COMBINATION OF ADOPTIVE CELL THERAPY AND CHEMOTHERAPY FOR ACUTE MYELOID LEUKEMIA
20230381234 · 2023-11-30
Assignee
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
A61K31/53
HUMAN NECESSITIES
A61L27/3604
HUMAN NECESSITIES
International classification
A61K35/17
HUMAN NECESSITIES
A61K31/706
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
Disclosed here are methods of treatment of cancers by administering autologous tumor infiltrating lymphocytes optionally in combination with chemotherapeutic and immunotherapeutic agents. Methods include treating acute myeloid leukemia with a combination of autologous TILs-based adoptive cell therapy and agents, such as a PD-1 inhibitor and a hypomethylating agent. The TILs can be bioengineered to modify the expression of function of a gene or a molecule of interest.
Claims
1. A method of treating acute myeloid leukemia in a subject, the method comprising the step of: administering a tumor infiltrating lymphocytes-based adoptive cell therapy along with therapeutically effective amounts of an inhibitor of programmed death-1 (PD-1 inhibitor) and a hypomethylating agent, thereby treating acute myeloid leukemia in the subject.
2. The method of claim 1, wherein the hypomethylating agent is 5-azacytidine (AZA).
3. The method of claim 1, wherein the tumor infiltrating lymphocytes-based adoptive cell therapy comprises an administration of ex vivo expanded autologous PD-1-inhibited-tumor infiltrating lymphocytes.
4. The method of claim 1, wherein the tumor infiltrating lymphocytes-based adoptive cell therapy comprises an administration of ex vivo expanded bioengineered tumor infiltrating lymphocytes exposed to a PD-1 inhibitor.
5. The method of claim 4, wherein the ex vivo expanded tumor infiltrating lymphocytes are bioengineered to express 25-hydroxyvitamin D-1 alpha hydroxylase (CYP27B1).
6. The method of claim 1, wherein the PD-1 inhibitor is an anti-PD-1 antibody.
7. A method of treating acute myeloid leukemia in a subject, the method comprising the steps of: isolating tumor infiltrating lymphocytes (TILs) from the subject; culturing the isolated TILs in a culture system to produce cultured TILs; and administering the cultured TILs to the subject, thereby treating acute myeloid leukemia in the subject.
8. The method of claim 7, wherein the TILs are isolated from a bone marrow sample from the subject.
9. The method of claim 7, wherein the TILs are isolated from a peripheral blood sample from the subject.
10. The method of any one of claims 7-9, wherein the TILs comprise CD3.sup.+ T cells.
11. The method of any one of claims 7-10, wherein the TILs comprise CCR7.sup.+CD95.sup.− and/or CD62L.sup.+CD45RA.sup.+ T cells.
12. The method of claim 10, wherein the step of culturing the isolated TILs comprises increasing the number of CD3.sup.+ TILs by about a 1000-fold relative to a sample from the subject.
13. The method of any one of claims 7-12, wherein the step of culturing the isolated TILs comprises exposing the isolated TILs to a PD-1 inhibitor.
14. The method of claim 13, wherein the PD-1 inhibitor is an anti-PD-1 antibody.
15. The method of any one of claims 7-14, wherein the step of culturing the isolated TILs comprises increasing the ratio of CD8.sup.+ TILs.
16. The method of any one of claims 7-15, further comprising the step of: bioengineering the isolated TILs to increase expression or function of a CYP27B1 gene.
17. The method of any one of claims 7-16, further comprising the step of: bioengineering the isolated TILs to reduce expression or function of a PD-1 gene.
18. The method of any one of claims 7-17, further comprising the step of: bioengineering the isolated TILs to increase expression or function of one or more of CD3, CCR7, CD62L, CD45RA, CD95, CD127, CD27, and CD28.
19. The method of any one of claims 7-18, further comprising administering a therapeutically effective amount of a PD-1 inhibitor and a hypomethylating agent to the subject, thereby treating acute myeloid leukemia in the subject.
20. The method of claim 19, wherein the hypomethylating agent is 5-Azacytidine (AZA).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] 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.
[0008] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements or procedures in a method. Embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings.
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[0014] FIGS. 6A1-6A4 and FIGS. 6B1-6B2 are photographic images of femoral bone marrow in naïve environment and when transplanted PD-1-inhibited-TILs, respectively.
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DETAILED DESCRIPTION
[0026] AML is a hematopoietic cancer that has a heterogeneous cell population and is an aggressive malignancy with poor prognosis. AML most commonly occurs in older adults. With multiple subtypes and heterogeneous genetic abnormalities, AML is extremely difficult to treat with the current chemotherapeutic regimens, and has the lowest survival rate amongst all types of leukemia. Disease relapse is the most important cause of treatment failure. The standard therapy for AML involves intensive chemotherapy induction with combination of daunorubicin or idarubicin and cytarabine. However, many elderly patients cannot tolerate such regimens. The mortality of elderly AML (at age or older), following relapse, remains at 95% within 5 years, and this has not changed much over the last 30 years. Therefore, there is a need for novel targeted therapies that are more active and less toxic for this patient population. Single agent 5-Azacytidine (AZA) is not effective in inducing remission, possibly due to the development of increased expression of PD-1/PD-L1. Leukemia stem cells (LSCs) are a key factor in the problem of relapse. These LSCs are characterized by self-renewal, cell-cycle quiescent and chemotherapy-resistance. Unfortunately, AZA-based cytotoxic therapy fails to completely eliminate the LSCs.
