one embodiment, freezing occurs by a controlled rate freezing process, for example reducing the temperature by 1° C. per minute to ensure the crystals formed are small and do not disrupt cell structure. This process may be continued until the sample has reached at least −80° C.
[0644] Controlled- or sustained-release formulations of the pharmaceutical composition of the disclosure may be made by adapting otherwise conventional technology. The term “controlled release” as used herein is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This includes immediate as well as non-immediate release formulations, with non-immediate release formulations including, but not limited to, sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) is used herein in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant levels of a drug over an extended time period. The term “delayed release” is used herein in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.” The term “long-term” release, as used herein, means that the drug formulation is constructed and arranged to deliver therapeutic levels of the active ingredient over a prolonged period of time, e.g., days.
[0645] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations may include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. For parenteral application, suitable vehicles consist of solutions, e.g., oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances, which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
[0646] According to some embodiments, the present disclosure provides a method of transporting a cell product comprising the expanded and enriched population of SCKTCs according to the present disclosure from the place of manufacture, or a convenient collection point, to a therapeutic facility. According to some embodiments, the temperature of the cell product is maintained during such transporting. According to some embodiments, for example, the pharmaceutical composition can be stored below 0° C., such as −135° C. during transit. According to some embodiments, temperature fluctuations of the pharmaceutical composition are monitored during storage and/or transport.
3. Administering the Pharmaceutical Composition Comprising the Cell Product
[0647] According to another aspect, the present disclosure provides a method of treating a virus infection, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutic amount of the cell product comprising superactivated cytokine killer T cells of the present disclosure.
[0648] According to some embodiments, the virus infection is an infection with a respiratory virus. According to some embodiments the respiratory virus is a respiratory syncytial virus (RSV), an Ebola virus, a cytomegalovirus, a Hanta virus, an influenza virus, a coronavirus, a Zika virus, a West Nile virus, a dengue virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a yellow fever virus, a rhinovirus, an adenovirus, a herpes virus, an Epstein Barr virus, a measles virus, a mumps virus, a rotavirus, a coxsackie virus, a norovirus, or an encephalomyocarditis virus (EMCV). According to some embodiments, the coronavirus is SARS-CoV-1, SARS-CoV-2 or MERS.
[0649] According to some embodiments, the respiratory virus infection is a severe viral infection. According to some embodiments, symptoms of the severe respiratory virus infection include one or more of: primary viral pneumonia; superimposed bacterial pneumonia; disruption or injury to alveolar epithelium, endothelium or both; acute lung injury (ALI); acute respiratory distress syndrome (ARDS); symptoms of shock; excessive complement activation; a pathological increase in vascular permeability; endothelial activation, loss of barrier function and consequent microvascular leak; thrombotic complications; kidney damage; or elevated concentrations of one or more inflammatory mediators in plasma (hypercytokinemia), compared to a normal healthy subject. 1%
[0650] According to some embodiments, symptoms of shock include low blood pressure, lightheadedness, shortness of breath, and rash. According to some embodiments, the thrombotic complications include one or more of formation of pulmonary microthrombi, acute pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic arterial embolism. According to some embodiments, the inflammatory mediator includes one or more of interferon α, interferon β, interferon-κ, interferon-γ, complement, prostaglandin D2, vasoactive intestinal peptide (VIP), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), IL-17, or tumor necrosis factor-alpha (TNF-α).
[0651] According to some embodiments, the severe viral infection is characterized by viral pathogen-infected cells.
[0652] According to some embodiments, the therapeutic amount reduces risk of the virus infection. According to some embodiments, the therapeutic amount reduces signs, symptoms, or both signs and symptoms of the viral infection. According to some embodiments, the therapeutic amount reduces extent of the viral infection where symptoms are not yet clinically recognized. According to some embodiments, the therapeutic amount reduces worsening or progression of the viral infection. According to some embodiments, the therapeutic amount reduces severity of the viral infection developed compared to an untreated subject. According to some embodiments, the therapeutic amount decreases viral burden. According to some embodiments, the therapeutic amount improves progression-free survival. According to some embodiments, the therapeutic amount improves overall survival.
[0653] According to some embodiments, the therapeutic amount destroys the infected cells through direct lysis or by effecting destruction of the infected cells indirectly, e.g., by mobilizing attracting cell cytotoxicity agents through secretion of cytokines.
[0654] According to some embodiments, the therapeutic amount mobilizes the patient's immune response to the viral pathogen, where the term “mobilizes” as used herein means to put into motion or use, become ready or capable of being moved quickly and with relative ease. stimulates activation of the patient's lymphocyte populations.
[0655] According to some embodiments, the term “a therapeutically effective amount” or dose does not necessarily mean an amount that is immediately therapeutically effective, but includes a dose which is capable of expansion in vivo (after administration) to provide a therapeutic effect. Thus, there is provided a method of administering to a patient a sub-therapeutic dose that nonetheless becomes a therapeutic amount after expansion and activation of SCKTCs in vivo to provide the desired therapeutic effect.
