Innate Immunity Killer Cells Targeting PSMA Positive Tumor Cells
20230096410 · 2023-03-30
Assignee
Inventors
- Yanwen Fu (San Diego, CA)
- Reyna Lim (San Diego, CA, US)
- Daniel Lee (San Diego, CA, US)
- Matthew Buschman (San Diego, CA, US)
- Tong Zhu (San Diego, CA, US)
- Alisher B. Khasanov (San Diego, CA, US)
Cpc classification
A61K35/15
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
Abstract
The present disclosure provides an innate immunity cell such as a gamma delta T (gdT) cell, Natural Killer (NK) cell, or macrophage having 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA) chemically conjugated to the cell surface. The DUPA-conjugated cells provided herein demonstrate increased cytotoxicity toward cancer cells expressing PSMA. DUPA-conjugated cells can be primary cells or cells of a cell line. Also provided are methods of conjugating DUPA to the surface of NK cells, gamma delta T (gdT) cells, or macrophages and methods of treating cancer using DUPA-conjugated NK cells, gamma delta T (gdT) cells, or macrophages.
Claims
1. A gamma delta T (gdT) cell, Natural Killer (NK) cell, or macrophage comprising 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) conjugated to the cell surface.
2. A cell according to claim 1, wherein DUPA is conjugated to the surface of the cell via a linker.
3. A cell according to claim 2, wherein the linker comprises a functional group that reacts with lysine.
4. A cell according to claim 3, wherein the functional group is N-hydroxysuccinimide (NHS), pentafluorophenyl, or p-nitrophenyl.
5. A cell according to claim 2, wherein the linker comprises one or more of (CH.sub.2).sub.n-, —(CH.sub.2CH.sub.2O).sub.n-, —CH.sub.2(C═O)—, —(C═O)—, —(C═O)CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2—, —(C═O)CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2NH—, —(C═O)CH.sub.2CH.sub.2(C═O)—, —(C═O)CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2NH(C═O)—, an amino acid, a dipeptide, a tripeptide, polyglycine, p-aminobenzyl (PAB), piperazine, piperidine, or a triazole, where n can be, independently, 1-30.
6. The innate immune system cell of claim 5, wherein the linker comprises (CH.sub.2).sub.n-, —(CH.sub.2CH.sub.2O).sub.n-, or —CH.sub.2(C═O)—.
7. A cell according to any of claims 1-6, wherein the cell is a gamma delta T (gdT) cell.
8. The gdT cell of claim 7, wherein the gdT cell is from a cell line.
9. The gdT cell of claim 8, wherein the gdT cell is irradiated.
10. The gdT cell of claim 7, wherein the gdT cell is a primary cell.
11. The gdT cell of claim 10, wherein the gdT cell is isolated from PBMCs or cord blood.
12. A population of DUPA-conjugated gamma delta T cells comprising a plurality of cells of claim 7.
13. A pharmaceutical composition comprising a population of gamma delta T cells according to claim 12.
14. The pharmaceutical composition of claim 13, wherein the cells are provided in a bag, vial, or tube, wherein the cells are optionally frozen.
15. A method of treating a subject having a PSMA-positive cancer, comprising administering one or more doses of an effective amount of the population of gamma delta T cells of claim 12 to the subject.
16. A method according to claim 15, wherein the PSMA-positive cancer is prostate cancer.
17. A method according to claim 15, wherein the population of cells is administered by injection or infusion.
18. A method according to claim 15, wherein more than one dose is administered.
19. A population of DUPA-conjugated gamma delta T cells according to claim 12 for use in a method of treating a subject having a PSMA-positive cancer, wherein the subject is administered one or more doses of an effective amount of the gamma delta T cells.
20. A population of DUPA-conjugated gamma delta T cells according to claim 19, wherein the PSMA-positive cancer is prostate cancer.
21. A population of DUPA-conjugated gamma delta T cells according to claim 19, wherein the population of cells is administered by injection or infusion.
22. A population of DUPA-conjugated gamma delta T cells according to claim 19, wherein the population of cells is administered more than once.
23. A cell according to any of claims 1-6, wherein the cell is a Natural Killer (NK) cell.
24. The NK cell of claim 23, wherein the NK cell is a primary cell.
25. The NK cell of claim 24, wherein the NK cell is isolated from PBMCs or cord blood.
26. The NK cell of claim 23, wherein the NK cell is from a cell line.
27. The NK cell of claim 23, wherein the NK cell is irradiated.
28. The NK cell of claim 26, wherein the NK cell is a KHYG cell.
29. A population of DUPA-conjugated NK cells comprising a plurality of cells of claim 23.
30. A pharmaceutical composition comprising a population of NK cells according to claim 29.
31. The pharmaceutical composition of claim 30, wherein the cells are provided in a bag, vial, or tube, wherein the cells are optionally frozen.
32. A method of treating a subject having a PSMA-positive cancer, comprising administering one or more doses of an effective amount of the population of NK cells of claim 29 or a pharmaceutical composition thereof to the subject.
33. A method according to claim 32, wherein the PSMA-positive cancer is prostate cancer.
34. A method according to claim 32, wherein the population of cells is administered by injection or infusion.
35. A method according to claim 32, wherein more than one dose is administered.
36. A population of DUPA-conjugated NK cells according to claim 29 for use in a method of treating a subject having a PSMA-positive cancer, wherein the subject is administered one or more doses of an effective amount of the NK cells.
37. A population of DUPA-conjugated NK cells according to claim 36, wherein the PSMA-positive cancer is prostate cancer.
38. A population of DUPA-conjugated gamma delta T cells according to claim 35, wherein the population of cells is administered by injection or infusion.
39. A population of DUPA-conjugated gamma delta T cells according to claim 35, wherein the population of cells is administered more than once.
40. A cell according to any of claims 1-6, wherein the cell is a macrophage.
41. The macrophage of claim 40, wherein the macrophage is from a cell line.
42. The macrophage of claim 40, wherein the macrophage is a primary cell.
43. The macrophage of claim 42, wherein the macrophage is isolated from PBMCs.
44. A population of DUPA-conjugated macrophages comprising a plurality of cells of claim 40.
45. A pharmaceutical composition comprising a population of macrophages according to claim
44.
46. The pharmaceutical composition of claim 45, wherein the macrophages are provided in a bag, vial, or tube, wherein the cells are optionally frozen.
47. A method of treating a subject having a PSMA-positive cancer, comprising administering one or more doses of an effective amount of the population of macrophages of claim 44 or a pharmaceutical composition thereof to the subject.
48. A method according to claim 47, wherein the PSMA-positive cancer is prostate cancer.
49. A method according to claim 47, wherein the population of macrophages is administered by injection or infusion.
50. A method according to claim 47, wherein more than one dose is administered.
51. A population of DUPA-conjugated macrophages according to claim 50 or a pharmaceutical composition thereof, for use in a method of treating a subject having a PSMA-positive cancer, wherein the subject is administered one or more doses of an effective amount of the macrophages.
52. A population of DUPA-conjugated macrophages or pharmaceutical composition according to claim 51, wherein the PSMA-positive cancer is prostate cancer.
