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  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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  • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
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  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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Definitions

• compositions which include T cells comprising chimeric antigen receptors (CARs) and checkpoint inhibitors (CPIs) and methods for using the compositions to treat cancer.
• CARs chimeric antigen receptors
• CPIs checkpoint inhibitors
• Adoptive cell transfer as a modality of immunotherapy for cancer, has demonstrated remarkable success in treating hematologic malignancies and malignant melanoma.
• An especially effective form of ACT which uses gene-modified T cells expressing a chimeric antigen receptor (CAR) to specifically target tumor-associated-antigen (TAA), such as CD 19 and GD2, has displayed encouraging results in clinical trials for treating such diseases as B cell malignancies and neuroblastoma.
• CAR chimeric antigen receptor
• TAA tumor-associated-antigen
• CARs are artificial receptor surface TAAs in a major histocompatibility class (MHC)-independent manner.
• MHC histocompatibility class
• CAR T cell therapy in patients with hematologic malignancies, only modest responses have been observed in solid tumors. This can be attributed, in part, to the establishment of an immunosuppressive microenvironment in solid tumors.
• Such milieu involves the upregulation of a number of intrinsic inhibitory pathways mediated by increased expression of inhibitory receptors (IRs) in T cells reacting with their cognate ligands within the tumor.
• IRs inhibitory receptors
• T cells such as CTLA-4, T cell Ig mucin-3 ( ⁇ -3), lymphocyte-activation gene 3 (LAG-3), and programmed death- 1 (PD-1).
• CTLA-4 T cell Ig mucin-3
• LAG-3 lymphocyte-activation gene 3
• PD-1 programmed death- 1
• PD-1 is upreguiated shortly after T ceil activation, which in turn, inhibits T cell effector function via interacting with its two ligands, PD-L1 or PD-L2.
• PD-L1 is constitutively expressed on T cells, B cells, macrophages, and dendritic cells (DCs).
• PD-L1 is also shown to be abundantly expressed in a wide variety of solid tumors. In contrast, the expression of PD-Ll in normal tissues is undetectable. As a consequence of its critical role in immunosuppression, PD-1 has been the focus of recent research, aiming to neutralize its negative effect on T cells and enhance antitumor responses. Clinical studies have demonstrated that PD-1 blockade significantly enhanced tumor regression in colon, renal and lung cancers and melanoma.
• CAR receptor
• CPI checkpoint inhibitor
• CAR-T cells secreting checkpoint inhibitors are provided.
• CAR-T ceils secreting checkpoint inhibitors (CPIs) targeting PD-1 are provided and shown of their efficacy in a human lung carcinoma xenograft mouse model.
• CPIs chimeric antigen receptor
• CAR T cells in order to overcome the inhibitor ⁇ ' effect of PD-1 signaling in CAR T cells, genetically engineered CAR T cells with the capacity to continuously produce a single-chain variable fragment (scFv) form of anti-PD-1 antibody are used.
• anti-PD-1 scFv expression and secretion interrupt the engagement of PD-1 with its ligand, PD-LI, and prevent CAR T cells from being inhibited and exhausted.
• the secretion of anti-PD-1 scFv by CAR T cells significantly improves the capacity of CAR T cells in eradicating an established solid tumor.
• CAR.aPDl -T cells demonstrate the effector function and expansion capacity, as measured by the production of IFN- ⁇ and T cell proliferation following antigen- specific stimulation.
• the antitumor efficacy of CAR.aPDl-T cells is superior than CAR-T ceils alone or CAR-T cells combined with anti-PD-1 antibody using a xenograft mouse model.
• the enhanced tumor eradication of CAR.aPDl-T cells is further supported by the expansion and functional capacity of tumor-infiltrating lymphocytes.
• CAR.aPDl-T ceils secrete human anti-PD-1 CPIs which efficiently bind to PD-1 and reverse the inhibitory effect of PD-1/PD-L1 interaction on T cell function.
• PD-1 blockade by continuously secreted anti-PD-1 prevents T cell exhaustion and significantly enhances T cell expansion and effector function both in vitro and in vivo.
• the secretion of anti-PD-1 enhances the antitumor activity of CAR-T cells and prolongs overall survival.
• CAR.aPDl-T cells are less exhausted, more functional and expandable, and more efficient at tumor eradication than parental CAR-T cells.
• CPI is administered to a subject in need thereof to enhance antitumor immunity and/or to treat cancer (especially reducing solid tumors),
• Figures 1A-1E depict construction and characterization of CAR19 and CAR19.aPDl .
• Figure 1A shows a schematic representation of parental anti-CD19 CAR (CAR 19) and anti-PD-1 -secreting anti-CD19 CAR (CAR19.ctPDl) constructs.
• Figure IB shows the expression of both CARs in human T cells. The two groups of CAR T cells were stained with biotinylated protein L followed by FITC-conjugated streptavidin to detect CAR expression on the cell surface. A viable CD3 + lymphocyte gating strategy was used. NT indicates nontransduced T cells, which were used as a control.
• Figures 1C and ID show the expression of secreted anti-PD-1 antibody in the supernatant from either CAR 19 or CAR19.aPDl T cell culture as analyzed by Western blot (1C) and ELISA (ID).
• Figures 2A-2D depict anti-PD-1 expression enhanced the antigen-specific immune responses of CAR T cells.
• Figure 2B shows cytotoxicity of both CARs against target cells. The two groups of CAR T cells were cocultured for 6 hours with H292-CD19 cells at 1 : 1 , 5: 1 , 10: 1 , and 20: 1 effector-to-target ratios, and cytotoxicity against H292-CD19 was measured.
• Nontransduced (NT) T cells were used as a control.
• Figure 2C shows proliferation of both CARs after antigen-specific stimulation. The two groups of CAR T cells were pre-stained with CFSE. The stained T cells were then cocultured for 96 hours with H292-CD19 cells at 1 : 1 effector-to-target ratio and the intensity of CFSE was measured.