[0656] According to some aspects, the pharmaceutical composition of the present disclosure supplements a biologically insufficient immune response of the subject at risk for a virus infection by stimulating one or more immune cell population of the subject. According to some embodiment, the immune cell population comprises a dendritic cell population. According to some embodiments, the immune cell population comprises a CD8+ T cell population. According to some embodiments, the immune cell population comprises an NK cell population. According to some embodiments, the immune cell population comprises an MHC-restricted T cell population. According to some embodiments, the MNC-restricted T cell population comprises an invariant NKT population.
[0657] According to some embodiments, the therapeutic amount stimulates an effector function of the patient's immune cells. According to some embodiments, the effector function of the immune cell includes one or more of cytokine secretion, cytotoxicity, or antibody-mediated clearance of the pathogen.
Additional Compatible Actives
[0658] According to some embodiments, the pharmaceutical compositions of the described invention can further include one or more compatible active ingredients which are aimed at providing the composition with another pharmaceutical effect in addition to that provided by the cell product of the described invention. “Compatible” as used herein means that the active ingredients of such a composition are capable of being combined with each other in such a manner so that there is no interaction that would substantially reduce the efficacy of each active ingredient or the composition under ordinary use conditions.
[0659] According to some embodiments, the pharmaceutical composition comprising the SCKTC cell product further comprises an enriched population of NK cells. According to some embodiments, the population of NK cells can be acquired by apheresis of peripheral blood from PBMCs. According to some embodiments, stem cell mobilization, a process whereby CD34+ hematopoietic stem cells are stimulated out of the bone marrow into the blood stream, may be used to harvest PBMCs. According to some embodiments, Plerixafor in combination with G-CSF may be used to mobilize the CD34+ stem cells into the blood before collection. According to some embodiments, the PBMCs are depleted of CD3+ T cells and/or CD19 B cells with magnetic beads. According to some embodiments, CD3−CD56+NK cells are positively selected with magnetic beads. According to some embodiments, the selected CD3−CD56+ cells or T cell and/or B cell depleted cells are differentiated to NK cells by culturing in NK cell medium containing high IL-2 (2813 U/mL) for 14 days. According to some embodiments, the population of enriched NK cells can be expanded using static cell culture bags or an automated bioreactor. [e.g., see Saito, S. et al. Human Gene Therapy Methods (2013) 24 (4): 241-52; Spanholtz, J. et al. PLoS One (2011) 6 (6): e20740]. According to some embodiments, the NK cells are characterized by flow cytometry for identity/activation markers, for IFN-γ expression and secretion; and for their cytolytic potential against an MHC class I null cell line, e.g., K562.
[0660] According to some embodiments, the pharmaceutical composition comprising the cell product containing the population of SCKTCs may be administered with a supportive therapy or an additional therapeutic agent, e.g., one or more of an immunomodulatory agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent or an anti-fibrotic agent.
[0661] According to some embodiments, the supportive therapy is therapeutic apheresis comprising a virion removing step. According to some embodiments, the therapeutic apheresis reduces viral load.
[0662] According to some embodiments, the additional agent regulates immune cell activation. According to some embodiments, the additional agent modulates T cell exhaustion pathways.
Immunomodulatory Agents
[0663] According to some embodiments, the immunomodulatory agent may comprise methotrexate; a glucocorticoid, cyclosporine, tacrolimus and sirolimus; a recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, IFN-ω; a recombinant IL-2 receptor inhibitor; a PDE4 inhibitor; a hyperimmune globulin prepared from a donor with high titers of a desired antibody; a TNFα inhibitor/antagonist; an IL-1β inhibitor; a chimeric IL-1Ra; an IL-6 inhibitor; an IL-12/IL-23 inhibitor selected from ustekinumab, briakinumab; an IL-23 inhibitor selected from guselkumab, tildrakizumab; a compound that targets TLR4 signaling; a p38 MAPK inhibitor, a compound that targets Janus kinase signaling; a compound that targets cell adhesion molecules to reduce leukocyte recruitment; a checkpoint inhibitor, or a recombinant anti-inflammatory cytokine.
[0664] According to some embodiments, a physiologic or supraphysiological dose of the recombinant interferon selected from IFN-α; IFN-α-2b, IFN-β, IFN-γ, IFN-κ, and IFN-ω or a PEGylated form thereof boosts immune defenses of the subject.
[0665] According to some embodiments, the glucocorticoid comprises a corticosteroid comprising prednisone, dexamethasone, azathioprine, mycophenolate, mycophenolate mofetil, or combinations thereof; or the recombinant IL-2 inhibitor comprises denileukin diftitox; or the PDE4 inhibitor comprises cilomilast; or the TNFα inhibitor/antagonist comprises etanercept; adalimumab; infliximab, certolizumab pegol, or golimumab; or the IL-1β inhibitor comprises rilonacept; canakinumab; or Anakinra; or the IL-6 inhibitor comprises tocilizumab, siltuximab, sarilumab, olokizumab, or sirukumab; or the compound that targets TLR4 signaling comprises (ethyl 4-(4′-chlorophenyl) amino-6 methyl-2-oxocyclohex-3-en-1-aote (enamionone E121), JODI 18b; JODI 19, resatorvid, TLR-C34; or C35; or the p38 MAPK inhibitor comprises 4-(4′-fluorophenyl)-2-(4′-methylsulfinylphenyl)-5-(4′-pyridyl)-imidazole (SB203580), trans-4-[4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol (SB239063), or 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ 67657); or the compound that targets Janus kinase signaling comprises tofacitinub, baricitinib, or upadacitinib; or the compound that targets a cell adhesion molecule to reduce leukocyte recruitment comprises an a4 integrin inhibitor comprising vedolizumab or natalizumab; or the recombinant anti-inflammatory cytokine comprises IL-4, IL-10, or IL-11; or the interferon is in a PEGylated form.