53. A population of DUPA-conjugated macrophages or pharmaceutical composition according to claim 51, wherein the population of macrophages is administered by injection or infusion.
54. A population of DUPA-conjugated macrophages or pharmaceutical composition according to claim 51, wherein the population of macrophages is administered more than once.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041] XCELLIGENCE® cytotoxicity assays: A) curve of cytolysis at 3:1 effector to target ratios; B) curve of cytolysis at 1:1 effector to target ratios; C) curve of cytolysis at 0.3:1 effector to target ratios; and D) curve of cytolysis at 0.1:1 effector to target ratios. Target cells were plated at time 0 and effectors were added approximately 25 hours later.
[0042]
[0043]
[0044]
DETAILED DESCRIPTION OF THE INVENTION
[0045] Throughout this application, including the Background section and Examples, various publications, patents, and/or patent applications are referenced. The disclosures of the publications, patents and/or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.
[0046] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure.
[0047] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.
[0048] The term “about” in relation to a reference numerical value can include a range of values plus or minus from that value. For example, the amount “about 10” includes amounts from 9 to 11, including the reference numbers of 9, 10, and 11. The term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
[0049] The term “primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of me in vitro.
[0050] The terms “subject,” “patient,” and “individual” are used herein interchangeably to include a human or animal. For example, the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
[0051] The term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Administration by injection can be, as nonlimiting examples, intravenous, intraperitoneal, intramuscular, intratumoral, or peritumoral. Other modes of delivery include, but are not limited to, the use of intravenous infusion or implantation of a matrix or polymer comprising the conjugated cells.
[0052] The term “treating” refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
[0053] The term “effective amount” or “sufficient amount” refers to the amount of an agent (e.g., DUPA-conjugated NK cells) that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, and the physical delivery system in which it is carried.
[0054] The term “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an agent (e.g., DUPA-conjugated NK cells) to a cell, an organism, or a subject. “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in a composition or formulation and that causes no significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable carrier include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, and the like. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present invention.
[0055] Headings are solely for the convenience of the reader, and do not limit the invention or its embodiments.
Cells Having DUPA Conjugated to the Cell Surface
[0056] Cells that exhibit HLA-independent cytotoxicity toward bacteria, virus-infected cells, and tumor cells that express abnormal molecules can be considered cells of the innate immune system and can be used in the compositions and methods provided herein. Exemplary cells considered for conjugation with DUPA include macrophages, Natural Killer (NK) cells, and gamma delta T (gdT) cells. Because these cells are activated by HLA-independent mechanisms, they are unlikely to generate graft-versus-host disease and cytokine release syndrome when delivered to a subject.
[0057] DUPA (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid) is a small molecule that specifically binds PSMA; binding assays using DUPA conjugated to a labeled moiety have provided a Kd for binding to PSMA of 14 nM (Kularatne et al. (2009) Mol Pharm. 6:780-789). DUPA can be conjugated to the surface of a cell, such as an NK cell, macrophage, or gdT cell, by means of a functional group attached to DUPA via a spacer. In various preferred embodiments, a DUPA compound is conjugated directly to the cell surface, where a “DUPA compound” refers to a compound that comprises DUPA and a linker, where the linker comprises a spacer and a functional group. In various examples, the functional group of the linker is a group that reacts with amines or sulfhydryls that may be present on the cell surface, such as exposed lysines or cysteines of cell membrane proteins.
[0058] Nonlimiting examples of functional groups that can react with sulfhydryls include, without limitation, maleimide, pyridyldithio, bromoacetyl, iodoacetyl, bromobenzyl, iodobenzyl, and 4-(cyanoethynyl)benzoyl. Functional groups used for conjugation to cell surface lysines include, as nonlimiting examples, N-hydroxysuccinimide (NHS), pentafluorophenyl, tetrafluorophenyl, tetrafluorobenzenesulfonate, nitrophenyl, isocyanate, isothiocyanate, and sulfonylchloride. In exemplary embodiments DUPA is conjugated to the surface of an NK cell, macrophage, or gdT cell, via lysine-reactive functional group such as NHS that is attached to DUPA via a spacer.
[0059] In some exemplary embodiments, NK cells, macrophages, or gdT cells as provided herein have DUPA conjugated to the cell surface via a linker that includes a functional group, e.g., such as but not limited to N-hydroxysuccinimide (NHS), that allows conjugation of DUPA to exposed lysine residues on the cell surface.
[0060] Linkers that include functional group for conjugation to the cell surface preferably also include a spacer between the functional group and the DUPA moiety. A spacer can be any composition or length, and in various exemplary embodiments can include, without limitation, any of the following, including any combinations of one or more of the following: an amino acid, a dipeptide, a tripeptide, polyglycine, p-aminobenzyl (PAB), a sugar, piperazine, piperidine, a triazoyl, (CH.sub.2).sub.n-, —(CH.sub.2CH.sub.2O).sub.n-, —(C═O)—, —CH.sub.2(C═O)—, —(C═O)—CH.sub.2—, —(C═O)CH.sub.2CH.sub.2O)—, —CH.sub.2CH.sub.2NH—, —(CH.sub.2CH.sub.2O).sub.n—CH.sub.2CH.sub.2NH—, —(C═O)CH.sub.2CH.sub.2(C═O), —(C═O)CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2NH(C═O)—, —(C═O)CH.sub.2CH.sub.2O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2NH—, and —(C═O)CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2—, where n can be, independently, 1-30. A linker used for attaching DUPA to a cell surface can include any combinations of the foregoing groups or moieties, optionally in combination with other chemical groups or moieties in the linker. In some embodiments the spacer will include at least one of (CH.sub.2).sub.n-, —(C═O)—, —(CH.sub.2CH.sub.2O).sub.n-, and —CH.sub.2(C═O)—. In some embodiments the spacer does not include a cleavage site for a protease or peptidase, for example, does not include a cathepsin B cleavage site.
[0061] In some embodiments, a linker can include a spacer that has a length of at least 25 Angstroms, at least 50 Angstroms, at least 75 Angstroms, or at least 100 Angstroms, or, for example, can have a linker with a chain length of at least 16 atoms, at least 32 atoms, at least 50 atoms, at least 65 atoms, at least 70 atoms, or at least 75 atoms. A spacer can be of any length, but in some embodiments a spacer of a linker of a DUPA compound that connects DUPA to the functional group used for conjugation to cells can in some embodiments have a length of from about 50 angstroms to about 400 angstroms or greater, for example from about 50 angstroms to about 300 angstroms, or from about 100 Angstroms to about 400 Angstroms, from about 100 Angstroms to about 350 Angstroms, from about 50 Angstroms to about 250 Angstroms, from about 80 Angstroms to about 250 Angstroms, from about 90 Angstroms to about 250 Angstroms, from about 100 angstroms to about 250 Angstroms, from about 70 Angstroms to about 200 Angstroms, from about 80 Angstroms to about 150 Angstroms, or from about 100 Angstroms to about 150 Angstroms. Examples of linkers are shown attached to DUPA (DUPA-Bis-Phe-L1 and DUPA-Bis-Phe-L2) in
[0062] While the disclosure provides efficient methods of one-step conjugation of DUPA to innate immune cells, the methods and compositions provided herein are not limited to the exemplified methods of attaching DUPA to a cell surface and resulting cell-conjugates. The inventors also contemplate that a cell, such as an NK cell, gdT cell, or macrophage can have DUPA conjugated to the cell surface by any feasible means. For example, an NK cell, macrophage, or gdT cell can have an alkyne-containing linker conjugated to the cell surface and DUPA may be attached to a linker that includes an azide (or vice versa), where the cell and antibody can be conjugated via a copper-free click reaction between the alkyne and azide.