• Nontransduced (NT) T ceils were used as a control.
• Figure 2D shows the summarized statistics in bar graphs of proliferation rate for nontransduced (NT) T cells, CAR19 T cells, and CAR19.aPDl T cells corresponding to Fig. exhausted. Both CAR 19 and CAR19.aPDl T cells were cocultured with H292-CD19 cells for 24 hours.
• Figure 3A shows PD-1 expression as measured by flow cytometry.
• CD8 + T cells were shown in each panel.
• PD-1 -expressing CDS T cells were gated, and their percentage over total CD8 + T cells was shown in each scatterplot.
• Figure 3D shows TIM-3 expression as measured by flow cytometiy.
• the percentages of PD-L1 -expressing CDS T cells over total CD8 + T cells (FIG. 3E) and PD-L1 -expressing CD4 T cells over total CD4 + T cells (FIG. 3F) were shown in bar graphs (n 3, mean ⁇ SEM, *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001).
• FIGS 4A-4D depict adoptive transfer of CAR T cells secreting anti-PD-1 scFv enhanced the growth inhibition of established tumor.
• Figure 4A shows schematic representation of the experimental procedure for tumor challenge, T cell adoptive transfer and antibody treatment.
• NSG mice were s.c. challenged with 3 I i of H292-CD19 tumor cells.
• 3 10 6 of CAR 19 or CAR19. ⁇ xPDl T cells were adoptively transferred through i.v. injection.
• Figure 4B shows tumor growth curve for mice treated with nontransdueed (NT), NT plus anti-PD-1 injection, CAR19, CAR19 plus anti-PD-1 injection, or CAR19.
• Figure 4C shows waterfall plot analysis of tumor reduction on day 17 post-therapy for various treatment groups.
• Figure 4D shows survival of H292-CD19 tumor-bearing NSG mice after indicated treatment. Overall survival curves were plotted using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test (/?
• FIGS 6A-6G depict CAR T cells secreting anti-PD-1 were more functional than parental CAR T cells at local tumor site.
• Figure 6A shows a schematic representation of the experimental procedure for tumor challenge, T cell adoptive transfer and antibody treatment.
• NSG mice were s.c. challenged with 3 x 10 6 of H292-CD19 tumor cells.
• 3 ⁇ 10 6 of CAR 19 or CAR 19 PI) i T cells were adoptively transferred through i.v. injection.
• One day post-T cell adoptive transfer, anti-PD-1 antibody treatment was initiated, and the treatment was continued on the indicated dates. The mice were then euthanized on day 8 for analysis.
• Figure 6B shows the percentage of human CD45 + T cells in the tumor, blood, spleen and bone marrow of H292-CD19 tumor-bearing mice that were adoptively transferred with CAR19 or CAR19.aPDl T cells, or treated with CAR 19 T cells along with injection of anti-PD-1 antibody, as characterized by flow cytometry.
• Figure 6C shows the ratio of CD8 "f versus CD4 + TILs in the tumor (n ------- 3, mean ⁇ SEM; ns, not significant, P>0,05; * ⁇ 0.05;
• Figure 6G shows the secreted anti-PD-1 scFvs and injected anti-PD-1 antibodies in the sera as evaluated using ELISA (n -------
• Figure 7A depicts the production of anti-PD-1 scFv from CAR19.aPDl T cells (1 x 10°) after 24-hour culture with or without Brefeldin A.
• Figure 7B depicts the expression of anti-PD-1 scFv during the course of C.AR19.aPDl T cell expansion. The concentration of secreted scFv was measured at four different time points post T cell transduction, including days 4, 7, 10 and 12. The cell density was maintained around 2-4 x 10 b per ml during T cell expansion.
• Figure 7C depicts human T ceils were activated with anti-CD3/CD28 beads for antibody or anti-PD-1 antibody.
• Figure 7D depicts the activated human T cells were incubate with lml of CA 19.aPDl T cell culture supernatant for 30 min. The T cells was washed once with PBS and then stained with anti-HA antibody.
• Figure 8 depicts the expression of PD-L1 on H292-CD 19 and SKOV3-CD19 as determined by flow cytometry.
• FIG 11A depicts the blocking activity of anti-PD-1 scFv on the binding of PD- 1 detection antibody.
• Human T cells were activated with anti-CD3/CD28 beads for 48 hours and then cultured in TCM supplemented with 10 ng/ml of human IL-2 for two weeks. The activated T cells were then incubated with 1 ml of CAR19.aPDl T cell culture supernatant or control medium for 30 min. The T cells were washed once with PBS and then stained with anti-PD- 1 antibody.
• Figures 12 A and 12B depict the representative gating schemes and plots for CD8 + PD-L1 + T cells (12A) and CD8 + LAG-3 + and CD8 + TIM-3 + T ceils (12B) after antigen- specific stimulation for 24 hours.
• FIGS 13A-13E depict that both CAR 19 and CAR19.aPDl T cells were cocultured with H292-CD19 ceils for 24 hours.
• the expression of PD-1 (13A), LAG- 3 (13B) and TIM-3 (13C) was measured by flow cytometry.
• Figure 14A depicts the ratio of CD8 + versus CD4 "f T cells before they were adoptively transferred into the mice.
• Figure 14C depicts the expression of IFN- ⁇ in the sera was measured by ELISA.
• the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
• the term "about” refers to a measurable value such as an amount, a time duration, and the like, and encompasses variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5% or ⁇ 0.1% from the specified value.
• CARs Chimeric antigen receptor
• CAE engineered receptors, which graft an antigen specificity onto cells
• CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.
• CARs are recombinant polypeptides comprising an antigen-specific domain (ASD), a hinge region (HR), a transmembrane domain (TMD), co-stimulatory domain (CSD) and an intracellular signaling domain (ISD).