[0027] The programmed cell death 1 (PD-1) receptor is expressed on activated T cells, B cells, macrophages, regulatory T cells (Tregs), and natural killer (NK) cells. PD-1 inhibitors and PD-L1 inhibitors are checkpoint inhibitors that interfere with the interaction between programmed cell death protein 1 (PD-1) and its B7 family of ligands, programmed death-ligand 1 (PD-L1 or B7-H1) or PD-L2 (B7-DC). Activation of PD-1/PD-L1 signaling serves as a principal mechanism by which tumors evade antigen-specific T-cell immunologic responses. Blocking either PD-1 or PD-L1 enhances T-cell-mediated anti-tumor activity. Cancer immunotherapy utilizes components of the immune system to eliminate cancer cells while sparing healthy cells. Among immunotherapies, tumor infiltrating lymphocyte (TIL) adoptive cell therapy is a therapy in which TILs, which are primarily T-cells, are isolated from patients' surgically removed tumors, primed ex vivo, and reintroduced into patients to eliminate tumor cells. As used herein, “tumor infiltrating lymphoycytes” (TILs) are anti-tumor lymphocytes, i.e., lymphocytic cell populations that invade a tumor tissue and have the ability to recognize and kill tumor cells. In some aspects, TILs include characteristics of naïve T cells. In some embodiments, TILs include CD3.sup.+ T cells. In some embodiments, TILs include CCR7.sup.+CD95.sup.− and/or CD62L.sup.+CD45RA.sup.+ T cells.
[0028] Addition of chemotherapeutic agents along with PD-1/PD-L1 inhibitors sensitizes tumors to respond to immunotherapies, and increases CD8.sup.+ T cell infiltration and cytotoxicity to tumors. PD-1 inhibitors and PD-L1 inhibitors activate immune responses from TILs, resulting in a reduction of tumor metastasis and growth.
[0029] The present disclosure describes various embodiments related to compositions and methods for management or treatment of cancers, such as AML, gastric cancer, or breast cancer. In the following description, numerous details are set forth in order to provide a thorough understanding of the various embodiments. Before the present methods and compositions are described, it is to be understood that these embodiments are not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present embodiments will be limited only by the appended claims. The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous.
[0030] A “subject” refers an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey) and a non-primate (such as a mouse). In some aspects of the invention, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.
[0031] A “patient” refers to a subject who shows symptoms and/or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and/or is at risk of developing a disease. A “patient” can be human and veterinary subjects. Any reference to subjects in the present disclosure, should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patient who has a liquid cancer, such as a leukemia.
[0032] As used herein, the terms “treating”, “treatment” and the like, shall include the management and care of a subject or patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, reduce at least one associated sign, symptom, or condition, or eliminate the disease, condition, or disorder. Treatment also refers to a prophylactic treatment, such as prevention of a disease (e.g., AML) or prevention of at least one sign, symptom, or condition associated with the disease (e.g., relapse of AML). Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
[0033] Administration of TIL Adoptive Cell Therapy with a PD-1 Inhibitor and Hypomethylating Agent
[0034] Most AML patients experience relapse 2-3 years after complete remission. Besides the report of dysfunctional immunity in AML patients, the existence of leukemia stem cells (LSCs) is also a key factor in the problem of relapse. These LSCs are quiescent, chemo-resistant, and they are not wiped out easily. In an embodiment, autologous transplantation of sufficient amount of ex vivo expanded PD-1-inhibited-TILs are administered to replenish impaired immune system in AML patients. The genetically manipulated TILs can specifically target LSCs. The combination of TIL adoptive cell therapy with a PD-1 inhibitor and AZA can reduce the likelihood of relapse of AML at the early stage. AML blasts express PD-L1 and are susceptible to PD-1 inhibitor treatment. In an embodiment, a combination immunotherapy of PD-1-inhibited-TILs, AZA and a PD-1 inhibitor results in potent anti-leukemic effects, and overcomes the immune-escape of LSCs and AML blasts ex vivo and in vivo.
[0035] Methods disclosed herein include administration of TIL adoptive cell therapy with a PD-1 inhibitor and AZA to the AML BM microenvironment which can then improve survival and reduce the leukemia burden endured by patients. In an embodiment, a combination of TILs, PD-1 inhibitors, and AZA are administered to treat AML and prevent or mitigate a relapse. CD3.sup.+ T-cells are isolated from AML patients' bone marrow (BM) samples. In some embodiments, these cells express PD-1. These primary CD3.sup.+ T-cells could be isolated and expanded ex vivo. In an embodiment, the PD-1 expression is inhibited in TILs to restrict the inhibitory signal of blast cells on the CD3.sup.+ T-cell population and lead to more effective killing of LSCs and AML blasts. In an embodiment, the ex vivo expanded autologous TILs in combination with anti-PD-1 antibodies and AZA is administered to produce a superior anti-leukemic therapeutic effect against LSCs and AML blasts and enhance the disease-free and overall survival. In an embodiment, the ex vivo expanded autologous TILs are first exposed to an inhibitor of PD-1 to generate PD-1-inhibited-TILs. These PD-1-inhibited-TILs are administered in combination with AZA to generate a cytotoxic effect and treat cancer. In an embodiment, autologous PD-1-inhibited-TILs are administered in combination with AZA and a PD-1 inhibitor to reduce the leukemic burden and increase survival of an AML patient. These combinations of TILs with chemotherapeutic agents prevent relapse and improve AML patient survival outcomes.
[0036] A PD-1 inhibitor can be a chemical agent or a biological agent. A PD-1 inhibitor will inhibit the expression or function of PD-1 by TILs by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%) as compared to expression or function of PD-1 in TILs that have not been treated by the PD-1 inhibitor. Examples of immunotherapeutic PD-1 inhibitors include one or more of atezolizumab, avelumab, cemiplimab, durvalumab, nivolumab, and pembrolizumab.
[0037] A “therapeutically effective amount” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. “Administering” refers to the physical introduction of a therapeutic agent to a subject in need thereof. Exemplary routes of administration for agents to inhibit proliferation of mesenchymal cancer stem cells. include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. A therapeutic agent may be administered via a non-parenteral route, or orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. Therapeutic agents can be constituted in a composition, e.g., a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
[0038] In an embodiment, in addition to the treatment of the TILs with a PD-1 inhibitor ex vivo, to ensure the inhibition of PD-1 expression in TILs during their expansion, a PD-1 inhibitor is administered to the patient preceding or along with administration of transplantation of the TILs. In an embodiment, administration of AZA can start ahead of the TIL transplantation. In an embodiment, combination treatment with autologous PD-1-inhibited-TILs and AZA shows superior therapeutic efficacy and prolonged survival as compared to the single treatment with PD-1-inhibited-TILs or AZA alone. In an embodiment, AZA is administered preceding the administration of PD-1-inhibited-TILs to condition the BM in preparation for the transplantation. Thus, AZA will simultaneously serve as a treatment and an agent to prime the BM. In an embodiment, appropriate dose of IL2 is administered to help T cell proliferation in vivo.