[0063] Also provided is a population of NK cells, gdT cells, or macrophages in which cells of the population have conjugation groups or linking moieties covalently bound to the cell surface. The NK cells, gdT cells, or macrophages can be human NK cells and can be primary cells derived from a single donor or multiple donors. Alternatively the NK cells, gdT cells, or macrophages may be from a cell line. The cells can be provided in buffers or cell media and can be provided as frozen formulations, and may be, for example, pharmaceutical formulations.
[0064] Further provided are NK cell, gdT cell, or macrophage populations that have DUPA conjugated to the cell surface via a linker that comprises a functional group for cell-surface conjugation, such as, for example, an amine reactive group such as NHS. The NK cells, gdT cells, or macrophages can be human cells and can be derived from a single donor or multiple donors. In an alternative the cells can be derived from a cell line. The cells can be provided in buffers or cell media and can be provided as frozen formulations, and may be, for example, pharmaceutical formulations. Pharmaceutical formulations comprising cells can be for intravenous administration or for injection, or a pharmaceutical formulation can be a formulation in which the conjugated NK cells, gdT cells, or macrophages are provided with a matrix, gel, fiber, structure, or polymer.
[0065] Cells conjugated with DUPA can be assessed for cytotoxicity toward PSMA-expressing tumor cells using any of various cytotoxicity assays. Examples of cytotoxicity assays are dye exclusion assays, for example using the dyes trypan blue or propidium iodide; assays that detect the reduction of tetrazolium dyes such as MTT, MTS, XTT, or WST; and assays that measure leakage of lactate dehydrogenase (LDH) from non-intact cells or assay proteases (Adan et al. (2016) Curr Pharm Biotechnol. 17:1213-1221). Other viability assays detect ATP (Nowak et al. (2018) Clin Hemorheol Microcirc 69:327-336) or use labeled Annexin V to detect phosphatidylserine (PS) on the outer membrane of cells undergoing apoptosis (e.g., Goldberg et al. (1999) J. Immunol. Methods 224:1-9). Cytotoxicity toward adherent cells can also be measured as changes in electric impedance measurements when the culture vessel includes electrodes over which the cells grow. In these assays measurements are made periodically, for example, every fifteen minutes, every thirty minutes, or every hour, to assess the degree of cell death over time. See, for example, Cerignoli et al. (2018) PLoS ONE 13(3): e0193498).
[0066] The DUPA-conjugated cells in various embodiments provided herein are not genetically modified, i.e., are not modified by molecular genetic techniques including the introduction of nucleic acid constructs, nucleic acids that target expression of endogenous genes, or enzymes that modify the genome. In other embodiments the conjugated cells may have one or more introduced genetic modifications.
DUPA-Conjugated NK Cells
[0067] Natural Killer (NK) cells are lymphocytes of the innate immunity system that are able to kill cancer cells without prior sensitization. The cytolytic behavior of NK cells is regulated by multiple receptor-mediated signals that individually may inhibit or promote cytolytic behavior. The inventors have found that conjugation of the small molecule DUPA to the surface of NK cells using a simple conjugation method results in efficient targeting and killing of prostate cancer cells by the conjugated NK cells.
[0068] An NK cell having DUPA conjugated to the cell surface can be a primary cell or a cell of a cell line. Primary cells can be cells isolated from peripheral blood or PBMCs of one or more individuals, or primary NK cells can be derived from placental tissue or umbilical cord blood. Isolation can include positively or negatively selecting NK cells from a sample, for example using antibodies bound to a solid support. The primary NK cells may be enriched following isolation from the donor source. As used herein, “enriched” can mean culturing the cells under conditions that promotes the growth of a particular cell type or subtype while not promoting the growth of another cell type or subtype that may be present in the culture. Methods of isolating and/or enriching Natural Killer cells are known in the art and some are described for example in Spanholtz et al. (2010) PLoS ONE 6 (2):e9221 (1-13); Kaur et al. (2017) Front Immunol. 8:297; Fujisaki et al. (2009) Cancer Res. 69:4010-4017; Leivas et al. (2016) Oncolmmunology 5: e1250051; U.S. Pat. Nos. 9,834,753; 9,193,953; and 9,109,202; US 2017/0029777; US 2015/0225697; US 2014/0080148; US 2013/0059379; US 2013/0011376; and WO 2020/014029, all of which are incorporated herein by reference in their entireties. Typically, one or more cytokines is included in the culture medium to promote the selective growth or survival of NK cells in culture. Such cytokines can include, without limitation, one or more of any of the following: thrombopoeitin, Flt-3L, SCF, G-CSF, GM-CSF, IL-2, IL-3, IL-6, IL-7, IL-13, IL-15, IL-17, and H9. For example, isolated NK cells Can be placed in an expansion/activation reaction mixture with any one or any combination of, for example, 2-3 cytokines, including IL-2, 1L12, IL21, IL15 and/or IL18, under conditions that are suitable for expanding and activating the isolated NK cells. In one embodiment, the expansion/activation reaction mixture also include any one or any combination of the following agents: anti-NKp46 antibody, B7-H6-Fc, anti-NKp30 antibody and/or 4-1BBL-Fc.
[0069] For example, NK cells can be directly isolated or enriched from PBMCs using density gradient centrifugation. For example, NK cells can be directly isolated/enriched from PBMCs using positive magnetic enrichment for CD56+ NK cells (e.g., using MACS separation technology including Whole Blood CD56 MicroBead Kit or Buffy Coat CD56 MicroBead Kit, both from Miltenyi BioTec). For example, a magnetic depletion step can be employed to remove CD3+T cells from PBMCs. In one embodiment, the magnetic depletion step can be employed using the MACSxpress NK Cell Isolation Kit (Miltenyi BioTec). In some embodiments, the PBMCs can be obtained from one or more healthy human donors via leukapheresis. The depleted cells can be stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2, for example for approximately 14 days, to generate a population of NK cells that are CD3-CD16+ CD56+.
[0070] Placental-derived NK cells can be isolated from umbilical cord blood or placental perfusate, or NK cells can be differentiated from CD34.sup.+ hematopoietic stem cells recovered from umbilical cord blood or placental perfusate. For example, human placenta-derived NK cells can be prepared by: culturing, hematopoietic stem or progenitor cells in a, first medium comprising a stein cell mobilizing agent and thrombopoietin (Trio), followed by culturing the cells in a medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and then culturing the cells in a third medium comprising IL-2 and IL-15 to produce a third population of cells. Human placenta-derived NK cells are prepared as described in U.S. published application No. 2019/0093081, entitled “Placental-Derived intermediate Natural Killer (PINK) Cells for Treatment of Glioblastoma”, incorporated herein by reference.