• Antigen-specific domain refers to the portion of the CAR that specifically binds the antigen on the target cell.
• the ASD of the CARs comprises an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof.
• the targeting regions may comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen.
• scFv single chain Fv
• divalent single chain antibodies or diabodies each of which are specific to the target antigen.
• almost any molecule that binds a given antigen with high affinity can be used as an ASD, as will be appreciated by those of skill in the art.
• the ASD comprises T cell receptors (TCRs) or portions thereof.
• Hinge region refers to the hydrophilic region which is between the ASD and the TMD.
• the hinge regions include but are not limited to Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences or combinations thereof.
• Examples of hinge regions include but are not limited to CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly3 or CHI and CH3 domains of IgGs (such as human IgG4).
• the hinge region is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD 8 a, (v) a hinge, CH2 and CH3 regions of IgGl, (vi) a hinge region of IgGl or (vi) a hinge and CH2 region of IgGl .
• Other hinge regions will be apparent to those of skill in the art and which crosses the plasma membrane.
• the transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof.
• a transmembrane protein for example Type I transmembrane proteins
• Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
• the TMD of the CAR comprises a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T-eeli receptor, CD28, CD 3 epsiion, CD45, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, CD154, KIRDS2, QX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, f !
• Co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival and/or development of memory ceils.
• the CARs of the invention may comprise one or more co-stimulatory domains.
• Each co-stimulatory domain comprises the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD 137 (4- IBB), CD 134 (OX40), Dap 10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD 11 a/CD 18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.
• Other co ⁇ stimulatory domains e.g., from other proteins will be apparent to those of skil l in the art and may be used in connection with alternate embodiments of the invention.
• ISD Intracellular signaling domain '
• cytoplasmic domain refers to the portion of the CAR which transduces the effector function signal and directs the cell to perform its specialized function.
• domains that transduce the effector function signal include but are not limited to the z chain of the T-cell receptor complex or any of its homologs (e.g., h chain, FceRlg and b chains, MB 1 (Iga) chain, B29 (Igb) chain, etc), human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention,
• Linker refers to an oligo- or polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains/regions of the CAR of the invention.
• Linkers may be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not stericaily interfere with one another.
• Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof.
• the linkers include the picomaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof.
• the linker sequences may comprise Asp-Val/Ile- Glu-X-Asn-Pro-Gly (2A)_ Pro (2B) (SEQ ID NO: 1) motif, which results in cleavage between the 2A glycine and the 2B proline.
• Other linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention,
• Genetically modified cells refer to cells that express the CARs and checkpoint inhibitors.
• the genetically modified cells comprise vectors that encode a CAR and vectors that encode one or more checkpoint inhibitors, wherein the two vectors are different.
• the genetically modified ceils comprise a vector that encodes a CAR and one or more checkpoint inhibitors.
• the genetically modified cells comprise a first vector that encodes a CAR and a second vector that encodes the checkpoint inhibitor.
• the genetically modified cell is a T-lymphocyte cell (T-cell).
• the genetically modified cell is a Natural Killer (NK) cells.
• Immunocell refers to the cells of the mammalian immune system including but not limited to antigen presenting cells, B-celis, basophils, cytotoxic T-cells, phagocytes, plasma cells and T-cells.
• Immunune effector cell refers to the T cells and natural killer (NEC) cells.
• Immuno response refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, i nil ani mators response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity.
• CD4 lymphocytes refer to lymphocytes that express CD4, i.e., lymphocytes that are CD4+.
• CD4 lymphocytes may be T cells that express CD4.
• antibody refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region, referred to herein as the "Fc fragment” or "Fc domain".
• Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
• Antigen-binding fragments include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
• the Fc domain includes portions of two heavy chains contributing to two or three classes of the antibody.
• the Fc domain may be produced by recombinant DNA techniques or by enzymatic (e.g. papain cleavage) or via chemical cleavage of intact antibodies.
• antibody fragment refers to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
• antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain, (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv;
• Single chain variable fragment ' refers to forms of antibodies comprising the variable regions of only the heavy (VH) and light (VL) chains, connected by a linker peptide.
• the scFvs are capable of being expressed as a single chain polypeptide.
• the scFvs retain the specificity of the intact antibody from which it is derived.
• the light and heavy chains may be in any order, for example, VH-linker-VL or VL -linker- VH, so long as the specificity of the scFv to the target antigen is retained.
• Therapeutic agents refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of and/or cure, a disease.
• Diseases targeted by the therapeutic agents include but are not limited to infectious diseases, carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune ceils, and antigens expressed on cells associated with various hematologic diseases, and/or inflammatory diseases.
• cancer and "cancerous” refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
• the term “cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
• solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial lung, cancer of the small intestine and cancer of the esophagus.
• the cancer is a melanoma, e.g., an advanced stage melanoma.
• Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention.
• cancers examples include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute ieukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphoc
• isolated refers to molecules or biological materials or cellular materials being substantially free from other materials.
• isolated refers to nucleic acid, such as DNA or RNA, or protein or polypeptide (e.g., an antibody or derivative thereof), or cell or cellular organelle, or tissue or organ, separated from other DNAs or RN As, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source.
• isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
• isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not recombinant polypeptides.
• isolated is also used herein to refer to cells or tissues that are isolated from other cells or ti ssues and is meant to encompass both, cultured and engineered ceils or tissues.
• naked DNA refers to DNA encoding a CAR cloned in a suitable expression vector in proper orientation for expression.
• Viral vectors which may be used include but are not limited SIN lentiviral vectors, retroviral vectors, foamy virus vectors, adeno-assoeiated virus (AAV) vectors, hybrid vectors and/or plasmid transposons (for example sleeping beauty transposon system) or integrase based vector systems.
• AAV adeno-assoeiated virus
• Other vectors that may be used in connection with alternate embodiments of the invention wi ll be apparent to those of skill in the art.