[0039] In addition, cell vehicles for TILs adoptive cell transfer could be further manipulated to create antigen specific to make chimeric antigen receptor (CAR)-TILs as the precision medicine for all cancer patients. Embodiments include methods for ex vivo expansion of tumor-infiltrating lymphocytes for use in adoptive cell therapy. In one such method, the steps include: (a) obtaining autologous tumor-infiltrating lymphocytes from the subject, (b) culturing the lymphocytes in a culture medium containing several interleukins to produce expanded lymphocytes, and (c) administering the expanded lymphocytes, a PD-1 inhibitor, and AZA to the patient.
[0040] In an embodiment, the combination of autologous CYP27B1-expressing TILs and PD-1 inhibitors are administered to treat AML by elongating the overall survival without significant side effects. TILs are an alternative cell vehicle to deliver transgenes or peptides to AML BM. CYP27B1, also known as 25-Hydroxyvitamin Dl-alpha-hydroxylase, VD 1A hydroxylase, cytochrome p450 27B1, 1-alpha-hydroxylase, is a cytochrome P450 enzyme encoded by the CYP27B1 gene. CYP27B1 catalyzes calcifediol to calcitriol, i.e., the bioactive form of Vitamin D. Local newly synthesized Vitamin D by ectopic CYP27B1 will not only contribute to BM regeneration but also suppress tumor lysis syndrome. Because TILs have been sensitized by surrounding AML antigens before isolation, the antigen (Ag)-specific property will direct TILs to locate AML cells in the BM or the peripheral tissues and kill them in situ. In addition, treatment of PD-1 inhibitors will prevent negative signaling from AML cells exhausting TILs in vivo. Therapeutic roles of TILs in vivo and how Vitamin D regenerates the healthy microenvironment for remission are illustrated in
[0041] CYP27B1-based vitamin D gene therapy provides several advantages. It enhances the differentiation of leukemic cancer cells because of vitamin D as a strong differentiator. It generates synergistic effect with 5-AZA, an FDA-approved epigenetic modulator to eliminate majorities of viable blasts. It serves as an immune-modulator to suppress Cytokine Release Syndrome, a major side effect of adoptive T cell therapies including CAR-T; and improves the regeneration of bone marrow microenvironment and healthy hematopoiesis through positively regulating stem cell properties of mesenchymal stem cells and hematopoietic stem cells. Further embodiments of TIL adoptive cell therapy are provided below.
[0042] Ex Vivo Isolation, Expansion, and Bioengineering of TILs from Bone Marrow or Peripheral Blood Samples of AML Subjects
[0043] In some aspects, methods of treating AML in a subject include the administration of an autologous TIL-based adoptive cell therapy. One such method includes the steps of isolating tumor infiltrating lymphocytes (TILs) from the subject, culturing the isolated TILs in a culture system to produce cultured TILs, and administering the cultured TILs to the subject. In some embodiments, TILs are CD3.sup.+ T cells that are isolated from bone marrow samples of the subject, peripheral blood samples of the subject, or both the bone marrow and peripheral blood samples of the subject. In some embodiments, CD3.sup.+ TILs are isolated, by e.g., using CD3 microbeads and a separator, e.g., MiniMACS™ Separator, with an MS Column. In some embodiments, selected CD3.sup.+ T cells comprise naïve T cells and are considered AML TILs.
[0044] In some embodiments, TILs, e.g., CD3.sup.+ T cells, isolated from peripheral blood samples and TILs, e.g., CD3.sup.+ T cells, isolated from bone marrow have similar characteristics with regards to cell surface molecule expression and/or cytotoxic function. In some embodiments, the use of peripheral blood samples as source of TILs provides several advantages. The peripheral blood-derived TILs can be acquired in large amount by leukapheresis and can be bioengineered (e.g., genetically engineered) to improve antigen-specificity.
[0045] In some embodiments, isolated TILs are cultured and expanded efficiently in the ex vivo culture system. In some embodiments, these isolated TILs are CD3.sup.+ T cells. The TILs are isolated and cultured at 37° C. and 5% CO.sub.2 in a RPMI 1640 culture medium containing 10% fetal bovine serum, antibiotics, IL2, and Dynabeads® Human T-Activator CD3/CD28 without feeder cells. In some embodiments, the TILs are expanded following the stages set forth below:
[0046] Stage 1 (Naïve TILs): The cell density of CD3.sup.+ TILs can be started around 300 μl of 20,000 cells/ml in appropriate wells of 48-well-plates, according to the cell count after pulling down CD3.sup.+ TILs. Fresh media is added at 1:1 ratio to each well every 2 days, and the cells/media are mixed. Based primarily on the growth of the TILs, medium change is performed every 5-7 days, and split cells at the ratio 1:3.
[0047] Stage 2 (Ready to grow): After 7 days, IL7 (25 ng/ml) and IL15 (25 ng/ml) are added to the media with IL2 (1000 U/ml). Although every patient's bone marrow mononuclear cells (BM-MNC) sample are different, TILs are raised in 48-well-plates for expansion to sufficient amount during the beginning 10-14 days, instead of large wells and flasks.
[0048] Stage 3 (Quickly expand and differentiate into T effectors): After 10-14 days, TILs can grow very fast. Medium change is performed every 2 days, and the cells were split quickly to expanded TILs at the ratio 1:4 to 1:8. TILs can be expanded in 24 or 12-well-plates. Dynabeads® Human T-Activator CD3/CD28 can be used once every 2 weeks for re-stimulation of TILs.
[0049] In some embodiments, TILs are expanded ex vivo, with the number of the TILs increased to about 1.1-10-fold, about 10-100-fold, about 100-1000-fold, about 1000-2000-fold, or more than about 2000-fold. In some embodiments, TILs are expanded ex vivo at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 10-fold, at least about 100-fold, at least about 1000-fold, or at least about 2000-fold.