[0071] NK cell lines can be, without limitation, KYHG cells, NK92 cells, YTS cells, NK3.3 cells, NK-YS, NK-YT, NKL, NKG, MOTN-1, HANK-1, SNK-6, IMC-1, NKL cells, or other NK cell lines. An NK cell line used in the methods or compositions of the present invention can be developed for the purpose of cell therapy as set forth herein. Preferably cells of a cell line that are used in cancer therapy (i.e., a cell line having DUPA conjugated to the cell surface) is irradiated prior to delivery to a patient, where irradiation is performed at a level that allows for viability of the cells but prevents the cells from dividing. In some embodiments, the DUPA conjugated NK cells is a KHYG or KHYG-1 NK cell. The KHYG-1 cell line mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin (Suck G et al., Exp Hematol 2005). KHYG-1 cells can be cultured (e.g., in RPMI 1640 medium containing 2 mM L-Glutamine, 20% FBS, 2 mM sodium pyruvate, supplemented with 450 U/ml rhlL-2) and irradiated, for example, at 10 Gy (Suck G et al. (2006) Int J Radiat Biol). Following irradiation, the cells are allowed to recover in culture for example, for twenty-four hours, and can then be frozen or used directly.
DUPA-Conjugated Gamma Delta T (gdT) Cells
[0072] Gamma delta T cells used for conjugation of DUPA to the cell surface can be isolated from blood samples, PBMCs, cord blood, or placental tissue. Methods for selecting and expanding gdT cells are known in the art and can be found in Wilhelm et al. (2014) J. Transl. Med. 12:45 as well as US 2017/0196910, WO2017/072367 and WO2018/212808, WO 2020172555, WO 2021032961, and US 20210030794, all of which are incorporated herein by reference. Commercial kits for gdT cell isolation and enrichment are also available (Stemcell Technologies, Vancouver, Calif.; Miltenyi Biotec, San Diego, Calif.). Alternatively, cells of a gamma delta T cell line may be used.
[0073] For example, gd T cells can be isolated from PBMCs using a commercially available kit such as the EasySep Human Gamma/Delta T Cell Isolation Kit (StemCell Technologies). Alternatively, gd T cells can be isolated by plating PBMCs in a culture medium containing Concanavalin A (Con A), IL-2, and IL-4 for about 1 week and culturing in a cultured medium that does not contain Con A for an additional 7 days. Another isolation method comprises plating PBMCs in a culture medium containing zolendronic acid (or another aminobisphoshonate) and IL-2 for approximately 2 days, The cells can be further cultured in a medium that does not contain zolendronic acid for an additional 12 days. Magnetic (or non-magnetic) cell sorting methods can be employed. In some cases, percent purity of the isolated gd T cell population can be determined using flow cytometry.
[0074] For example, isolation can be carried out during culturing by the addition of one or more components such as aminobisphosphonate (e.g., pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, or a salt or hydrate thereof) that allows the gamma delta T cells to be selectively expanded in a culture. Purification during cell culture may also be achieved by the addition of synthetic antigens such as phosphostim/bromohalohydrin pyrophosphate (BrHPP), synthetic isopentenyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) or co-culture with antigen presenting cells (APCs). The addition of such components can provide a culturing environment which allows for positive selection of gamma delta T cells typically at 70% or greater by number of total cells in the purified sample after a culture period of from 5 to 15 days, for example.
[0075] Additional factors that may be used to proliferate gamma delta T cells such as IL-2, IL-15 or IL-18 may be provided in the step of culturing the blood cells. For example, IL-2, IL-15 or IL-18 or combinations thereof may be provided in the range of 50-2000 U/ml, for example, 400-1000 U/ml, to the culturing medium.
[0076] Following isolation, gd T cells may be stimulated according to any appropriate protocol. In some cases, isolated gd T cells are stimulated using Con A. Alternatively or in addition, isolated gd T cells can be stimulated with CD3, or CD3/CD28 antagonists which promote rapid replication and expansion of the cells. Further alternatively, gd T cells can be activated through direct stimulation with ligand or antibody that binds to the gd T cell receptor (TCR).
DUPA-Conjugated Macrophages
[0077] Macrophages are mononuclear phagocytes that are widely distributed throughout the body, where they participate in innate and adaptive immune responses. Human macrophages can be isolated by flow cytometry in view of their specific expression of proteins such as CD14, CD40, Cd11b and CD64.
[0078] Macrophages used for conjugation of DUPA to the cell surface can be derived from monocytes isolated from blood samples or PBMCs. For example, culturing of monocytes for differentiation into macrophages can be done using the cytokine M-CSF or GM-CSF in the culture medium, optionally in combination with IFNγ or IL-4. Antibodies that may be useful in enriching macrophages in a cell culture include anti-CD14, anti-CD40, anti-CD11b, and anti-CD64 antibodies. Commercial kits are available for isolating macrophages from primary monocytes (e.g., Stemcell Technologies, Vancouver, Calif.). See also Elkord et al Immunology. February 2005; 114(2):204-212); Repnik et al Journal of Immunological Methods Vol. 278, Issues 1-2, July 2003, pages 283-292); and Zhang et al (Curr. Protoc. Immunol. 83:14,1.1-14.1.14, 2008), Alternatively, macrophages may be isolated from tissue samples or may be cells of a macrophage cell line, such as U937 (Vogel et al. (2005) Environ Health Persp. 113:1536-1541), THP-1, or m2.
Conjugation Methods
[0079] Further included herein are methods for producing DUPA-conjugated cells, such as NK cells, gdT cells, or macrophages. The methods include covalently attaching a DUPA compound that includes a DUPA moiety and a linker that comprises a functional group to NK cells, gdT cells, or macrophages. The functional group can be a functional group that reacts with thiols or amines. For example, functional groups that can be used for reaction with cell-surface sulfhydryls include, without limitation, maleimide, pyridyldithio, bromoacetyl, iodoacetyl, bromobenzyl, iodobenzyl, and 4-(cyanoethynyl)benzoyl. Functional groups used for conjugation to cell surface lysines include, as nonlimiting examples, N-hydroxysuccinimide (NHS), pentafluorophenyl, tetrafluorophenyl, tetrafluorobenzenesulfonate, nitrophenyl, isocyanate, isothiocyanate, and sulfonylchloride.
[0080] In exemplary embodiments, the DUPA compound that is conjugated to the cell surface includes an NHS functional group for conjugation to the cell surface. The linker includes a spacer that links the DUPA moiety to the NHS functional group.