• Target ceil refers to ceils which are involved in a disease and can be targeted by the genetically modified cell s of the invention (including but not limited to genetically modified T-eeils, NK cells, hematopoietic stem ceils, pluripotent stem cells, and embryonic stem cells). Other target cells will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.
• T-celi and "T-lymphocyte” are interchangeable and used synonymously herein. Examples include but are not limited to naive T cells, central memory T cells, effector memory T cells or combinations thereof.
• Vector refers to the vehicle by which a polynucleotide sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
• Vectors include plasmids, phages, vimses, etc.
• administering refers to the placement an agent as disclosed herein into a subject by a method or route which results in at least partial localization of the agents at a desired site.
• “Beneficial results” may include, but are in no way limited to, lessening or al leviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient' s stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer,
• the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slowdown or stop the progression or severity of a condition associated with, a disease or disorder.
• the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, such as cancer.
• Treatment is generally “effective” if one or more symptoms or clinical markers are reduced.
• treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
• treatment of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
• treatment of cancer includes decreasing tumor volume, decreasing the number of cancer cells, inhibiting cancer metastases, increasing life expectancy, decreasing cancer cell proliferation, decreasing cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
• Conditions and “disease conditions,” as used herein may include, cancers, tumors or infectious diseases.
• the conditions include but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases.
• conditions include any one or more of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer.
• an effective amount refers to the amount of a pharmaceutical composition comprising one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of disorder, at a reasonable benefit/risk ratio applicable to any medical treatment.
• a therapeutically or prophylactically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering the oligopeptides described herein.
• Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for diabetes.
• “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species: farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
• farm animals such as cattle, sheep, pigs, goats and horses
• domestic mammals such as dogs and cats
• laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
• the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
• CAR-T cells with antitumor activity are frequently exhausted in the immunosuppressive tumor microenvironment.
• the PD-1 receptor is a major effector in mediating T cell exhaustion.
• a previous study demonstrated that anti-PD-1 antibody treatment enhanced antitumor activity when combined with anti-HER2 CAR-T cells in a syngeneic breast carcinoma mouse model.
• achieving a substantial and sustained efficacy requires continuous admini stration and a large amount of antibodies, often leading to severe systemic toxicity.
• a cell for example, a genetically modified cell containing a nucleic acid encoding both a chimeric antigen receptor (CAR) and a checkpoint inhibitor, or nucleic acids encoding a CAR and a CPI, respectively.
• the cell expresses a CAR and a checkpoint inhibitor.
• the cell is a T- iymphocyte ceil (T-ceil).
• the cell is a Natural Killer (NK) cells.
• the checkpoint inhibitor for example, anti-PD-1 scFv
• the checkpoint inhibitor is constitutively expressed.
• the cell expresses a CAR that targets any one or more of targets expressed on disease causing or disease associated cells including but not limited to CD 19, CD22, CD23, MPL, CD30, CD32, CD20, CD70, CD79b, CD99, CD 123, CD138, CDI79b, CD200R, CD276, CD324, , FcRH5, CD171, CS-1, CLL-1 (CLECLl), CD33, CDH1, CDH6, CDH 16, CDH17, CDH19, EGFRviii, FcRH5, GD2, GD3, HLA-A2, BCMA, Tn Ag, PSMA, ROR1 , FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, ILl lRa, Mesothelin, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, PR
• the cell (for example, a genetically modified cell) expresses a CAR that targets CD 19.
• the cell expresses a checkpoint inhibitor target any one or more of PD-1, LAG-3, TIMS, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), TIGIT, CTLA-4, BTLA, and LAIRl .
• a checkpoint inhibitor target any one or more of PD-1, LAG-3, TIMS, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), TIGIT, CTLA-4, BTLA, and LAIRl .
• the checkpoint inhibitors are antibodies or fragments thereof that target any one or more of PD-1 , LAG-3, TIM 3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), TIGIT, CTLA- 4, BTLA, and LAIRl .
• CEACAM e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5
• TIGIT e.g., CTLA- 4, BTLA, and LAIRl .
• the cell expresses the checkpoint inhibitor that targets PD-1.
• the checkpoint inhibitor is an anti-PD-1 scFv.
• the cell expresses a CAR that targets CD19 and a checkpoint inhibitor that targets PD-1, wherein the checkpoint inhibitor that targets PD-1 is an anti-PD-1 -scFv.
• nucleic acid comprising a first polynucleotide encoding the CAR described herein and a second polynucleotide encoding the checkpoint inhibitor described herein.
• polypeptides encoded by the one or more nucleic acids described herein are also provided herein.
• a vector comprising the one or more nucleic acids described herein.
• the methods comprise administering to a subject in need thereof, a therapeutically effective amount of a cell comprising a nucleic acid encoding a chimeric antigen receptor and a checkpoint inhibitor (or nucleic acids encoding a CAR and a CPL respectively), so as to treat, inhibit, prevent metastasis of and/or reduce severity of cancer in the subject.
• a therapeutically effective amount of a cell comprising a nucleic acid encoding a chimeric antigen receptor and a checkpoint inhibitor (or nucleic acids encoding a CAR and a CPL respectively).
• the methods include administering a therapeutically effective amount of a composition including a cell that contains a nucleic acid encoding both a chimeric antigen receptor (CAR) and a checkpoint inhibitor, or a cell that contains nucleic acids encoding a CAR and a checkpoint inhibitor, respectively, to the subject so as to treat, inhibit, prevent metastasis of and/or reduce severity of cancer in the subject.
• the cancer is lung cancer.
• the methods comprise administering a therapeutically effective amount of a composition comprising a cell comprising a nucleic acid encoding both a CD 19 specific chimeric antigen receptor and a PD- 1 specific checkpoint inhibitor (for example, anti-PD-l-scFv), or nucleic acids encoding a CD 19 specific CAR and a PD-1 specific checkpoint inhibitor, respectively, to the subject so as to treat, inhibit, prevent metastasis of and/or reduce severity of lung cancer in the subject.