[0666] The term “immune checkpoint molecules” as used herein refers to ligand-receptor pairs that exert inhibitory or stimulatory effects on immune responses. Examples include programmed cell death 1 receptor (PD-1, also known as CD279), thought to regulate T cell proliferation later in the immune response, and its ligand programmed cell death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG3), which suppresses T cells activation and cytokine secretion, thereby ensuring immune homeostasis and shows synergy with PD-1 to inhibit immune responses (Long, L. et al. Genes Cancer (2018) 9 (5-6): 176-89. and cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152), a negative regulator of T cell immune function thought to regulate T cell proliferation early in an immune response [Buchbinder, E I, and Desai, A. Am. J. Clin. Oncol. (39) (10: 98-106). In addition, glucocorticoid-induced TNFR family related gene (GITR), a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes activation; GITR activation by its ligand (GITRL) influences the activity of effector and regulatory T cells, thus participating in the development of immune response against tumors and infectious agents, as well as in autoimmune and inflammatory diseases. [Nocentini, G. et al. Br. J. Pharmacol. (2012) 165 (7): 2089-99] T-cell immunoglobulin and mucin domain 3 (Tim-3) is a checkpoint receptor expressed by a wide variety of immune cells as well as leukemic stem cells. [Acharya, N. et al. J. Immunother. Cancer (2020) 8(10: e000911). T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is an immune checkpoint receptor that can suppress T-cell activation and promote T-cell exhaustion. Inhibition of TIGIT may increase cytotoxic T-cell proliferation and function. Inducible T cell costimulator (ICOS, cluster of differentiation (CD278)) is an activating costimulatory immune checkpoint expressed on activated T cells. Its ligand, ICOSL is expressed on antigen-presenting cells and somatic cells, including tumour cells in the tumour microenvironment. [Solinas, C. et al. ESMO Open. (2020) 5(1): e000544].
[0667] According to some embodiments, the immunomodulator is an immune checkpoint inhibitor. The term “immune checkpoint inhibitor” as used herein refers to a molecule that can block immune checkpoint molecules. Specific immune checkpoint inhibitors, including antibodies against CTLA-4, PD-1 receptor or its ligand PD-L1 include YERVOY™ (Ipilimumab; CTLA-4 antagonist), OPDIVO™ (Nivolumab; PD-1 antagonist) and KEYTRUDA™ (Pembrolizumab; PD-1 antagonist) in multiple tumor indications, with ongoing registration trials in many more.
[0668] According to some embodiments, the immunomodulatory agent comprises recombinant IL-37. According to some embodiments, the immunomodulatory agent comprises recombinant CD24. According to some embodiments, the immunomodulatory agent comprises rIL-37 and rCD24.
[0669] IL-37 is a member of the IL-1 family, which includes IL-1α, IL-1β, IL-18, IL33, IL36α, IL-36β, IL-36γ, IL-37 and IL-38. [Mantovani, A. et al. Immunity (2019) doi.10.1016/j.immuni.2019.03.012]. The IL-1 family of cytokines is divided into three subgroups on the basis of the Il-1 consensus sequence and the signaling receptor chain. These include secreted molecules with agonistic activity [IL-1α, IL-1β, IL-18, IL33, IL36α, IL-36β, IL-36γ], receptor antagonists [IL-1Ra, IL36Ra, and IL-38] and an anti-inflammatory cytokine (IL-37) [Id., citing Dinarello, C A Immunol. Rev. (2018) 281: 8-27]. There is no murine counterpart to human IL-37. [Id.].
[0670] In humans, production of IL-37 is activated by pro-inflammatory stimuli, including cytokines, as a protective mechanism to prevent runaway inflammation and excessive damage. Five transcripts for the human IL-37 gene have been identified (IL-37 a-e). IL-37b is the most complete of these isoforms, is the most abundant and studied, and includes 5 of the 6 exons of the IL-37 gene (all but exon 3). Exons 4,5, and 6 encode for the sequence required for the beta-fold barrel structure and account for the extracellular activity of recombinant IL-37. Conversely, exons 1 2, and 3 may be cleaved in the extracellular environment by unknown proteases. IL-37 isoforms a, b and d share exons 4, 5, and 6 and encode functional proteins. The IL-37 isoforms c and e lack one or more of these exons and likely encode non-functional proteins. [Cavalli, G. and Dinarello, C A. Immunological Revs. (2017) 281: 1-12].