[0081] The methods can include contacting the DUPA compound that includes a functional group with a population of NK cells, gdT cells, or macrophages under conditions that allow chemical conjugation of the functional group to the surfaces of the NK cells. The cells can be in an isotonic medium that may be buffered such as PBS. Reaction conditions such as concentration of reagents and reaction time and temperature can be determined empirically, but as general guidance only, the temperature can be any that allows for viability of the cells and is permissive for the conjugation reaction, for example, the conjugation reaction can be performed at temperatures ranging from about 4° C. to about 37° C., or from about 15° C. to about 37° C. In illustrative embodiments, the reaction can be performed from about 18° C. to about 32° C. Optimal concentrations of cells and DUPA compound can be determined empirically. As nonlimiting examples, the cells can be provided in the reaction at concentrations of from about 10.sup.5 per mL to about 10.sup.8 per mL, for example from about 10.sup.6 per mL to about 5×10.sup.7 per mL, and the DUPA compound can be provided at a concentration of from about 5 μM to about 1 mM, or from about 10 μM to about 800 μM, or from about 30 μM to about 600 μM. In illustrative embodiments, the DUP compound can be present in the conjugation reaction at a concentration of from about 40 μM to about 400 μM, or from about 50 μM to about 250 μM. The reaction can be incubated for minutes to hours, for example, from about 10 min to about 16 hours, and can in some exemplary embodiments be performed from about 15 min to about 2 h. The cells can be washed after the conjugation reaction from one to multiple times using a buffer such as PBS or culture medium.
Pharmaceutical Compositions
[0082] Pharmaceutical compositions of the present invention may comprise a DUPA-conjugated cell, or a population of DUPA-conjugated cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline (PBS) and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are in some embodiments formulated for intravenous administration.
[0083] For example, the pharmaceutical composition may be formulated for parenteral, systemic, intracavitary, intravenous, intra-arterial or intratumoral routes of administration which may include injection or delivery by catheter. Suitable formulations may comprise the cells in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid form suitable for injection, e.g. as a liquid, solution, suspension, or emulsion, or may be formulated as a depot or reservoir, e.g. suitable for implantation in the subject's body, from which the rate of release of the cells may be controlled. Depot formulations may include gels, pastes, boluses or capsules. The preparation may be provided in a suitable container or packaging. Fluid formulations may he formulated for administration by injection or via catheter to a selected region of the human or animal body.
[0084] The term “pharmaceutically acceptable” as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts.
Methods of Treating Cancer
[0085] Further provided is a method of treating cancer, comprising delivering a population of NK cells, gdT cells, or macrophages having DUPA covalently attached to the cell surface via a linker to a subject with cancer. In some embodiments, the method includes delivering a population of NK cells, gdT cells, or macrophages having DUPA covalently attached to the cell surface to a subject with cancer.
[0086] A cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinurn, mesentery, myometrium, nasopharynx, omentume, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.
[0087] Tumors to be treated may be nervous or non-nervous system tumors that express PSMA. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancer and sarcoma.
[0088] The cancer may be a solid tumor that expresses PSMA, such as but not limited to PSMA-positive prostate cancers which may be metastatic to other organs or tissues. Other types of cancer that may express PSMA such as but not limited to colorectal cancer, gliomas, lung cancer, breast cancer, pancreatic cancer (Galina Barbosa et al. (2020) Cancer Imaging 20:23), may also be treated with the cells and methods provided herein.
[0089] Delivery can be, for example, by intravenous administration or injection. The DUPA-conjugated cells can be infused into the bloodstream or can be delivered into a body cavity. The conjugated cells can be delivered peritumorally or intratumorally, for example by injection.
[0090] The methods can be used to deliver an effective amount of DUPA conjugated cells to a patient having cancer, for example, having prostate cancer, including metastatic prostate cancer. An effective amount is an amount that provides a therapeutic benefit. The method can comprise giving a single does or multiple doses of DUPA-conjugated cells, where a dose can include, for example, from 10.sup.4 to 10.sup.12 cells per kg of body weight.
[0091] Cell-based immunotherapy can include the transfer of primary NK cells, gdT cells, or macrophages isolated from one or multiple donors. Autologous cell-based immunotherapy can include transfer of primary cells isolated from the patient. Isolation of NK cells from PBMCs is disclosed, for example in Example 2, and in numerous references in the art. Cell-based immunotherapy can alternatively include the transfer of NK cells, gdT cells, or macrophages from cell lines, such as for example KHYG cells or NK92 cells where the NK cells of the cell lines have cell surface-conjugated DUPA. Cell lines used for adoptive cell immunotherapy can be irradiated prior to transfer to the patient so that the transferred cells do not proliferate in the patient.
[0092] Pharmaceutical compositions of DUPA-conjugated cells as provided herein may be administered in a manner appropriate to the disease to be treated. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials. The subject may be a human patient. For example “an effective amount”, “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “a therapeutic amount” can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising the cells, e.g., gdT cells, NK cells, or macrophages described herein may be administered at a dosage of 10.sup.4 to 10.sup.9 cells/kg body weight, in some instances 10.sup.5 to 10.sup.6 cells/kg body weight, including all integer values within those ranges. In some embodiments, the cells, e.g., T cells described herein may be administered at 3×10.sup.4, 1×10.sup.6, 3×10.sup.6, or 1×10.sup.7 cells/kg, body weight. The cell compositions may also be administered multiple times at these dosages. The cells can be administered for example by using infusion techniques that are commonly known in immunotherapy.
[0093] The compositions described herein may be administered to a patient by intravenous infusion, trans-arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. The administration of the subject compositions may also be carried out by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In some embodiments, the cell compositions, e.g., macrophage, gdT cell, or NK cell compositions, of the present invention may be administered to a patient by intradermal or subcutaneous injection. In some embodiments, the cell compositions e.g., DUPA conjugated macrophage, gdT cell., or NK cell compositions of the present invention may be administered by i.v. injection. The compositions of cells e.g., macrophage, gdT cell, or NK cell compositions, of the present invention are administered to a patient by intradermal or subcutaneous compositions, and may be injected directly into a tumor, lymph node, or site of infection.
[0094] In some embodiments, the subject (e.g., human subject) receives an initial administration of DUPA-conjugated cells, e.g., macrophages, gdT cells, or NK cells as provided herein, and one or more subsequent administrations of the DUPA-conjugated cells, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the DUPA-conjugated cells, e.g., macrophages, gdT cells, or NK cells as provided herein are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the DUPA-conjugated cells, e.g., are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the DUPA-conjugated (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no cells, and then one or more additional administration of the DUPA-conjugated cells, (e.g., more than one administration of the DUPA-conjugated cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of DUPA-conjugated cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the DUPA-conjugated cells are administered every other day for 3 administrations per week. In one embodiment, the DUPA-conjugated cells are administered for at least two, three, four, five, six, seven, eight or more weeks. The foregoing schedules are exemplary and not limiting to the methods provided herein.
EXAMPLES
Example 1
Synthesis of DUPA-BisPhe-L1
[0095] For conjugation of DUPA to the surface of NK cells, the DUPA-BisPhe-L1 was synthesized by the following method depicted below.
##STR00001##
[0096] Starting material DUPA-BisPhe (1) was synthesized as described in Bioorganic & Medicinal Chemistry Letters (2017), 27(24), 5490-5495.
[0097] To a solution of compound 1 (20 mg, 15.7 μmol) in 2 mL of dimethyl formamide (DMF) was added activated ester 2 (26 mg, 79.8 μmop and 6 μL of N,N-Diisopropylethylamine (DIEA). The reaction mixture was stirred for 30 min until all of compound 1 was consumed. Then the mixture was purified by reverse phase (RP) HPLC (0.1% trifluoroacetic acid (TFA) in water/acetonitrile). The fractions containing the product were lyophilized to give DUPA-BisPhe-L1 (3) (13.3 mg) as a glassy solid.