• a composition comprising a cell comprising a nucleic acid encoding both a CD 19 specific chimeric antigen receptor and a PD- 1 specific checkpoint inhibitor (for example, anti-PD-l-scFv), or nucleic acids encoding a CD 19 specific CAR and a PD-1 specific checkpoint inhibitor, respectively, to the subject so as to treat, inhibit, prevent metastasis of and/or reduce severity of lung cancer in the subject.
• the methods further comprise administering the subject a therapeutically effective amount of existing therapies (existing therapeutic agents), wherein the existing therapies are administered sequentially or simultaneously with the compositions described herein.
• the cells (genetically modified cells) described herein may be used in a treatment regimen in combination with existing therapies including but not limited to surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoabiative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fiudarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.
• immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoabiative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fiudarabine,
• a CAR-expressing cell described herein can be used in combination with a chemotherapeutic agent.
• chemotherapeutic agents include an anthracyciine (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bor
• the precise amount of the compositions of the present invention to be administered 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).
• the therapeutically effective amount of the genetically modified cells is administered at a dosage of 10 4 to 10 9 cells/kg body weight in some instances 10 5 to I 0 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.
• the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
• the cells can be administered by injection into the site of the lesion (e.g., intra-tumoral injection).
• the CAR and CPI are introduced into immune effector ceils (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI of the invention, and one or more subsequent administrations of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI of the invention, 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.
• the subject e.g., human
• more than one administration of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI of the invention are administered per week.
• the subject e.g., human
• administrations of the immune effector cells e.g., T cells, NK cells
• the subject receives more than one administration of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI of the invention per week (e.g., 2, 3 or 4 administratio s per week) (also referred to herein as a cycle), followed by a week of no immune effector ceils (e.g., T cells, NK cells) administrations, and then one or more additional administration of the immune effector cells (e.g., T cells, NK ceils) comprising the CAR and CPI of the invention (e.g., more than one administration of the immune effector cells (e.g., T cells, NK cells) comprising the CAR and CPI, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
• the immune effector cells e.g., T cells, NK cells
• the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
• the immune effector cells e.g., T cells, NK cells
• the immune effector cells comprising the CAR and CPI are administered every other day for 3 administrations per week.
• the immune effector cells e.g., T cells, NK cells
• the immune effector cells comprising the CAR and CPI of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.
• the therapeutic methods described herein further comprise administering to the subject, sequentially or simultaneously, existing therapies.
• existing cancer treatment include, but are not limited to, active surveillance, observation, surgical intervention, chemotherapy, immunotherapy, radiation therapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and fractionated stereotactic radiotherapy (FSR)), focal therapy, systemic therapy, vaccine therapies, viral therapies, molecular targeted therapies, or combinations thereof.
• methods for preparing the genetically modified cells include obtaining a population of ceils and selecting ceils that express any one or more of CD3, CD28, CD4, CDS, CD45RA, and/or CD45RO.
• the population of immune effector cells provided are CD3+ and/or CD28+.
• the method for preparing the genetically modified ceils include obtaining a population of cells and enriching for the CD25+ T regulatory cells, for example by using antibodies specific to CD25, Methods for enriching CD25+ T regulator ⁇ ' cells from the population of cells will be apparent to a person of skill in the art.
• the Treg enriched cells comprise less than 30%, 20%, 10%, 5% or less non-Treg ceils.
• the vectors encoding the CARs and CPIs described herein are transfected into Treg-enriched cells. Treg enriched cells expressing a CAR and a CPI may be used to induced tolerance to antigen targeted by the CAR.
• the method further includes expanding the population of cells after the vector(s) comprising nucleic acid(s) encoding the CARs and CPIs described herein have been transfected into the cells.
• the population of cells is expanded in culture for 9 days under the same culture conditions.
• the population of cells is expanded in culture for 5 days show at least a one, two, three or four fold increase in cell doublings upon antigen stimulation as compared to the same ceils expanded in culture for 9 days under the same culture conditions.
• the population of cells is expanded in an appropriate media that includes one or more interleukins that result in at least a 200-fold, 250-fold, 300- fold, or 350-fold increase in cells over a 14 day expansion period, as measured by flow cytometry.
• the expanded cells comprise one or more CARs and one or more CPIs as descri bed herein.
• the present invention provides a pharmaceutical composition.
• the pharmaceutical composition includes a cell comprising nucleic acids encoding a CAR and a checkpoint inhibitor, as described herein.
• the pharmaceutical compositions according to the invention can contain any pharmaceutically acceptable excipient.
• “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
• excipients include but are not limited to starches, sugars, macrocrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.
• the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration.
• Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral.
• Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.
• the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
• the compositions are administered by injection. Methods for these administrations are known to one skilled in the art.
• compositions according to the invention can contain any pharmaceutically acceptable carrier.
• “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
• the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
• Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
• compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
• Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
• Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water.
• Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
• the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
• the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
• milling, mixing, granulation, and compressing when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
• the pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount.
• the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, phannacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
• formuiants may be added to the rAAV vector, the cell transfected with the rAAV vector, or the supernatant conditioned with the transfected ceil.
• a liquid formulation may be preferred.
• these formuiants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents or combinations thereof.
• Carbohydrate formuiants include sugar or sugar alcohols such as monosaccharides, di saccharides, or polysaccharides, or water soluble glucans.
• the saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcelluiose, or mixtures thereof.
• “Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an -OH group and includes galactitol, inositol, mannitoi, xyiitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. In one embodiment, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more betaine, however, other amino acids may be added,
• polymers as formulants include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.
• PVP polyvinylpyrrolidone
• PEG polyethylene glycol
• a buffer in the composition it is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution.