[0671] Low concentrations of recombinant IL-37 most effectively suppress cytokine production in vitro. In nature, IL-37 is a dual function cytokine exerting potent anti-inflammatory effects via two distinct mechanisms, either extracellular (receptor-mediated) or intracellular (nuclear function). As shown in
[0672] Intracelluar/endogenous activity of IL-37. The IL-37 precursor is synthesized in human blood monocytes following stimulation by IL-1 or TLR agonists. in human blood monocytes, Pro-inflammatory stimuli induce an increase in intracellular IL-37 precursor while also triggering the activation of caspase-1, which cleaves the carboxyldomain of the IL-37 precursor. Mature Il-37 then associates with phosphorylated Smad-3, which enables nuclear translocation and regulation of gene transcription. Both the mature and precursor forms of IL-37 are released into the extracellulular space upon cell death or secreted by an unknown mechanism.
[0673] Extracellular/exogenous activity of IL-37. Both the mature and precursor forms of IL-37 are released into the extracellular space upon cell death or secreted by an unknown mechanism. Extracellular proteases process IL-37 precursor into the mature form. IL-37 binds to the IL18Rα and recruits IL1R8. IL-1R8 has a mutated TIR domain, which functions as a sink for MyD88; as a result, there is a weak or no transduction of pro-inflammatory signals, while anti-inflammatory pathways are activated. [Cavalli, G. and Dinearello, C A. Immunological Revs. (2018) 281 (1): 179-90].
[0674] Li, et al. (accepted manuscript) examined early response of IL-37 in 254 SARS-CoV-2 infected patients prior to any clinical intervention and determined that higher early IL-37 plasma responses correlated with earlier viral RNA negative conversion, chest CT image improvement and cough relief, resulting in earlier hospital discharge. Higher IL-37 was associated with lower IL-6 and IL-8 and higher IFN-α in these patients. In contrast, low early IL-37 plasma responses predicted severe clinical prognosis in combination with IL-8 and C-reactive protein (CRP), a blood test for inflammation. They reported that IL-37 administration attenuated lung inflammation and alleviated respiratory tissue damage in human angiotensin-converting enzyme 2 (hACE2)-transgenic mice infected with SARS-CoV2.
[0675] CD24, also known as Heat Stable Antigen (HSA) or Small Cell Lung Carcinoma Cluster 4 Antigen, is a heavily glycosylated glycophosphatidylinositol (GPI)-anchored surface protein [Barkal, A. A. et al. Nature (2019) 572 (7769): 392-96, citing Pirruccello S J, LeBien T W The human B cell-associated antigen CD24 is a single chain sialoglycoprotein. J. Immunol. 136, 3779-3784 (1986), Chen G Y, Brown N K, Zheng P, Liu Y Siglec-G/10 in self-nonself discrimination of innate and adaptive immunity. Glycobiology 9, 800-806 (2014)]. Several signal transduction proteins are associated with CD24 activity, including the Src-family protein tyrosine kinases Lyn, Fyn, Fgr, Lck snf Hck, but how these are activated is unknown. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146]. Many ligands have been identified for CD24, including P-, L- and E-Selectin, High Mobility Group Box 1 (HMGB1), Li cell adhesion molecule (L1CAM), Neural cell adhesion molecule (NCAM1) and Siglec-G. [Id., citing Aigner, et al. 1995; Myung, et al. 2011; Tan et al 2016]. To explain the contradictory nature of the processes regulated by CD24, its apparent lack of intrinsic signaling capability or its diverse collection of reported ligands, It has been proposed that CD24 functions as a rheostat to modulate responses transduced by partnered cell surface receptor(s), and that these partners define the biological outcomes observed [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146]. Mechanistically, the variable nature of CD24-mediated effects can be explained by its in cis association with unique cell-type specific signaling partners through direct physical interaction mediated by its modifiable glycosylations. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146].
[0676] It has been hypothesized that the DAMPS released during cell death in viral infection may cause a self-propagating inflammatory response with lasting lung damage. [Tian, R-R et al. Cellular & Molec. Immunol. (2020) 17: 887-888]. Therefore, it has been proposed that the CD24-mediated Siglec10/G interaction is an immune checkpoint that regulates inflammation caused by DAMPS. [Id., citing Chen, G. et al. Science (2009) 323: 1722-25; Liu, Y. et al. Trends Immunol. (2009) 30: 557-61; Fang, X. et al. Cell Mol. Immunol. (2010) 7: 100-103].