Example 2
Conjugation of DUPA to NK Cells
[0098] KHYG cells (Yagita et al. (2000) Leukemia 14:922-930; Suck et al. (2005) Exp. Hematol. 33:1160-71) are CD3− cells of a Natural Killer leukemia cell line having a p53 point mutation. KHYG cells were cultured in RPMI 1640 medium including 10% FBS. Two conjugation conditions were tested, one in which the concentration of DUPA-BisPhe-L1 in the KHYG cell conjugation reaction was 200 μM, and one in which the concentration of DUPA-BisPhe-L1in the cell conjugation reaction was 353 μM.
[0099] To conjugate DUPA to the surface of KHYG cells, the DUPA-BisPhe-L1 having the NHS functional group (
[0100] A stock of 35 mM DUPA-BisPhe-L1 (
[0101] The plate was incubated for 30 min at room temperature with gentle shaking (60 rpm). After the reaction, the cells were washed twice with 200 μL PBS and once with 200 μL cell growth media (cells were spun down each time at 1500 rpm) to remove the excess unreacted DUPA-BisPhe-L1. After the washing step, the DUPA KHYG cell conjugates were resuspended in cell growth media awaiting in vitro analysis.
Example 3
Cytotoxicity of DUPA-conjugated KHYG Cells
[0102] Cytotoxicity assays were performed following the manufacturer's protocols for the XCELLIGENCE® Real Time Cell Analyzer (Acea Biosciences, San Diego, Calif.) that allows for real-time monitoring of NK cell-mediated cytolysis of target cells. In these assays, LNCaP cells (of a human PSMA-positive prostate carcinoma cell line) were used as target cells to test the cytotoxicity of DUPA-conjugated KHYG NK cells as effectors.
[0103] LNCaP cells for use as target cells in the assay were harvested in growth phase, counted, washed, and resuspended to a cell density of 3×10.sup.5 per mL. The target cells (100 μL) were added to the wells of the XCELLIGENCE® 96-well plate and incubated at 37° C. Cell growth was monitored as impedance values by the XCELLIGENCE® analyzer until the cells reached growth phase with a cell index value greater than 0.5, approximately 24 hours after plating.
[0104] Effector cells (KHYG cells with DUPA conjugated to the cell surface) were then added to the wells containing target cells. Target cells were either PSMA-positive LNCaP cells or PSMA-negative PC3 cells used as controls. Natural Killer KHYG cells that had the DUPA compound conjugated to the cell surface using the methods described in Example 2 were washed in culture medium (RPMI 1640 containing 10% FBS) and adjusted to a density of 6×10.sup.6 cells per mL. As additional controls, KHYG NK cells that had not been DUPA-conjugated were used as effector cells in additional wells. Assays were performed in duplicate.
[0105] Effector cells were added in 50 μL volumes at cell number ratios of 10:1 and 5:1 with respect to the target cells. Following addition of effector cells, the 96 well plate continued to be incubated at 37° C. and impedance measurements were taken every 15 min.
[0106]
[0107] The results are even more striking in
[0108]
Example 4
Cytotoxicity of DUPA-Conjugated KHYG Cells at Low Effector:Target Ratios
[0109] Further cytotoxicity assays were performed to confirm and extend the results of Example 3. These assays also used LNCaP cells (human PSMA-positive prostate carcinoma cell line) as target cells and DUPA-conjugated KHYG NK cells as effectors, where the KHYG NK cells were conjugated under reaction conditions that included either 200 μM or 50 μM DUPA. In an additional control, the conjugation moiety dibenzocyclooctyne (DBCO), which does not specifically bind PSMA, was conjugated to KHYG cells (with a concentration of 200 μM DBCO in the conjugation reaction).
[0110] Conjugation of DUPA to KHYG NK cells was performed as described in Example 2, except that the two concentrations of DUPA-BisPhe-L1 in the conjugation reaction were 200 μM and 50 μM.
[0111] For conjugation of DBCO to KHYG cells, cells were first extensively washed with PBS to remove components in the cell media that might interfere with the first reaction step. Aliquots of 5×10.sup.6 cells were added to the wells of a U-bottom 96-well plate, the plate was centrifuged to pellet the cells, and excess supernatant was removed. Then, 200 μL of 200 μM of DBCO-Sulfo-NHS (
[0112] The cytotoxicity assays were performed as described in Example 3, except that the number of target cells per well was 2.5×10.sup.4 (in 100 μl growth medium) and effector cells were added at ratios of 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1 by diluting the effectors to the appropriate cell concentration prior to adding them to the assay wells that included targets. Natural Killer KHYG cells that had the DUPA compound conjugated to the cell surface using the methods described in Example 2 were washed in culture medium (RPMI 1640 containing 10% FBS) and adjusted to a density of 2.5×10.sup.5 cells per mL prior to plating. For the assays, effector cells (unconjugated KHYG cells or KHYG cells with either DUPA or DBCO conjugated to the cell surface) were added in a volume of 50 μl to the wells containing LNCaP target cells, and as controls were also added to wells that included PC3 (PSMA negative) target cells. As additional controls, some target cell wells did not receive effector cells (“Targets Only” wells). Following addition of effector cells, the 96 well plate continued to be incubated at 37° C. and impedance measurements were taken every 15 min.
[0113]
[0114] At a 5:1 effector:target ratio (
[0115] At ratios of effector to target cells of 2.5:1 and below (
[0116] The same assays conducted with PSMA-negative PC3 cells as the target cells showed that these cells are not killed by unconjugated KHYG cells or KHYG cells conjugated with either DUPA or DBCO regardless of the effector to target cell ratio (
Example 5
Synthesis of DUPA-BisPhe-L2
[0117] To determine whether compounds that included linkers of different lengths were also effective when conjugated to NK cells, DUPA-BisPhe-L2, having an additional PEG sequence proximal to the NHS group, was prepared as shown.
##STR00002##
[0118] Briefly, to a solution of compound 1 (5.0 mg, 3.9 μmol) in 1.5 mL of DMF was added activated ester 4 (10 mg, 17 μmop and 3.5 μL of DIEA. The reaction mixture was stirred for 10 min until all compound 1 was consumed. Then the mixture was purified by RP HPLC (0.1% TFA in water/acetonitrile). The fractions containing the product were lyophilized to give DUPA-BisPhe-L2 (5) (5.7 mg) as a glassy solid.
Example 6
Cytotoxicity of DUPA-BisPhe-L2 Conjugated KHYG Cells
[0119] Cytotoxicity assays were performed on KYHG cells conjugated with either 200 μM DUPA-BisPhe-L1 linker, having a spacer length of 55 atoms or approximately 82 Angstroms (
[0120]
[0121]
[0122] At target:effector ratios of 5:1 and 2.5:1 (
[0123] At the 1.25:1 target: effector ratio, KYHG cells conjugated with DUPA-BisPhe-L2 clearly killed a higher percentage of target cells than KYHG cells conjugated with DUPA-BisPhe-L1 (
[0124] At the lowest effector:target cell ratio, 0.625:1, non-conjugated KHYG cells are not effective against the PSMA-positive LNCaP target cells (
Example 7
Cytotoxicity of KHYG Cells Conjugated with DUPA-BisPhe-L1 and DUPA-BisPhe-L2 Conjugated at Different Concentrations.