[0050] In some embodiments, the CD3.sup.+ cells can be considered AML TILs due to the lack of specific biomarker. Some AML patient BM samples had low CD3.sup.+ TIL populations. The availability of CD3.sup.+ TILs can be a good prognostic marker of survival for cancer patients. High percentages of CD8.sup.+ TILs were also required to monitor the disease relapse. Accordingly, the present disclosure provides methods of producing at the clinical scale of, e.g., therapeutically appreciable quantity of, TILs. In specific embodiments, the method for expansion of TILs can produce 10×10.sup.7 TILs within 3-4 weeks after the bone marrow aspiration to meet the minimum requirement of 10.sup.6 TILs/kg for a 60 kg AML patient.
[0051] The complexity of AML suggests that AML patients require personalized therapies to achieve long term remission. In some embodiments, availability of CD3.sup.+ TILs and high percentages of CD8.sup.+ TILs in situ are essential in preventing disease progression or relapse, and prolonging the survival in cancer patients.
[0052] In some embodiments, TILs comprise naïve T cells, i.e., immature cells. In some embodiments, TILs are characterized by the surface expression of CD62L (L-selectin) and CCR7 (C—C Chemokine receptor type 7). In some embodiments, TILs comprise CD62L.sup.+ memory T cells, i.e., a subset of a naïve T cell with stemness including self-renewal and multipotent capabilities in vivo. In some embodiments, TILs isolated from bone marrow or peripheral blood samples contain CCR7.sup.+CD95.sup.− and/or CD62L.sup.+CD45RA.sup.+ naïve T cells. In some embodiments, these cells are isolated and expanded by a modified ex vivo culture system. By using the TIL culture methods of the present disclosure, TILs can be expanded ex vivo over a three log-fold (e.g., a 1000-fold or a 2000-fold), which is useful for studying subsets of TILs and bioengineering them to fit potential clinical applications for AML. The TIL protocol of the present disclosure has incorporated co-stimulation from anti-CD3/CD28 microbeads supplemented with cytokines i.e., IL-7 and IL-15. In some embodiments, such co-stimulation increases the viability and induce expansion naïve T cells for sustainable expansion.
[0053] In some embodiments, inhibitors are added to block the PD-1, which facilitates TILs to perform potent anti-leukemic effect to AML blasts ex vivo and in vivo. In some embodiments, TILs are cultured ex vivo in the presence of a PD-1 inhibitor, e.g., an anti-PD-1 antibody, such as atezolizumab, avelumab, cemiplimab, durvalumab, nivolumab, and pembrolizumab. In some embodiments, the ratio of CD8.sup.+ TILs as compared to the native sample is increased in the step of culturing TILs. CD8.sup.+ TILs can infiltrate tumors and kill cancer cells by exerting cytotoxicity.
[0054] Bioengineering of TILs Ex Vivo
[0055] In some embodiments, the methods of the present disclosure further comprise bioengineering TILs or T cells isolated from the subject. Bioengineering can be pharmacological intervention, e.g., culturing in the presence of a PD-1 inhibitor, or genetic engineering, e.g., gene editing using a CRISPR-Cas9 system.
[0056] In some embodiments, the TILs are bioengineered to increase expression or function of a CYP27B1 gene. In some embodiments, the expression or function of the CYP27B1 gene can be increased by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%) as compared to expression or function of CYP27B1 in TILs that have not been bioengineered. In some embodiments, the TILs are bioengineered to suppress expression or function of a PD-1 gene. In some embodiments, the expression or function of the PD-1 is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% as compared to expression or function of PD-1 in TILs that have not been bioengineered. Such modulation of expression or function of the CYP27B1 gene and/or a PD-1 gene can be useful for combination therapies of TIL adoptive cell therapy with PD-1 inhibitor and a hypomethylating agent (e.g., AZA).
[0057] In some embodiments, the TILs are bioengineered to improve antigen-specificity. In some embodiments, the TILs are engineered to increase expression or function of one or more of CD3, CCR7, CD62L, CD45RA, CD95, CD127, CD27, and CD28. In some embodiments, expression or function of one or more of CD3, CCR7, CD62L, CD45RA, CD95, CD127, CD27, and CD28 is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200% as compared to expression or function of these molecules in TILs that have not been bioengineered. Increasing expression or function of one or more of CD3, CCR7, CD62L, CD45RA, CD95, CD127, CD27, and CD28 can confer the TILs sternness and/or proliferative and functional capability as in naïve TILs.
[0058] In some embodiments, the bioengineered TILs have increased horning, proliferating, cytotoxic and/or therapeutic capabilities compared to TILs that have not been bioengineered. In some embodiments, the horning, proliferating, cytotoxic, and/or therapeutic capabilities are increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200% in bioengineered TILs compared to TILs that have not been bioengineered.
[0059] TIL-based immunotherapy disclosed herein can be administered in combination with other therapy to treat AML in subjects. Accordingly, in some embodiments, the methods include administering autologous PD-1-inhibited-TILs in combination with AZA and PD-1 inhibitors as a viable combination therapy to treat AML and prevent relapse. Autologous PD-1-inhibited-TILs in combination with AZA generate a potent anti-leukemic effect and overcome the immune-escape of leukemia blasts by reducing the amount of LSCs and AML blasts thereby extending the survival rate of AML subjects. Additionally, specificity of using the same patient's cells will minimize the likelihood of unwanted side effects of combination treatment for elderly AML patients in the clinic. Accordingly, in some embodiments, the methods include administering a therapeutically effective amount of a PD-1 inhibitor, e.g., atezolizumab, avelumab, cemiplimab, durvalumab, nivolumab, and pembrolizumab, and a hypomethylating agent, e.g., 5-Azacytidine (AZA).
EXAMPLES
[0060] The following examples are offered by way of illustration and not by way of limitation.