[0677] CD24 is known to interact with Sialic Acid Binding Ig Like Lectin 10 (Siglec-10) on innate immune cells in order to dampen damaging inflammatory responses to infection [Id., citing Chen W et al. Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation. (2013) Cell 152(3), 467-478], sepsis [(Id, citing Chen G Y et al. Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. Nature Biotechnology (2011) 29, 428-435), liver damage [Id., citing Chen G Y et al. CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science (2009) 323 (5922), 1722-1725], and chronic graft v. host disease [Id., citing Toubai T et al. Siglec-G-CD24 axis controls the severity of graft-versus-host disease in mice. Blood (2014) 123(22), 3512-3513]. The binding of CD24 to Siglec-10 elicits an inhibitory signaling cascade mediated by SHP-1 and/or SHP-2 phosphatases associated with the two immunoreceptor tyrosine-based inhibition motifs (ITIMS) in the cytoplasmic tail of Siglec-10, thereby blocking TLR-mediated inflammation and the cytoskeletal rearrangement required for cellular engulfment by macrophages [Id., citing Crocker P R, Paulson J C, Varki A Siglecs and their roles in the immune system. Nature Reviews Immunology (2007) 7, 255-266; Abram C L, Lowell C A Shp1 function in myeloid cells. J. Leukoc. Biol (2017) 102(3), 657-675 Dietrich J, Cella M, Colonna M Ig-Like Transcript 2 (ILT2)/Leukocyte Ig-Like Receptor 1 (LIR1) Inhibits TCR Signalling and Actin Cytoskeleton Reorganization. J. Immunol. (2001) 166(4), 2514-2521].
[0678] It has been reported that a recombinant fusion protein CD24-Fc (an agonist of Sioglecs, which can fortify the CD24-Siglec innate immune checkpoint) had a therapeutic effect on SIV-induced lung inflammatory lesions. [Tian, R-R et al., Cellular & Molec. Immunol. (2020) 17: 887-88].Chinese rhesus macaques were infected with simian immunodeficiency virus SIVmac239 via intravenous infusion received either three injections of a recombinant fusion protein CD24-Fc or normal saline on day 56 of infection. Five months later, another cycle of treatment was given to the surviving animals, which were terminated one week after the last dosing. Previous studies had shown that lung lesions developed within 2-4 weeks in SIV-infected rhesus monkeys. By 8 weeks, essentially all monkeys developed lung pathology. The data showed that CD24Fc not only reduced the incidence of viral pneumonia but also qualitatively altered the nature of the pathology in the lung.
[0679] It has also been reported that CD24 is important in regulating T cell survival. T cells must regulate their proliferation to support a long-lived cell population, but can expand their numbers during immune activation. [Ayre, D C and Christian, S L, Front. Cell & Devel. Biol. (2016) 4: 1146, citing Boyman, O. et al Eur. J. Immunol. (2009) 39: 2088-94]. In the absence of CD24, homeostatic proliferation of T cells is markedly reduced, however immune-driven proliferation is less affected [Id., citing Li. O. et al. J. Exp. Med. (2004) 200: 1083-89], likely because it depends on TCR co-receptors [Id., citing Chen, L. and Flies, D B Nat. Rev. Immunol. (2013) 13: 227-42]. When CD24+ T cells are transferred to CD24-knockout mice, excessive and destructive homeostatic T cell proliferation occurs, but CD24 expressed on dendritic cells is sufficient to ameliorate this effect [Id., citing Li, O. et al J. Exp. Med. (2006) 203: 1713-20]. This suggested that CD24 can act in cis on the T cell to regulate TCR signaling, or in trans, where DC-expressed Cd24 can bind and modulate its partner(s) on the T cell.
Other Compatible Actives
[0680] According to some embodiments, the anti-inflammatory agent may comprise aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabunetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sarilumab (Kevzara®) sulindac, tolmetin, or combinations thereof.
[0681] According to some embodiments, the anti-infective agent may comprise amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazle and trimethoprim; sulfasalazine, amoxicillin and clavulanate; vancomycin, dalbavancin, oritavancin, telavancin, gentamycin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem, or ertapenem.
[0682] According to some embodiments, the anti-malarial agent may comprise quinine, quinidine, chloroquine, hydroxychloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; or atovaquone.
[0683] According to some embodiments, the anti-viral agent may comprise acyclovir, gancidovir, foscamet; ribavirin; amantadine, azidodeoxythymidine/zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, lopinavir/ritonavir, umifenovir, favipiravir, ivermectin, or delavirdine. According to some embodiments, the anti-viral agent is an agent that inhibits viral entry and decreases viral load.
[0684] According to some embodiments, the anti-fibrotic agent may comprise nintedanib, pirfenidone, or combinations thereof.
Treating Regimens
[0685] The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
[0686] The administration of the pharmaceutical compositions containing the cell product may be carried out in any manner appropriate to the particular disease, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The pharmaceutical compositions of the present disclosure may be administered to a patient parenterally, e.g., subcutaneously, intradermally, intramuscularly, by intravenous (i.v.) injection, intraperitoneally, or by infusion techniques. According to some embodiments, the pharmaceutical compositions of the described invention also can be administered to a subject by direct injection to a desired site, or systemically.
[0687] According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient daily. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient by continuous infusion. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient twice daily. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient more than twice daily. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient every other day. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient twice a week. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient every other week. According to some embodiments, the pharmaceutical composition containing the population of SCKTCs can be administered to a patient every 30 days, or every 1, 2, 3, 4, 5, or 6 months.