[0125] An additional set of assays was performed to compare the cytotoxicity of KHYG cells conjugated with different concentrations of DUPA-BisPhe-L1 and DUPA-BisPhe-L2 toward PSMA-positive target cells. KHYG cells were conjugated in reactions that included either 1) 50 μM DUPA-BisPhe-L1 linker (
[0126] Assays were performed on the XCELLIGENCE® Real Time Cell Analyzer (Acea Biosciences, San Diego, Calif.) as described in Example 3.
Example 8
Isolation and Expansion of Primary NK Cells
[0127] PBMC are isolated from human blood by density gradient centrifugation using Lymphoprep™ (StemCell Technologies). The isolated PBMCs are resuspended in OpTmizer™ T Cell Expansion Medium (Thermo Fisher) with 5% CTS immune cell serum replacement (Thermo Fisher)(SR) or, alternatively, 5% human AB serum (AB). The cells are cultured in coated T25 flasks (10×10.sup.6 cells in 10 mL/flask) in OpTmizer™ medium supplemented with cytokines. On day 4 the medium is removed and replaced with fresh 10 ml of medium plus cytokines containing either 5% SR or 5% AB. On day 7, cells are counted and evaluated for NK cell content by staining with anti-human CD3 conjugated to APC and anti-CD56 conjugated to PE, after which the cells are re-plated with fresh culture medium with cytokines in fresh coated T75 flasks. The medium is replenished again as before on day 10, and on day 14 the cells are harvested from the flasks and collected by centrifugation at 400×g for 5 minutes. The pelleted cells are resuspended, counted, and phenotyped.
[0128] The culturing enriches CD56-positive NK cells in the culture significantly by day 12, such that the NK cells may make up at least 90% or at least 95% of the culture, with a concomitant essentially complete loss of T cells (CD3-positive cells) from the culture. Such primary NK cells may be used for conjugation of DUPA to the cell surface for cell-based therapies.
Example 9
Isolation of gdT Cells
[0129] Gamma delta T cells (gdT cells) were isolated from peripheral blood mononuclear cells (PBMCs) using the Stemcell Technologies Human Gamma/Delta T Cell Isolation Kit (Stemcell Technologies, Seattle, Wash.). PBMCs were isolated from Leukopaks ordered through HemaCare and frozen at a concentration of 1×10.sup.8 cells per ml in vials. Freshly thawed PBMC suspensions were suspended in 30 mL Dulbecco's Phosphate Buffered Saline (DPBS) containing 25% fetal bovine serum (FBS). Ten vials were thawed into 30 mL DPBS medium containing 25% FBS in a single 50 mL centrifuge tube, resulting in approximately 8×10.sup.8-1×10.sup.9 cells per 50 mL tube. The PBMCs were passed through a 40 μm cell strainer and cell number was determined. Approximately 3×10.sup.5 cells were reserved for flow cytometry and the remainder were harvested by centrifugation. The supernatant was removed and the cell pellet was resuspended in 60 μL MACS separation buffer (Miltenyi Biotech, San Diego, Calif.) per 10.sup.7 cells in a 50 mL tube, to which 20 μL FcR blocking reagent per 10.sup.7 cells was added. The cells were incubated with blocking reagent for 5 minutes at room temperature, after which 12.5 μL of EasySep™ Human Gamma/Delta T Cell Isolation Cocktail (Stemcell Technologies, Seattle, Wash.) was added per 5×10.sup.7 cells. After mixing briefly, the cells were incubated a further 15 min at room temperature with mixing on a plate shaker. The cells were then washed to remove unbound primary antibody by adding 1-2 mL of buffer per 10.sup.7 cells and centrifuged at 1400 rpm for 5 min. The supernatant was aspirated and the cell pellet was resuspended in MACS separation buffer (80 μL per 10.sup.7 cells).
[0130] For pan cell depletion, magnetic particles (anti-biotin microbeads, Miltenyi Biotech) were vortexed before removing 12.5 μL of suspended beads per 5×10.sup.7 cells and adding to the suspended cell preparation. The cells and magnetic beads were incubated for 10 min without shaking at room temperature, and then additional MACS separation buffer was added to bring the volume up to 25 mL (if the original volume was less than 10 mL) or 50 mL (if the original volume was greater than 10 mL). The cells were pipeted up and down gently 2-3 times to mix and the tube (without lid) was placed into the magnet stand (MACS Column Separator, Miltenyi Biotec) for 10 min at RT. The enriched cell suspension was carefully pipeted into a new 50 mL tube. The cells were centrifuged at 1400 rpm for 5 min, after which the supernatant was removed. The cells were then resuspended at approximately 10.sup.7 cells/mL in MACS separation buffer.
[0131] 2.5 μL anti-TCR α/β-biotin human antibodies (Miltenyi Biotec) were added per 10.sup.7 cells to the pan cell depleted cells and the antibodies and cells were mixed with pipette tips and then incubated for 10 min at 4° C. in the dark. The cells were then washed by adding 13 mL MACS separation buffer, transferring the suspended cells to a 15 mL tube, centrifuging at 1400 rpm for 5 min, and aspirating the supernatant. The wash was repeated and the final pellet was resuspended in 97.5 μK MACS separation buffer per 10.sup.7 cells and 2.5 ul/10.sup.7cells anti-biotin Microbeads were then added to the cells. The suspension was pipeted a few times to mix, mixed with pipet tips, and incubated 15 min in the dark at 4° C.
[0132] The cells were then washed by adding 13 mL buffer and centrifuging the sample at 1400 rpm for 5 min. The supernatant was aspirated completely and the cells were resuspended in up to 500 μL MACS separation buffer/10.sup.8cells.
[0133] For depletion of α/β T cells, the LD column (Miltenyi Biotec) was rinsed with 2 mL of MACS separation buffer and the cell suspension was applied to the column. The flow through of unlabeled cells was collected and the column was washed 5 times with 1 mL of buffer each time. The washes were added to the flow through and the cell number was determined. An aliquot of 3×10.sup.5 cells was removed for flow cytometry to assess cell purity. The remaining cells were spun down and resuspended in T cell medium to a concentration of 2×10.sup.6 cells per mL and dispensed into wells of a 6 well culture plate.
[0134] For expansion of T cells, T cell TransAct solution (Miltenyi Biotec, 5 μL per 10.sup.6 cells) was added to the cells in T Cell Medium, which was T cell OpTmizer™ CTS™ Medium (Fisher Scientific) modified to include 1% glutamax, 5% human serum, 26 ml of OpTmizer™ T-Cell Expansion Supplement, 1:1000 gentamicin, and 300U IL-2. The plate was placed in a cell incubator for 2-3 days. The culture medium was then exchanged with fresh T cell medium without added T cell TransAct solution (Miltenyi Biotec).