Example 1: Isolation of TILs from AML Patient Bone Marrow Samples
[0061] In an example, human CD8.sup.+CD3.sup.+ cytotoxic T effector cells were enriched from AML BM and their PD-1 expression was inhibited for generating PD-1-inhibited-TILs. This method generated PD-1-inhibited-TILs in sufficient amounts and in a quicker time frame. Separation of CD3.sup.+ T cells from bone marrow mononuclear cells (BM-MNC) was performed by using CD3 microbeads (Cat. #130-050-101, Miltenyi Biotech, Germany) and a MiniMACS™ Separator with an MS Column according to the manufacturer's protocol. Selected CD3.sup.+ T cells were considered AML TILs.
[0062] Following ex-vivo expansion of low number TILs using media with cytokines, AML patient BM samples were screened and CD3.sup.+ T cells were present in some samples (N=8,
[0063] Sufficiently large amounts of CD3.sup.+ TILs were isolated and enriched ex vivo to serve as alternative candidate cell vehicles for autologous treatment for AML. PD-1-inhibited-TILs were generated by treating ex vivo expanded TILs with PD-1 inhibitors. There were PD1.sup.+CD3.sup.+T cells inside the AML BM. First, AML patient BM samples were screened for CD3.sup.+T cells (
[0064] Conventionally, T-cell culture protocols are experimentally established in a hierarchy through proliferation of CD3.sup.+T cells, differentiation into CD8.sup.+ T cells, and functional assay. Disclosed here are efficient methods of isolating and expanding CD3.sup.+ TILs ex vivo, thus isolating and expanding BM TILs. The AML patient shown in
Example 2: Anti-Leukemic Effect of TILs in MOLM-14 Ex Vivo
[0065] The function of ex vivo expanded TILs was examined. Patient-derived CD3.sup.+TILs were co-cultured with MOLM-14 cells. MOLM-14 is a human AML cell line with the FLT3 ITD mutation. CFSE was used to label the MOLM-14 blasts. After a 4-hour co-culture, the cells were collected and stained for FACS analyses to examine the anti-leukemia response. A clear population of dead cells was observed within the CFSE.sup.+MOLM-14 population (0.06% to 0.4%). The quantitative data suggested a significant difference between the TIL treated and non-treated experimental groups. The efficacy of TIL treatment was also observed. About 58.7-82% of primary AML CD117.sup.+ cells were PD-L1.sup.+ (n=3). In addition, expanded CD3.sup.+ TILs were found to have a large fraction of PD-1.sup.+ cells. MOLM-14-derived PD-L1 ligands might protect blasts through immune-escape by blocking the function of PD1.sup.+TILs, as other cancer cells did. Nivolumab, a PD-1 inhibitor, was applied to treat expanded primary TILs.
[0066] Vitamin D is known to play essential roles in bone metabolism and calcium homeostasis, and recognized to be involved in new immune-modulatory roles and cancer treatment through the extra-renal synthesis. A gene therapy system was developed using CYP27B1, encoding an enzyme of producing active 1.25(OH)2D3, combined with 5-Azacitidine (5-AZA, a hypomethylating agent) to improve clinical symptoms and prolong the survival of AML mice without hypercalcemia. Vitamin D combined with 5-AZA can not only reduce LSCs' frequency, but also maintain the stemness of human Mesenchymal Stem Cells (MSCs) and HSCs for regenerating healthy microenvironment and hematopoiesis. TILs consisting of CD3.sup.+T-cells were present in some AML patients, and they were isolated, quickly expanded, pharmaceutically and genetically bioengineered to perform cytotoxic functions ex vivo (N=8 among 11 samples).
[0067] Blocking PD-1 was very effective, a significant reduction of PD-1.sup.+CD3.sup.+ TILs from 62.8% to 1.8% (
Example 3: Anti-Leukemic Effect of TILs in Autologous AML Blasts Ex Vivo
[0068] Expanded TILs were evaluated for their ability to kill same patient derived AML blasts ex vivo Killing tests were performed by co-culturing Programmed-TILs with primary AML blasts from the same patient (isolated by CD33-microbeads pull down). About 1×10.sup.5 primary AML blasts per well (24-well-plate) are treated for different time points including 4 hours and 16 hours before analyzing cell viability and programmed cell death by FACS. The ratio of TILs to AML blasts will be in the range of Five experimental groups are evaluated to test the anti-leukemic effects of Programmed-TILs: (1) Group 1: Primary AML blasts with no treatment; (2) Group 2: Primary AML blasts treated with TILs (ratio: 10:1); (3) Group 3: Primary AML blasts treated with CYP27B1+TILs (ratio: 10:1); (4) Group 4: Primary AML blasts treated with Nivo-TILs (Nivolumab-treated; ratio: 10:1); (5) Group 5: Primary AML blasts treated with Nivo-CYP27B1.sup.+TILs (ratio: 10:1). After the treatment, the cells are collected and stained for FACS analyses according to manufacturers' protocols to examine the anti-leukemic effect of bioengineered-TILs. For cytotoxic studies, the cell viability-dye, and Annexin V are applied. Analyses and graphs are generated using the GraphPad Prism software to evaluate significance.
Example 4: AML PDX Models
[0069] ML PDX models have been developed to investigate therapies in immune-deficient mice (NSG or NRG). Previously, an AML xenograft mouse model was established by transplanting human AML cell line MOLM-14 cells into NRG mice. To determine whether ex vivo expanded TILs still maintain their Ag-specific, proliferation and cytotoxic capability in vivo, a localized AML PDX mouse model was generated to create an AML or non-AML microenvironment to illustrate the function of bioengineered-TILs. The AML PDX mouse model was generated by intrafemoral transplantation of AML cells.
[0070] To generate AML PDX mice, adult NRG mice (8-10 weeks old) are sub-lethally irradiated with 250 cGy of total body irradiation 24 hours before tail vein injection of T cell depleted AML cells (5×10.sup.6 per mouse). Daily monitoring of mice for symptoms of disease (ruffled coat, hunched back, weakness, motility) are performed. Mice are euthanized 10-12 weeks after AML injection. Femoral BM, splenocytes and peripheral blood (PB) are harvested. Human AML engraftment is assessed by FACS analyzing percentage of human (h) CD45.sup.+ CD33.sup.+ cells. In addition, percentage or absolute number of AML cells positive for hCD34, hCD38, hCD117, hCD13 and etc. is determined at time of euthanasia respectively. Primary AML cells from either fresh samples or cryopreserved samples are obtained with informed consent and IRB approval from LLU.