[0688] According to some embodiments, the pharmaceutical composition comprising a cell product containing the population of SCKTCs can be administered to a patient in a dosing regimen (dose and periodicity of administration) sufficient to maintain function of the administered SCKTCs in the bloodstream of the patient over a period of 2 weeks to a year or more, e.g., one month to one year or longer, e.g., at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, a year, 2 years.
[0689] The frequency of the required dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
[0690] Alternatively, the additional therapeutic agent(s) may be administered an hour, a day, a week, a month, or even more, in advance of the pharmaceutical composition, or any permutation thereof. Further, the additional therapeutic agent(s) may be administered an hour, a day, a week, or even more, after administration of the pharmaceutical composition, or any permutation thereof. The frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the additional therapeutic agent or agents being administered, the route of administration and the pharmaceutical composition comprising the population of SCKTCs, and the like.
[0691] According to some embodiments, a “subject having an infection” is a subject that has been exposed to an infectious pathogen with acute or chronic detectable levels of the microorganism in his/her body or has signs and symptoms of the infectious pathogen. Methods of assessing and detecting infections in a subject are known by those of ordinary skill in the art. A “subject at risk of an infection” is a subject that may be expected to come in contact with an infectious pathogen. Examples of such subjects are medical workers or those traveling to parts of the world where the incidence of infection is high. According to some embodiments, the subject is at an elevated risk of an infection because the subject has one or more risk factors to have an infection. Examples of risk factors to have an infection include, for example, immunosuppression, immunocompromise, age, trauma, burns (e.g., thermal burns), surgery, foreign bodies, cancer, newborns especially newborns born prematurely. The degree of risk of an infection depends on the multitude and the severity or the magnitude of the risk factors that the subject has. Risk charts and prediction algorithms are available for assessing the risk of an infection in a subject based on the presence and severity of risk factors. Other methods of assessing the risk of an infection in a subject are known by those of ordinary skill in the art. According to some embodiments, the subject who is at an elevated risk of an infection may be an apparently healthy subject. An “apparently healthy subject” is a subject who has no signs or symptoms of disease.
[0692] According to some embodiments, factors other than age associated with the target population for treatment are considered. These factors include, but are not limited to, comorbidities, geographic factors (including microbial endemicity), nutritional status, and iatrogenic immune suppression.
Subjects
[0693] The methods described herein are intended for use with any subject that may experience the benefits of these methods. Thus, “subjects,” “patients,” and “individuals” (used interchangeably) include humans as well as non-human subjects, particularly domesticated animals.
[0694] According to some embodiments, the subject and/or animal is a mammal, e g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions, which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
[0695] In other embodiments, the subject and/or animal is a non-mammal. According to some embodiments, the subject and/or animal is a human. According to some embodiments, the human is a pediatric human. According to other embodiments, the human is an adult human. According to other embodiments, the human is a geriatric human. According to other embodiments, the human may be referred to as a patient.
[0696] According to certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
[0697] According to some embodiments, the subject is a non-human animal, and therefore the disclosure pertains to veterinary use. According to some such embodiments, the non-human animal is a household pet. According to some such embodiments, the non-human animal is a livestock animal.
[0698] According to some embodiments, the susceptible subject includes a very young subject, an elderly subject, a subject who is ill; an immunocompromised subject, a subject with long term health conditions, a subject who is obese, or a subject that is physically weak due to malnutrition or dehydration.
[0699] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
[0700] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, exemplary methods and materials have been described.
[0701] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
[0702] All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.
[0703] It is also to be understood that throughout this disclosure where the singular is used, the plural may be inferred and vice versa and use of either is not to be considered limiting.
EXAMPLES
[0704] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1. Process Flow, Run 5 (Tissue Culture Flasks)
[0705] A flow chart depicting the process flow in Run 5 for stimulation of superactivated cytokine killer cells in tissue culture flasks is shown in
[0706] As shown in
[0707] The dendritic process flow is shown in
Characterization of SCKTCs, Run 5
[0708] Characteristics of representative SCKTCs produced by the process of
[0709] Cell Morphology.
[0710] Growth curves.
[0711] Cell Identity.
[0712] Cytokine production.
[0713] In vitro cytotoxicity.
Example 2. Superactivated Cytokine Killer Cell Amplification and Stimulation
[0714]
[0715] The process flow for the SCKTC cells is shown in
[0716] The process flow for the DCs is shown in
[0717] Characteristics of representative SCKTCs produced by the process of
[0718] Cell morphology. The morphology of the Run 14 cultures produced by the process flow of
[0719] Growth curve.
[0720] Cell identity.
[0721] Cytokine production.
[0722] Cytotoxicity.
Example 3. Use of K18-hACE2 Mice as a Model of SARS-CoV2 Infection
[0723] A transgenic mouse model that expresses the hACE2 gene under the control of the human cytokeratin 18 promoter will be used to test the efficacy of the pharmaceutical composition of the present disclosure as described by Moreau, G B et al. Am. J. Trop. Med. Hyg. (2020) 103 (3): 1215-19. Mice (K18-hACE2Prlmn/J, Jax #034860; available from Jackson Laboratories) will be infected with median tissue culture infected dose (TCID50) of 104 plaque-forming units (PFUs) of SARSCoV-2. The pharmaceutical composition comprising a cell product containing SSCKTCs will be administered by an intranasal route, intravenously, and/or intramuscularly in groups of 5 mice. Five mock-infected mice will receive 50 μl DMEM. Mice will be followed twice daily for clinical symptoms until day 5. Categories included in clinical scoring will include weight loss; posture and appearance of fur (piloerection), activity; eye closure, and respiratory rate.