• Most any physiological buffer may be used including but not limited to citrate, phosphate, succinate, and glutamate buffers or mixtures thereof.
• the concentration is from 0.01 to 0,3 molar.
• Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268, 1 10.
• liposome Another drug delivery system for increasing circulatory half-life is the liposome.
• Methods of preparing liposome delivery systems are discussed in Gabizon et al ,, Cancer Research (1982) 42:4734; Cafiso, Biochem Biophys Acta (1981) 649: 129; and Szoka, Ann Rev Biophys Eng (1980) 9:467.
• Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al., DRUG DELIVERY SYSTEMS (R. L. Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, Pharm Revs (1984) 36:277.
• the liquid pharmaceutical composition may be lyophilized to prevent degradation and to preserve sterility.
• Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art.
• the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients.
• a sterile diluent Finger's solution, distilled water, or sterile saline, for example
• the composition is administered to subjects using those methods that are known to those skilled in the art.
• the present invention provides a kit for treating cancer comprising a composition that includes cells comprising nucleic acids encoding one or more CARs and one or more CPIs, as described herein.
• the kit is an assemblage of materials or components, including at least one of the inventive compositions (for example, genetically modified ceils comprising nucleic acids encoding one or more CARs and one or more CPIs, as described herein).
• inventive compositions for example, genetically modified ceils comprising nucleic acids encoding one or more CARs and one or more CPIs, as described herein.
• the kit is configured particularly for human subjects.
• the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.
• Instructions for use may be included in the kit, "Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to treat, reduce the severity of, inhibit cancer in a subject. Still in accordance with the present invention, "instructions for use” may include a tangible expression describing the preparation of the composition and/or at least one method parameter, such as the relative amounts of composition, dosage requirements and administration instructions, and the like, typically for an intended purpose.
• the kit also contains other useful components, such as, measuring tools, diluents, buffers, pharmaceutically acceptable carriers, syringes or other useful paraphernalia as will be readily recognized by those of skill in the art.
• the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
• the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures.
• the components are typically contained in suitable packaging material(s).
• packaging material refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like.
• the packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
• the term "package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
• a package can be a glass vial used to contain suitable quantities of a composition containing a volume of the AAV1-P0-ICE vector.
• the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
• mice Six- to eight-week-old female NOD Cu-P! kdc ⁇ li f ! .2I1 ⁇ 2 ! " , l 3 ⁇ 4j i .S/ (NSG) mice were purchased from Jackson Laboratory (Farmington, CT). All animal studies were performed in accordance with the Animal Care and Use Committee guidelines of the NIH and were conducted under protocols approved by the Animal Care and Use Committee of the NCI.
• Ceil lines SKOV3 and 293T were obtained from ATCC.
• the lung cancer line NCI-H292 was kindly provided by Dr. Ite Laird-Offringa (University of Southern California, Los Angeles, CA).
• the H292-CD19 and SKOV3-CD19 cell lines were generated by the transduction of parental NCI-H292 and S OV3 cells with a lenti viral vector encoding the cDNA of human CD 19.
• the transduced H292 and SKOV3 cells were stained with anti-human CD 19 antibody (BioLegend, San Diego, CA) and sorted to yield a relatively pure population of CD19-overexpressing ceils.
• SKOV3, SKOV3-CD19, NCI-H292, and H292-CD19 cells were maintained in R10 medium consisting of RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-giutamine, 10 mM HEPES, 100 U/ml penicillin and 100 g ml streptomycin.
• the 293T cells were cultured in D10 medium consisting of DMEM medium supplemented with 10% FBS, 2 mM L- glutamine, 10 mM HEPES, 100 U/ml penicillin and lOO ⁇ ig/ml streptomycin. All above cell culture media and supplements were purchased from Hy clone (Logan, UT).
• T cell medium which is composed of X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (GemCell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax (Gibco), and 0,2% N-Acetyl Cysteine (Sigma-Aldrich, St. Louis, MO).
• the secondary antibodies used were FITC- conjugated streptavidin (BioLegend, San Diego, CA) and goat anti-rabbit IgG-FDRP (Santa Cruz, San Jose, CA).
• the SuperSignal® West Femto Maximum Sensitivity Substrate used for Western blot analysis was from Thermo Fisher Scientific (Waltham, MA).
• the retroviral vector encoding anti-CD 19 CAR was constructed based on the MP71 retroviral vector kindly provided by Prof. Wolfgang IJckert, as described previously (Engeis B, et al. 2003. Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther 14: 1 155-68.
• the vector encoding anti-CD19 CAR with anti-PD-1 scFv was then generated based on the anti-CD 19 CAR.
• the insert for CAR.aPDl vector consisted of the following components in frame 5' end to 3' end: the anti-CD 19 CAR, an EcoRI site, a leader sequence derived from human IL-2, the anti-PD-1 scFv light chain variable region, a GS linker, the anti-PD-1 scFv heavy chain variable region, the HA-tag sequence, and a Not! site.
• the anti-PD-1 scFv portion in the CAR.aPDl vector was derived from the amino acid sequence of human monoclonal antibody 5C4 specific against human PD-1 (Alan J. Korman MS, Changyu Wang, Mark J. Selby, Bingliana Chen, Josephine M. Cardarelli. 2011. United States.
• the corresponding DNA sequence of the scFv was codon-optimized for its optimal expression in human cells using the online codon optimization tool and was synthesized by Integrated DNA Technologies (Coralvserver, LA).
• the anti-PD-1 scFv was then ligated into the CD 19 CAR vector via the EcoRI site through the Gibson assembly method.