Example 5: Evaluation of Therapeutic Effect of Autologous TILs and Chemotherapy in AML PDX Mice In Vivo
[0071] The therapeutic effect of autologous bioengineered-TILs in combination with Nivolumab on leukemic cell burden and survival of AML PDX mice are evaluated in vivo. In preliminary studies (
Example 6: Effect of AZA and 1.25(OH)2D3 on AML Cells
[0072] Experiments are done to determine whether the regaining of CYP27B1 and newly synthesized Vitamin D rescue bone structures in AML BM. Bone structures in AML BM and non-AML BM before and after treatment were evaluated by Micro-computed Tomography (μCT) for bone volume, bone mineral density, connectivity density and spaces. To understand the mechanism of 1.25(OH).sub.2D.sub.3 based treatment for AML, in context of combination with AZA, the RNA-Seq transcriptome profiling of different AML cells lines were performed, which were pre-treated with either 1.25(OH).sub.2D.sub.3 alone, AZA alone or their combination in vitro for 48 h.
Example 7: Confirmation of the Presence of TILs in Bone Marrows of AML Patients
[0073] Autologous TILs based therapies could be a novel therapeutic strategy for AML if the following is done: 1) phenotypically identify TILs, 2) expand TILs ex vivo to sufficient numbers for clinical use, 3) demonstrate cytotoxic effect to autologous AML blasts and 4) bioengineer TILs to restore their Ag-specific cytotoxic functions. To this end, AML patient BMMNC (Patients #1-10, Table 1) were screened. A different degree of CD3.sup.+ T cells infiltration could be detected in all the tested samples.
Example 8: Ex Vivo Expansion of TILs from AML BMs Using a Modified Protocol
[0074] The ex vivo expandability of AML TILs was examined using the T cell culture system of the present disclosure (
[0075] From the “High” group, 0.5 to 2×10.sup.6 CD3.sup.+ T cells/ml were obtained using CD3 microbeads. After magnetic separation, these cells (Patient #10, Table 1) were cultured with supporting feeder cells in RPMI-1640 supplemented with IL-2. A 4-fold increase of the CD8.sup.+CD3.sup.+ T cell population (red arrow) was obtained after the 5-day culture (30.4% on Day 5 vs 7.6% on Day 0, P<0.01,
Example 9: Bioengineering Expanded TILs Pharmaceutically and Genetically Ex Vivo
[0076] The possibility of pharmaceutically and genetically bioengineering expanded TILs ex vivo were investigated (
Example 10: Difficulties with Expansion of CD3.SUP.+ TILs in Some AML Patients
[0077] During the culture of TILs from 10 AML patient samples, one consistent aspect for TIL cultures (n=10) was that the proliferation status of TILs during the early stages (days 3-5) was a good predictor for whether TILs (representative images of early TIL clusters,
[0078]
[0079] To investigate the mechanism underlying differential growth capabilities of TILs, an immunophenotypic comparison of these AML BMMNC by using biomarkers for naïve T cells, including CD62L, CD45RA, CCR7, CD95, were performed. No significant difference of CD62L.sup.+CD45RA.sup.+ naïve TILs was found between the no/slow growth BMMNC and the quick growth BMMNC (P=0.38,
[0080] However, there was a significant loss of CCR7.sup.+CD95− naïve T cell population (red arrow,
[0081] The data suggest that to effectively expand TILs to a sufficient amount, the CCR7.sup.+CD95− naïve T cell population in AML patient BM are needed to support the quick expansion ex vivo. Alternative sources of T cells for TILs therapy in patients with low BM TILs were developed, including peripherally isolated T cells expanded by the methods disclosed herein. Similar patterns of naïve T cells and differentiated T cells were identified in the peripheral blood (PB) and BM samples of same patients (
Example 11: Functional Characterization of Ex Vivo Expanded TILs from AML Patients Using Ex Vivo Cytotoxic Assays and In Vivo Homing Assays
[0082] To examine the function of ex vivo expanded TILs, cytotoxic tests were performed. CD33, a surface biomarker, is expressed on leukemia blasts from the majority of AML patients. Thus, CD33.sup.+ AML blasts were isolated from BM samples by using CD33 antibodies with microbeads. Then, 2×10.sup.4-10.sup.5 autologous CD33.sup.+ blasts were co-cultured with 2×10.sup.5-10.sup.6 isolated and ex vivo expanded TILs (E:T ratio 10:1). After 18 hours, cells were collected and stained for FACS analysis. A significant decrease of viable CD33.sup.+ blast population were observed in TIL treatment versus the control of no treatment group (90.6% vs. 1.89%; p<0.01) (
[0083] To investigate whether ex vivo expanded TILs will home to the BM and maintain their proliferation and functional capabilities in vivo, the following experiments were conducted.
[0084] To examine the ex vivo expandability of TILs for the possibility of autologous transplantation, a novel ex vivo culture system was developed to expand TILs from AML patient BM samples with low numbers of CD3.sup.+ T cells to clinical scales. Furthermore, it was immunophenotypically determined that these TILs expressed either CCR7.sup.+ CD95−/or CD62L.sup.+CD45RA.sup.+, which are makers for naive T cells. Some patients have high numbers of CD3.sup.+ TILs while others have low numbers of CD3.sup.+ TILs in their BM. The presence of naïve T cells is the hallmark of expandability of T cells, even in patients with low initial CD3.sup.+ TILs. Finally, the data demonstrate that TILs can cause cytotoxicity to autologous blasts ex vivo, can be engineered to express desirable genes, and are able to migrate to BM after being transplanted into immunodeficiency mice in vivo. An in vivo experiment showed that transplantation of expanded TILs is feasible and that IV injected cells can be tracked to the bone marrow. These ex vivo expanded TILs are likely to maintain their BM homing capability, proliferation and therapeutic capabilities in vivo. The in vivo data also suggested that primary TILs could be engineered to overexpress a desirable gene for therapeutic purpose (
[0085] Materials and Methods
[0086] Human Samples. AML BM samples (Patients #1-10, Table 1) were obtained from Loma Linda University Cancer Center Biospecimen Laboratory (LLUCCBL). AML Peripheral Blood and BM samples (Patients #11-20, Table 1) were obtained from the City of Hope National Medical Center (COHNMC). All donor patients signed an informed consent form. Sample acquisition was approved by the Institutional Review Boards at the LLUMC and the COHNMC in accordance with an assurance filed with and approved by the Department of Health and Human Services, and it met all requirements of the Declaration of Helsinki.