[0135] On day 9, during the exponential phase of T cell growth, the gdT cells were transferred to a suitable tissue culture bag. The cells were maintained at a density of 0.5×10.sup.6 cells/ml. On day 13, the cells were counted after resuspension and transferred to a tissue culture bag to keep the cell density at 0.5×10.sup.6 cells/ml. On day 16, the cells were again counted and thereafter the cell density was maintained at 1×10.sup.6 cells/ml in culture medium containing 300 U/ml rIL-2 in a tissue culture bag. On day 20, the cells were counted and frozen.
[0136] Example 10. Conjugation of DUPA to gdT Cells.
[0137] gdT cells were cultured in RPMI 1640 medium including 10% FBS. All gdT cells were reacted with 200 μM of DUPA-BisPhe-L1 (short linker), DUPA-BisPhe-L2 (long linker), or with the non-PSMA targeting small molecule compound as control, EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) (
[0138] To conjugate DUPA to the surface of gdT cells, the DUPA-BisPhe-L1, DUPA-BisPhe-L2, or Sulfo-NHS-LC-Biotin having NHS functional group was covalently attached to the cells via exposed lysine residues on the cell surface as follows.
[0139] Stocks of 36-50 mM small molecule compounds (DUPA-BisPhe-L1, DUPA-BisPhe-L2, or Sulfo-NHS-LC-Biotin) in DMSO were further dissolved in PBS to final concentrations of 200 μM (0.4-0.55% final DMSO concentration in solution). The gdT cells were extensively washed with PBS to remove components in the cell media that might interfere with the conjugation reaction. Aliquots of 5×10.sup.6 gdT cells were added to the wells of a U-bottom 96-well plate, the plate was centrifuged to pellet the cells, and excess supernatant was removed. After the final wash, the pelleted cells were resuspended in either 200 μL per well of 200 μM DUPA-BisPhe-L1, 200 μM DUPA-BisPhe-L2, or 200 μM Sulfo-NHS-LC-Biotin. An additional control well was also included in which the same number of cells (5×10.sup.6) were resuspended in PBS containing 0.4-0.55% final DMSO concentration but no compound for conjugation.
[0140] The plate was incubated for 30 min at room temperature with gentle shaking (60 rpm). After the reaction, the cells were washed twice with 200 μL PBS and once with 200 μL cell growth media (cells were spun down each time at 1500 rpm) to remove the excess unreacted small molecules. After the final washing step, the DUPA-gdT cell conjugates and Biotin-gdT cell conjugates were resuspended in cell growth media awaiting in vitro analysis.
[0141] To confirm the conjugation of DUPA to the cell surface of gdT cells, gdT cells that had been reacted with DUPA-BisPhe-L1 and DUPA-BisPhe-L2 were analyzed by flow cytometry. Aliquots of 1×10.sup.6 unconjugated and DUPA-conjugated gdT cells were labeled with or without 40 μL of 25 μg/mL PSMA-Fc in 90 μL PBS by incubation at room temperature for 30 min in individual microcentrifuge tubes. Cells not treated with PSMA-Fc (resuspended in PBS only) served as controls. After incubation, the cells were washed with 400 μL PBS and spun down at 150×g for 3 min. After removal of PBS, cells were resuspended in the presence or absence of 90 μL 200 μg/mL APC-anti-human IgG Fc antibody for detection. Cells not labeled with APC-anti-human IgG Fc (resuspended in PBS without APC-anti-human Fc) were included as further controls. The cells were incubated for 30 min at room temperature, washed with 400 μL PBS, spun down at 150×g for 3 min, and resuspended in PBS.
[0142] For gdT-biotin reaction confirmation, aliquots of 1×10.sup.6 unconjugated and biotin-conjugated gdT cells were labeled with 1 μL of 500 μg/mL Streptavidin-FITC in 90 μL PBS by incubation at room temperature for 30 min in individual microcentrifuge tubes. Cells incubated in PBS in the absence of Streptavidin-FITC served as controls. Cells were incubated for 30 min at room temperature, washed with 400 μL PBS, and spun down at 150×g for 3 min. Finally, all cells, including controls receiving no treatment, were resuspended in PBS and analyzed by flow cytometry.
Example 11
Cytotoxicity of DUPA-Conjugated gdT Cells
[0143] Cytotoxicity assays were performed following the manufacturer's protocols for the XCELLIGENCE® Real Time Cell Analyzer (Acea Biosciences, San Diego, Calif.) that allows for real-time monitoring of gdT cell-mediated cytolysis of target cells. In these assays, LNCaP cells (of a human PSMA-positive prostate carcinoma cell line) were used as target cells to test the cytotoxicity of DUPA-conjugated gdT cells as effectors.
[0144] LNCaP cells for use as target cells in the assay were harvested in growth phase, counted, washed, and resuspended to a cell density of 5×10.sup.5 cells per mL. The target cells (50 μL) were added to the wells of the XCELLIGENCE® 96-well plate and incubated at 37° C. PC-3 cells (a human PSMA-negative prostate carcinoma cell line) were used as a negative control cell line in the assay, resuspended at a cell density of 2×10.sup.5 cells per mL. Cell growth was monitored as impedance values by the XCELLIGENCE® analyzer until the cells reached growth phase with a cell index value greater than 0.5, approximately 24 hours after plating.
[0145] Effector cells (gdT cells with DUPA conjugated to the cell surface, gdT-DUPA-L1 and gdT-DUPA-L2) were then added to the wells containing target cells. Target cells were either PSMA-positive LNCaP cells or PSMA-negative PC3 cells used as control. gdT cells that had the DUPA compound conjugated to the cell surface using the methods described in Example 10 were washed in culture medium (RPMI 1640 containing 10% FBS) and adjusted to a density of 5×10.sup.6 cells per mL. As controls, gdT cells that had not been conjugated to either DUPA or biotin (gdT) as well as gdT cells that had been conjugated with the non-PSMA targeting compound (gdT-biotin) were used as effector cells in additional wells. Assays were performed in duplicate.
[0146] Effector cells were added in 50 μL volumes at cell number ratios starting at 3:1 with respect to the target cells. A three-fold serial dilution was made in cell growth media for a total of four Effector:Target ratio treatments, namely 3:1, 1:1, 0.3:1, and 0.1:1. Following addition of effector cells, the 96-well plate continued to be incubated at 37° C. for at least 72 h and impedance measurements were taken every 15 min.
[0147]
[0148] The gap between the higher cell killing capabilities of gdT cells conjugated with DUPA and the lower killing capabilities of gdT cells not conjugated with DUPA (unconjugated gdT cells and gdT cells conjugated with non-PSMA targeting small molecule) was even more pronounced at ET ratios lower than 3:1. At lower Effector:Target ratios, both L1 and L2 DUPA-conjugated cells remained highly potent with maximum cytolysis at greater than 95% and 80% for 1:1 and 0.3:1 ET ratios, respectively, compared to the maximum cell killing achieved by both gdT cells and gdT-biotin cells, which behave almost identically, at 45-48% (1:1 ET ratio) and 19% (0.3:1 ET ratio) (
[0149]
[0150]
[0151] PSMA-expressing tumor was observed for the gdT cells conjugated with DUPA and the cells were highly effective at killing PSMA-positive tumor cells at ratios at or below 1:1.
[0152] Nonetheless, both the DUPA-conjugated gdT cells have demonstrated high selectivity towards the PSMA positive cancer cell line (