• Retroviral vector production Retroviral vectors were prepared by transient transfection of 293T cells using a standard calcium phosphate precipitation protocol. 293T cells cultured in 15-cm tissue culture dishes were transfected with 37.5 ⁇ ig of the retroviral backbone plasmid, along with 18.75 ⁇ g of the envelope plasmid pGALV and 30 ⁇ g of the packaging plasmid encoding gag-pol. The viral supematants were harvested 48 h post- transfection and filtered through a 0.45 ⁇ filter (Corning, Corning, NY) before use,
• Activated PBMCs were resuspended at the concentration of 5 x 10 5 cells/ml with fresh TCM complemented with 10 ng/ml recombinant human IL-2 and added to the vector-loaded plates. The plates were spun at lOOOxg at 32°C for 10 minutes and incubated overnight at 37°C and 5% CC . The same transduction procedure was repeated on the following day. During ex vivo expansion, culture medium was replenished, and cell density was adjusted to 5 ⁇ lOVml every two days.
• T cells (1 x 10 6 ) were cultured with target cells at a ratio of 1 : 1 for 6 hours at 37°C and 5% CO? with GolgiPlug (BD Biosciences, San Jose, CA) in 96-well round bottom plates.
• PE-Cy5.5-anti-CD3, FITC-anti-CD4, Pacific blue-CD8, PE-anti-IFN- ⁇ and PE-anti-Ki67 antibodies were used for the intracellular staining.
• Cytofix/Cytoperm Fixation and Permeabilization Kit (BD Biosciences) was used to permeabilize the cell membrane and perform intracellular staining according to the manufacturer's instruction.
• ELISA IFN- ⁇ was measured using a human IFN- ⁇ ELISA kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. Briefly, 96-well ELISA plates (Thermo Scientific, Waltham, MA) were coated with 20()ng/well of capture antibodies against the indicated proteins at 4°C overnight. On the next day, plates were washed with wash buffer (PBS containing 0.05% Tween 20) and blocked with assay buffer (PBS containing 10% FBS) for 2 hours at room temperature. Equal volume of serum, or ceil culture supernatant was added to the plate and incubated for 2 hours at room temperature. Plates were then washed and incubated with detection antibodies for 1 hour at room temperature.
• wash buffer PBS containing 0.05% Tween 20
• assay buffer PBS containing 10% FBS
• recombinant human PD-1 (rhPD-1) was used to pre-coat the plate.
• Goat anti-mouse IgGl-HRP and anti-HA tag antibodies were used as detection antibodies, respectively.
• the assay plate was incubated for 4 hours at 37°C, Human T cells were harvested, washed once and then resuspended to 1 ⁇ 10° cells/ml in TCM. The wells of the assay plate were aspirated . Then, 100 ⁇ of human T-cell suspension (1 ⁇ 10 3 ) and 100 ⁇ of supernatant of CAR or CAR.aPDl T cell culture 3-day post-transduction, supplemented with GolgiPlug (BD Biosciences), were added to each well. The plate was covered and incubated at 37°C and 5% CO?, overnight. After incubation, T cells were harvested and stained with IFN- ⁇ intracellular! ⁇ '.
• H292-CD 19 cells were labeled by suspending them in PBS+0.1 % BSA with 5 ⁇ Carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye at a concentration of 1 ⁇ 10 6 cells/mL. The cells were incubated for 30 minutes at 37°C. After incubation, the same volume of FBS was added into the cell suspension and then incubated for 2 minutes at room temperature. The cells were then washed twice and suspended in fresh RI O medium. Equal amounts of NCI-H292 and H292-CD19 ceils (5 ⁇ 10 4 each) were combined in the same well for each culture with effector CAR-T cells.
• CFSE Carboxyfluorescein succinimidyl ester
• Cocultures were set up in round bottom 96-well plates in triplicate at the following effector-to-target ratios: 1 : 1 and 5: 1. The cultures were incubated for 4 hours at 37°C, followed by 7-AAD labeling, according to the manufacturer's instructions (BD Biosciences). Flow cytometric analysis was performed to quantify remaining live (7-AAD-negative) target cells. For each coculture, the percent survival of H292-CD19 cells was determined by dividing the percentage of live H292-CD19 cells by the percentage of live NCI-H292 cells.
• the ratio of the percentage of H292-CD19 ceils to the percentage of NCI-H292 cells was calculated and used to correct the variation in the starting cell numbers and spontaneous cell death.
• the cytotoxicity was determined in triplicate and presented in mean ⁇ SEM.
• CAR expression was normalized to 20% in both CAR groups by addition of donor-matched nontransduced T cells.
• Tumor growth was monitored twice a week. Tumor size was measured by calipers and calculated by the following formula: W 2 x L / 2. Mice were euthanized when they displayed obvious weight loss, ulceration of tumors, or tumor size larger than 1000 mmA
• FIG. 1A The schematic representation of the retroviral vector constructs used in this study is shown in Fig, 1A.
• the retroviral vector encoding both anti-CD19 CAR and secreting anti-PD-1 scFv was designated as CAR19.
• PDl Human PBMCs were transduced with each construct to test the expression of CAR in primary lymphocytes.
• CAR19.aPDl T cells secreted significantly higher IFN- ⁇ compared to the parental CAR19 T cells after stimulation with H292-CD19 cells (Fig. 2A).
• CAR19 T cells secreting anti-PD-1 expressed significantly more IFN- ⁇ than that expressed by the parental CAR! 9 T cells (Fig, 2 A and Fig. 9A).
• cytolytic function of engineered T cells was examined by a 6-hour cytotoxicity assay.
• the cytotoxic activity of CAR19 and CAR19.aPDl T cells against H292- CD19 cells was evaluated at effector/target (E/T) ratios of 1, 5, 10 and 20.
• E/T effector/target
• little difference was found between CAR 19 and CAR19.aPD i T cells in terms of cytolytic activity (Fig.
• PD-1 expression on human GD2 and mouse HER2 CAR T ceils has been shown to increase following antigen-specific activation, and PD-1 blockade was found to downreguiate PD-1 expression in T cells.