[0724] Blood samples will be collected by standard procedures. Neutralizing and immunogen-specific antibody titers and isotypes produced by vaccinated mice in serum will be determined by measuring inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively.
[0725] For histology, the tissues of euthanized mice will be fixed in formaldehyde. Histopathological scoring for lung tissue will be performed according to the guidelines of the American Thoracic Society. Statistical significance will be determined by standard methods.
[0726] Viral titers will be determined by homogenizing the left lobe of the lung in 1 mL serum-free DMEM with a disposable tissue grinder and plaque assays performed. In brief, Vero cells grown in DMEM with fetal bovine serum will be seeded into multiwell plates at a concentration of 2×10.sup.5 cells/well the night before the assay. Serial dilutions will be added to the wells. The plate will be incubated at 37° C., 5% CO.sub.2 for 2 hr, shaking the plates every 15 minutes. After 2 hr the plate media will be replaced with a liquid overlay of DMEM, 2.5% FBS containing 1.2% Avicel PH-101 (Signa-Aldrich, St. Louis, Mo.) and incubated at 37° C., 5% CO.sub.2. After 3 days, the overlay will be removed, wells will be fixed with 10% formaldehyde and stained with 0.1% crystal violet to visualize plaques. Plaques will be counted, and PFUs calculated according to the following equation: average # plaques/dilution factor x volume diluted virus added to the well.
Example 4. Use of NSG Mice Reconstituted with Human Immune System Components for Evaluation of the Cell Product of the Present Disclosure
[0727] NSG (NOD-scid 11.2 Rγnull) mice (from The Jackson Laboratory, jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio) will be engrafted with human PBMC as follows. Fresh whole blood from healthy adult donors collected with preservative free heparin will be diluted (1:3) with low endotoxin PBS (PBSle) (Biochrom) and the leukocyte fraction enriched using standard ficoll gradient centrifugation. The interface will be harvested and washed twice with PBSle. For a 9 week reconstitution protocol, mice will be irradiated with a sub-lethal dose of 100 cGy one day before intravenous injection of 1×10.sup.6 human PBMCs; a 4-week protocol will use a single intravenous injection of 10×10.sup.6 PBMC, without irradiation.
Example 5. Evaluation of Immune Response and Selective Expansion of Immune Cell Subtypes and Cytokines
[0728] A therapeutic amount of the pharmaceutical composition comprising the cell product comprising human SKCTs will be administered to the reconstituted NSG mice and the response to this administration will be evaluated.
[0729] Briefly, PBMCs, splenocytes, or bone marrow cells of human or murine origins will be isolated and stained for 1 hr at 4° C. in the dark with the appropriate antibody cocktail. Following washing (1% (v/v) FBS in PBS), cells will be fixed with fixation buffer (1% (v/v) FBS, 4% (w/v) PFA in PBS) for 30 min at 4° C. in the dark. Flowcytometric analysis will be performed, and flow cytometry data will be analyzed using FlowJo software (TreeStar, Ashland, Oreg.). Chimerism of all humanized mice model will be assessed prior to each experiment by quantifying the following human populations: Human CD45+, human CD45+ murine CD45−; T-cells, CD45+CD3+; CD4+ T cells, CD45+CD3+CD4+; CD8+ T cells, CD45+CD3+CD8+; CD45+CD16+ leukocytes; B-cells, CD45+CD19; conventional dendritic cells, CD45+CD11c+; NK/NKT cells, CD45+CD56+; Monocytes, CD45+CD14+. Mouse immune cell subsets will be gated as followed: Murine CD45+, Human CD45− Murine CD45+; Conventional dendritic cells, CD45+CD3−CD19−NK1.1−TER119−Ly-6G/Gr1−CD11c+; Plasmacytoid dendritic cells, CD45+CD3−CD19−NK1.1−TER 119−Ly-6G/Gr1−CD317+; Monocytes, CD45+CD3−CD19−NK1.1−TER 119−Ly-6G/Gr1−CD11b+CD11c−F4/80−; Macrophages, CD45+CD3−CD19−NK1.1−TER119−Ly-6G/Gr1−CD11b+F4/80+. Human immune cell subsets will be gated as follows: Human CD45+, human CD45+ murine CD45−; T-cells, CD45+CD3+; CD4+ T cells, CD45+CD3+CD4+; CD8+ T cells, CD45+CD3+CD8+; Myeloid cells, CD45+CD3−CD19−(CD56+) CD33+; Granulocytes, CD45+CD66b+; B cells, CD45+CD3−CD19+; Natural Killer cells, CD45+CD3−(CD19−) CD56+; Natural Killer T cells and γδ T cells, CD45+CD3+(CD19−) CD56+; Conventional dendritic cells, CD45+CD3−CD19−(CD56−) (CD33+) CD11c+(BDCA1/3+); CD45+CD3−CD19 CD123+, group composed of monocytes, plasmacytoid dendritic cells, basophils and myeloid precursors; Plasmacytoid dendritic cells, CD45+CD3−CD19−(CD56−) BDCA-2+CD123+; Monocytes, CD45+CD3−CD19−(CD56−) CD14+; Macrophages, CD45+CD3−CD19−(CD56−) CD68+.