TABLE-US-00001 TABLE 1 List of AML patients for the FACS screening and ex vivo/in vivo studies Disease Cytogenetics No. Diagnosis Status Age Sex (Karyotype) Gene Mutation 1 AML Newly 32 F Normal FLT3, CEBPA and NPM1: Diagnosed NEGATIVE 2 AML Newly 70 M Normal The molecular analyses of CEBPA, Diagnosed FLT3 and NPM1 mutations show only positive for FLT3 internal tandem duplication mutation (FLT3-ITD) 3 AML Diagnosed 35 M Normal FLT3, CEBPA and NPM1: NEGATIVE 4 AML Newly 59 M Normal Molecular Markers Diagnosed From OSH Quest Diagnostic CEBPA negative NPM mutation not detected FLT3 ITD not detected FLT3 TKD not detected 5 AML Newly 53 M inv(16)(p13.1q22) FLT3, CEBPA and NPM1: Diagnosed MYH11/CBFB NEGATIVE 6 AML Diagnosed 65 M Normal FLT3 ITD (.sup.+), CEBPA (−), NPM 1 (.sup.+), c kit (−), PML RARA 7 AML Newly 45 M Normal CEBPA, DNMT3A, FLT3, IDH1/2, Diagnosed KIT, KRAS, NRAS, RUNX1 TP53 8 AML Diagnosed 38 F t(8;21) RUNX1- Mutations noted in KRAS, NF1, and RUX1T1 TP53; Negative for IDH 1, IDH2 and FLT3, RUNX1 9 AML Diagnosed 30 M Normal Intermediate Risk (Wild type NPM1 without FLT3-ITD without adverse risk genetic lesions) 10 AML Newly 33 F Normal DNMT3A, NRAS, NPM1 Diagnosed 11 AML Newly 78 M Normal TP53,U2AF1,ASXL1,RUNX1, FLT3- Diagnosed ITD 12 AML Newly 53 M 46,XY,r(3)(p26q29),del TP53 Diagnosed (5)(q22q35),der(7)t(7;?;3) (q22;?;p11)[17] 13 AML Newly 37 F Normal WT1, FLT3-ITD Diagnosed 14 AML Newly 40 M Normal DNMT3A, IDH2, KRAS, NRAS,NPM1 Diagnosed 15 AML Newly 72 F Normal DNMT3A, TET2 Diagnosed 16 AML Newly 67 M Normal IDH2, NPM1 Diagnosed 17 AML Newly 20 M 46,XY,inv(16) KIT Diagnosed (p13.1q22.1)[23] 18 AML Newly 63 M 46,XY,i(21)(q10)[20] RUNX1, WT1 Diagnosed 19 AML Newly 63 F Normal NPM1, SF3B1 Diagnosed 20 AML Newly 38 F Normal FLT3, WT1, NPM1 Diagnosed
[0087] Mice. NRG (OD-Rag1.sup.null IL2rg.sup.null) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free animal facility at Loma Linda University (LLU). All mice were used at the age of 8 weeks. All experiments were performed in compliance with an Institutional Animal Care and Use Protocol approved by LLU Animal Care and Use Committee.
[0088] Isolation of TILs from AML patient bone marrow samples. CD3.sup.+ T cells from bone marrow mononuclear cells (BMMNC) were separated by using CD3 microbeads (Miltenyi Biotech, Germany) and a MiniMACS™ Separator with an MS Column according to the manufacturer's protocol. Selected CD3.sup.+ T cells were considered AML TILs.
[0089] Ex-Vivo Expansion of high number TILs by a Traditional T cell Protocol. CD3.sup.+ TILs were isolated from AML BMMNC by the pull-down through CD3 microbeads and magnetic separation. The non-CD3.sup.+ cells (feeder cells) were pre-treated with 10 mitomycin-C for 2 hours to arrest cell proliferation. CD3.sup.+ TILs and feeder cells were co-cultured at 37° C. and 5% CO.sub.2 in a RPMI 1640 culture medium containing 10% fetal bovine serum (FBS, HyClone), 100 μg/ml penicillin/streptomycin, and Interleukin (IL-2) (1000 U/ml, Peprotech). Seeding cell density was 300 μl of 100,000 cells/ml in each well of 48-well-plates. For the maintenance of quickly expanded TILs, medium was changed every 2-3 days, and split cells at the ratio of 1:4 when reaching 80% confluent. TILs were stimulated with 30 ng/mL human anti-CD3 (OKT3, Biolegend). Around 10-14 days, cultures of TILs were started in 12-well-plates or T25 flasks for expansion of large amounts before further analyses.
[0090] Ex-Vivo Expansion of low number TILs by a Modified T cell Protocol. (1) Media with Cytokines: CD3.sup.+ T cells were cultured at 37° C. and 5% CO.sub.2 in a RPMI 1640 culture medium containing 10% fetal bovine serum (FBS, HyClone) with penicillin/streptomycin (100 μg/ml), IL-2 (1000 U/ml, Peprotech), and Dynabeads® Human T-Activator CD3/CD28 (Gibco™) without feeder cells. (2) Timeline of TIL expansion: Stage 1 (Naïve TILs): The seeding cell density of CD3.sup.+ TILs was around 300 μl of 20,000 cells/ml in appropriate wells of 48-well-plates. Due to the low cell density of TILs, fresh media was added at a 1:1 ratio to each well every 2 days and mixed the cells/media. Based primarily on the growth of the TILs, media was changed every 5-7 days and split cells at the ratio 1:3. Stage 2 (Ready to grow): After 7 days, IL-7 (25 ng/ml, Peprotech) and IL-15 (25 ng/ml, Peprotech) were added to the media along with IL-2 (1000 U/ml). Every patient BMMNC sample was different; however, TILs were preferably raised in 48-well-plates for expansion to sufficient amounts during the beginning 10-14 days instead of in large wells and flasks. Stage 3 (Quickly expand and differentiate into T effectors): After 10-14 days, TILs grew very fast. Media change was performed every 2 days and split quickly expanded TILs at the ratio 1:4 to 1:8. Then, TILs were expanded in multiple 48 or 24 well-plates. Dynabeads® Human T-Activator CD3/CD28 was used once for re-stimulation of TILs.