• the engineered CAR T cells were cocultured with either H292-CD 19 or SKOV3-CD19 target cells for 24 hours and then stained for the T cell exhaustion marker PD-1.
• lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain-limiting the antitumor efficacy of CAR-T cell therapy.
• LAG-3 lymphocyte activation gene 3 protein
• T cell immunoglobulin domain T cell immunoglobulin domain
• mucin domain mucin domain-limiting the antitumor efficacy of CAR-T cell therapy.
• CAR19.aPDl T cells expressed slightly lower LAG-3 and TIM-3 after stimulation with H292-CD19 cells. Moreover, upon SKOV3-CD19 stimulation, CAR19.aPDl T cells had significantly lower LAG-3 expression than CAR19 T cells, whereas they had similar TIM-3 expression ( Figures 3C, 3D, 12A, 13A-13C). In comparison, without antigen-specific stimulation, LAG-3 in CAR19 and CAJR.19.aPDl T ceils was expressed at a similar level and remained stable over the course of T cell expansion ( Figures 13D and 13E).
• PD-1 blockade could promote the survival of GD2 CAR T cells after activation with the PD-L1 -negative target ceils, indicating that the interaction between PD-1 -expressing T ceils and T cells expressing PD-1 ligands, such as PD-L1, might contribute to the suppression of T cell function (Garget ⁇ T, et al 2016. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade. Molecular Therapy 24; 1135-49).
• Anti-PD-1 engineered CAR T cells exhibit enhanced antitumor reactivity
• Anti-PD-1 engineered CAR T ceils can expand more in vivo tha parental CAM T ceils
• T ceils in all groups had barely expanded and that less than 2% of T cells could be observed in all examined tissues.
• Anti-PD-1 engineered CAR T cells lead to reversal of T cell exhaustion and higher T cell effector junction at the established tumor site
• mice were challenged with H292-CD19 tumors before receiving 3 x iO 6 CAR T ceils.
• the experimental design is shown in Fig. 6A. Eight days after T cell infusion, we euthanized the mice and analyzed T cells in tumor, blood, spleen and bone marrow, using flow cytometry. Compared to the CAR T cell treatment, we observed that the injected anti- CA 19.oPDl regimen expanded at a higher rate in tumor, blood, and spleen (Fig.
• Adoptive T cell therapy has become a promising method of immunotherapy. It has achieved successful responses in patients with hematopoietic malignancies. However, the outcome has been less promising in the treatment of solid tumors, partly owing to the immunosuppressive properties and establishment of an immunosuppressive microenvironment.
• the PD-1/PD-L1 regulatory pathway has demonstrated particularly antagonistic effects on the antitumor response of TILs. Solid tumors with poor prognosis showed upregulation of PD-L1 expression, while TILs were shown to have PD-1 result, the ensuing research was designed to investigate the effects of PD-1/PD-L1 blockade in infused CAR T cells, which showed upregulation of PD-1 after activation.
• CAR19.aPDl T cells when compared to parental CAR19 T cells, further enhanced antitumor activity and prolonged overall survival. Mechanistically, we observed that CAR19.aPDl T cells had greater in vivo expansion. In addition, at the local tumor site, CARJ 9.aPDl T cells were shown to be less exhausted and more functional than parental CAR19 T cells.
• the PD-l/PD-Ll pathway involves the regulation of cytokine production by T cells, inhibiting production of IFN- ⁇ , TNF-a and IL-2.
• PD-1 expression of human GD2 and anti-HER2 CAR T cells has been shown to increase following antigen-specific activation, and PD-1 blockade has been shown to enhance T cell effector function and increase the production of IFN- ⁇ in the presence of PD-L1 + target cells. Therefore, in this study, to compare the functional capacity of CAR!
• CAR 19 T cells and CAR19.aPDl T cells to PD-Ll + target cells and examined the expression of T ceil exhaustion markers, including PD-1, LAG-3 and TTM-3.
• T ceil exhaustion markers including PD-1, LAG-3 and TTM-3.
• the decreased expression of PD-1 in CAR19.aPDl T cells may be caused by the dual effects of antibody blockade and downregulation of PD-1 surface expression.
• PD-1 upregulation on tumor-infiltrating T cells was reported to be a major contributor to T cell exhaustion in high PD-Ll -expressing tumors.
• Downregulation of PD-1 may contribute to reversion of T ceil exhaustion and enhanced T cell effector function, which is supported by CAR19.aPDl T cells upon antigen stimulation.
• CAR19.aPDl T cells upon antigen stimulation Our observation is consistent with a recent study, demonstrating that co-expression of multiple inhibitory receptors is a cardinal feature of T cell exhaustion.
• PD-L1 expression was significantly increased on CAR T cells with antigen-specific stimulation, which may also contribute to T cell exhaustion through T cell - T cell interaction.
• the anti-PD-1 systemic toxicity may be required to achieve substantial antitumor efficacy.
• both administered and self- secreting anti-PD-1 antibodies efficiently decreased and blocked the PD-1 expression in CD8 + T cells in vivo
• systemically injected anti-PD-1 antibody had little effect on increasing the population of cytolytic CD8 + TILs or enhancing IFN- ⁇ production of TILs upon ex vivo stimulation.
• the injected antibody has little effect on augmenting infused T cell function at the present dose. It also explains our observed failure of injected PD-1 blockade in enhancing the antitumor activity of CAR T cell therapy.
• the anti-PD-1 secreted by CAR T ceils may provide a safer and more potent approach in blocking PD-1 signaling and enhancing the functional capacity of CAR T cells,
• CAR19.oPDl T cells exhibited alleviated T cell exhaustion, enhanced T ceil expansion, and improved CAR T ceil treatment of human solid tumors in a xenograft mouse model .
• anti-PD-1 - engineered CAR T cells might be more powerful in inducing tumor eradication given the durable effect of PD-1 blockade on modulating the tumor microenvironment.

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