[0730] Flow cytometry fluorophor compensation for antibodies will be performed using AbC™ Anti-Mouse Bead Kit (Life Technologies, Invitrogen, Foster City, Calif., USA). Counting beads will be added to each sample prior to flow-cytometry analysis (AccuCheck Counting Beads, Life Technologies, Invitrogen, Foster City, Calif., USA).
[0731] The frequency of each cell fraction will be shown as a percentage of CD45+ cells, with the exception of CD4+ and CD8+ T cells, which will be shown as a percentage of CD3+ T cells. The frequencies of myeloid subsets (e.g., CD14+ monocytes and CD11c+ dendritic cells) and CD56+NK cells will also be determined.
IFN-γ ELISpot Assay
[0732] An exemplary ELISPOT assay protocol is as follows. Enzyme-linked immunosorbent spot (ELISpot) assays are conducted using mouse IFN-γ ELISpot kit (BD Bioscience, Cat #551083). Control animals or animals receiving the SCKTCs of the present disclosure are sacrificed and bronchoalveolar lavage cells and splenocytes were isolated. 2×10.sup.5 splenocytes are plated in triplicate in 96-well plates pre-coated with 5 pg/ml of purified anti-mouse IFN-γ and subsequently stimulated with a peptide specific for a viral immunogen at a final 5 pg/ml concentration. After 24 hours of stimulation, the cells are washed with deionized water and exposed to 100 μl biotinylated anti-mouse IFN-γ (2 μg/ml) for 2 hours at room temperature, followed by extensive washing prior to the addition of 100 μl Streptavidin-HRP. After 1 hour incubation at room temperature, the cells are washed and 100 μl of substrate solution is added to develop spots. The reaction is stopped with water and the number of spot-forming cells (SFCs) is determined using an automated ELISPOT software.
Cytokine Bead Assay.
[0733] Bead populations with distinct fluorescence intensities are coated with capture antibodies specific for IFN-γ and IL4 and mixed together to form a bead array that is resolved in a flow cytometer. During the assay procedure, the inflammatory cytokine capture beads are mixed with recombinant standards or SCKTCs and incubated with PE-detection antibodies. The intensity of PE fluorescence of each complex reveals the concentration of that cytokine.
Example 6. Infection of NSG Mice Reconstituted with Human Immune System Components with Highly Pathogenic H7N9 Influenza Virus and Low Pathogenicity/Mild H9N2 Influenza Virus
[0734] Reconstituted NSG mice are anesthetized with ketamine (40 μl/mouse) before infection, and then infected with an influenza virus H7N9 virus strain (3.5×10.sup.5 of 50% tissue culture infective dose TCID50/50 ul of volume) and an H9N2 virus strain (1.7×10.sup.7 of 50% egg infective dose EID50/50 μl of volume) at a high dose by nasal drip. The mice are placed in an IVC cage. For 14 consecutive days, the mice are weighed, and the survival and survival status of the mice is observed. Lung tissues are taken at 6 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after infection, and quick-frozen in liquid nitrogen for use. Weight and mortality will be determined.
[0735] Reconstituted NSG mice in groups of 6 will be dosed once by nasal dripping 2 hours before infection with as well as 3 days and 8 days after infection, respectively with the cell product of the present disclosure comprising superactivated cytokine killer T cells or a control carrier) supernatant. Mice will be weighed continuously, and the survival status of the mice observed.
[0736] For histology, the tissues of euthanized mice will be fixed in formaldehyde. Histopathological scoring for lung tissue will be performed according to the guidelines of the American Ihoracic Society. Statistical significance will be determined by standard methods.
[0737] Viral titers will be determined by homogenizing the left lobe of the lung in 1 mL serum-free DMEM with a disposable tissue grinder and plaque assays performed. In brief, Vero cells grown in DMEM with fetal bovine serum will be seeded into multiwell plates at a concentration of 2×10.sup.5 cells/well the night before the assay. Serial dilutions will be added to the wells. The plate will be incubated at 37° C., 5% CO.sub.2 for 2 hr, shaking the plates every 15 minutes. After 2 hr the plate media will be replaced with a liquid overlay of DMEM, 2.5% FBS containing 1.2% Avicel PH-101 (Signa-Aldrich, St. Louis, Mo.) and incubated at 37° C., 5% CO.sub.2. After 3 days, the overlay will be removed, wells will be fixed with 10% formaldehyde and stained with 0.1% crystal violet to visualize plaques. Plaques will be counted, and PFUs calculated according to the following equation: average # plaques/dilution factor x volume diluted virus added to the well.
[0738] While the present disclosure has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.