[0091] Flow Cytometry (FACS). Expanded TILs were harvested and examined for the expression of cell surface biomarkers (CD) and intracellular proteins for T cells by multichromatic FACS. Briefly, about 1×10.sup.4˜10.sup.6 cells in 100 μl FACS buffer (PBS containing 1% FBS and 0.05% sodium azide) were stained with various fluorescence-conjugated antibodies specific for the desired cell surface proteins at 4° C. for 30 min. The surface-stained cells were then fixed and permeabilized using the appropriate reagents (e.g. the BD Pharmingen Cytofix/Cytoperm buffer) and stained with different fluorescence-conjugated antibodies specific for the desired intracellular proteins at 4° C. for 30 minutes in the permeabilizing buffer (e.g. the BD Perm/Wash buffer). Finally, the cells were washed twice in the permeabilizing buffer and twice in the FACS buffer before being analyzed on the BD FACSAria II. Data was analyzed using the FlowJo software (Treestar).
[0092] Cytotoxicity Assay. The cytotoxicity assays were performed by co-culturing engineered TILs with primary AML blasts from the same patient (isolated by CD33-microbeads pull-down) in 24-well plates. AML blasts from BMMNC were separated using APC anti-human CD33 antibody (Biolegend), anti-APC microbeads (Miltenyi Biotech, Germany), and a MiniMACS™ Separator with an MS Column. The ratio of autologous TILs to AML blasts were in the range of 5:1 to 10:1 according to a previous report [20]. After overnight incubation, cells were collected, stained, and processed for FACS assay of biomarkers including viability dyes (Invitrogen™) and CD33 according to manufacturers' protocols. Analyses and graphs will be generated using the GraphPad Prism software to evaluate significance.
[0093] Adoptive Cell Transplantation of engineered human AML cells and TILs in immune-deficient NRG mice. Ex vivo expanded TILs (2×10.sup.6 cells/mouse) were pre-labeled to be red fluorescent with Qtracker™ 655 (Molecular Probes) and intravenously (IV) injected into NRG mice through the tail vein. To help the engraftment, 10 mg/kg of Azacitidine was intraperitoneally injected one day before the injection. TILs-engrafted mice were sacrificed at different time points. In another experiment, AML cells (non-CD3.sup.+ cells from BMMNC) were transduced with GFP lentivirus to generate GFP.sup.+ AML cells. Fourteen days after transplantation of GFP.sup.+ AML cells (1×10.sup.6 cells/mouse), Qtracker™ 655 labelled TILs were IV injected into these AML NRG mice. The detailed protocol and plasmids for generating lentivirus and generating GFP.sup.+ AML cells can be found in our previous report [21]. On day 10 after TILs' engraftment, mice were sacrificed for FACS analyses. Immunofluorescent histology was performed to visualize TILs and GFP.sup.+ AML cells inside of the bone marrow.
[0094] Histology. Preparation of undecalcified frozen sections from bone tissues was performed according to the protocol reported in Xu, Y., et al., 2020 Transl Oncol 13(12): p. 100869. Briefly, specimens were fixed in 4% paraformaldehyde, freeze-embedded with an embedding medium (SCEM), and frozen in pentane cooled with liquid nitrogen. The frozen specimen block was fixed to the cryostat and trimmed with a disposable blade. The block's surface was then covered with a pressure sensitive adhesive film (Cryofilm) and cut into 10 μm-thick frozen sections which were stored at −20° C. The frozen sections were immunohistochemically stained and photographed for further analyses.
[0095] Reagents
[0096] Table 2 shows exemplary reagents that can be used according to the present disclosure.
TABLE-US-00002 TABLE 2 List of Reagents Species Antibody/Reagents Color Cat. # Company Reactivity CD3 PE/Cyanine7 300420 Biolegend Human CD3 FITC 300406 Biolegend Human CD4 APC 17-0048-41 eBioscience Human CD4 PERCP 300527 Biolegend Human CD8 PerCP/Cy5.5 302922 Biolegend Human CD8 FITC 130-110-815 Milteny Biotec Human CD95 APC 305611 Biolegend Human CCR7 (CD197) PE 353203 Biolegend Human CCR7 (CD197) APC 352313 Biolegend Human CD62L FITC 304803 Biolegend Human CD45RA PE/Cyanine7 304125 Biolegend Human PD-1 (CD279) FITC 367411 Biolegend Human PD-1 (CD279) PE 12-2799-42 eBioscience Human PD-L1 (CD274) PerCP- 46-5983-42 eBioscience Human eFluor710 CD33 APC 303408 Biolegend Human Anti-APC Microbeads 130-090-855 Milteny Biotec Annexin V FITC 640906 Biolegend Viability Dye eFluor ™ 780 65-0865-14 eBioscience DAPI D9542-1MG Sigma Aldrich Qtracker ™ 655 Q25029 Molecular Probes CD3 MicroBeads 130-050-101 Milteny Biotec Human Dynabeads ® Human T- 11161D Gibco Human Activator CD3/CD28 IL2 200-02 Peprotech Human IL7 200-07 Peprotech Human IL15 200-15 Peprotech Human KAWAMOTO Film Kit Section-Lab Co.ltd, Japan Azacitidine (AZA) Celgene Nivolumab (Opdivo) Bristol-Myers Squibb
[0097] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.