Chimeric antigen receptors for treating myeloid malignancies

Abstract

To disclose compositions and methods for treating acute myeloid leukemia (AML) in subjects, in particular, disclose chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to treat AML, and also disclose immune effector cells, such as T cells or Natural Killer (NK) cells, that are engineered to express these CARs.SOLUTION: There are disclosed methods of treating AML in a subject that involves adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CARs.SELECTED DRAWING: Figure 1A-1E

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 888,072, filed Aug. 16, 2019, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted in electronic format as an ASCII.txt file named "320803 - 2410_ST25", created on Aug. 12, 2020. The contents of the sequence listing are hereby incorporated by reference in their entirety.

Background Art

[0003] Acute myeloid leukemia (AML) is a type of blood cancer in which the bone marrow makes abnormal myeloblasts. AML accounts for nearly one - third of all new leukemia cases each year. The American Cancer Society estimates that in 2017, 21,380 patients will develop AML and 10,590 AML patients will die.

[0004] The standard treatment for AML has not changed much over the past 40 years. Intensive chemotherapy followed by hematopoietic stem cell transplantation remains the most effective treatment. However, most newly diagnosed elderly patients are ineligible for intensive chemotherapy, and there is no effective second - line treatment for patients with relapsed / refractory disease. As a result, the 5 - year overall survival rate is 27%, and less than 10% for patients over 60 years old. Approximately 40 - 60% of hematopoietic stem cell transplant recipients develop graft - versus - host disease (GVHD). 30% of GVHD cases are fatal.

Summary of the Invention

Problems to be Solved by the Invention

[0005] According to long-term data from the Center for International Blood and Marrow Transplant Research (CIBMTR), more than 1,000 patients undergo allogeneic HCT for high-risk AML each year (Gupta, V. et al., Blood 117:2307-2318 (2011)). Even when patients can tolerate myeloablative preconditioning therapy, relapse-free survival is limited to 67.8%, compared to 47.3% after reduced-intensity pre-transplant therapy (Scott B.L. et al., J Clin Oncol 35:1154-1161 (2017)). Therefore, strategies to prevent AML relapse are urgently needed.

Means for Solving the Problems

[0006] Chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer for treating myeloid malignancies are disclosed. The disclosed CAR polypeptides contain, in an extracellular domain, an anti-CD83 binder that can bind to CD83-expressing cells. Immune effector cells genetically modified to express the disclosed CAR polypeptides are also disclosed. Also disclosed is a method of treating myeloid malignancy in a subject, the method comprising administering to the subject an effective amount of immune effector cells genetically modified with the disclosed CD83-specific CAR.

[0007] Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells. These are the result of genetic and epigenetic changes that disrupt important processes such as self-renewal, proliferation, and differentiation. These include chronic and acute stages, such as myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). In some embodiments, the subject has AML. In some embodiments, the subject has Hodgkin lymphoma.

[0008] Relapse remains an important cause of post-transplant failure and death, yet allogeneic HCT is often necessary to treat high-risk AML. Unlike classical GVL mediated by HLA, CD83 CAR T cells selectively destroy CD83-expressing malignant cells. Thus, the disclosed CD83 CAR T cells can be effective in treating myeloid malignancies independently of allogeneic HCT. In some embodiments, the subject is being treated with a hematopoietic stem cell transplant. In other embodiments, the subject is not being treated with a hematopoietic stem cell transplant. In some embodiments, the subject is not eligible for allogeneic HCT.

[0009] The anti-CD83 binding substance is, in some embodiments, an antibody fragment that specifically binds to CD83. For example, the antigen-binding domain can be a Fab or single-chain variable fragment (scFv) of an antibody that specifically binds to CD83. The anti-CD83 binding substance is, in some embodiments, an aptamer that specifically binds to CD83. For example, the anti-CD83 binding substance can be a peptide aptamer selected from a random sequence pool based on its CD83-binding ability. The anti-CD83 binding substance can also be a natural ligand of CD83 or its variants and / or fragments that is capable of binding to CD83.

[0010] In some embodiments, the anti-CD83 scFv comprises a variable heavy chain (V H ) domain having CDR1, CDR2, and CDR3 sequences and a variable light chain (V L ) domain having CDR1, CDR2, and CDR3 sequences.

[0011] For example, in some embodiments, the CDR1 sequence of the V H domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 1), SDGIS (SEQ ID NO: 7), or SNAMI (SEQ ID NO: 13); the CDR2 sequence of the V H domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 2), IISSGGNTYYASWAKG (SEQ ID NO: 8), or AMDSNSRTYYATWAKG (SEQ ID NO: 14); the V HThe CDR3 sequence of the domain contains the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 3), VVGGTYSI (SEQ ID NO: 9), or GDGGSSDYTEM (SEQ ID NO: 15); V L The CDR1 sequence of V contains the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 4), QSSQSVYNNDFLS (SEQ ID NO: 10), or QSSQSVYGNNELS (SEQ ID NO: 16); V L The CDR2 sequence of the domain contains the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 5), YASTLAS (SEQ ID NO: 11), or QASSLAS (SEQ ID NO: 17); and V L The CDR3 sequence of the domain contains the amino acid sequence GSSDSSGYV (SEQ ID NO: 6), TGTYGNSAWYEDA (SEQ ID NO: 12), or LGEYSISADNH (SEQ ID NO: 18).

[0012] For example, in some embodiments, V H The CDR1 sequence of the domain contains the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 1), V H The CDR2 sequence of the domain contains the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 2), V H The CDR3 sequence of the domain contains the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 3), V L The CDR1 sequence of V contains the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 4), V L The CDR2 sequence of the domain contains the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 5), and V L The CDR3 sequence of the domain contains the amino acid sequence GSSDSSGYV (SEQ ID NO: 6).

[0013] For example, in some embodiments, V H The CDR1 sequence of the domain contains the amino acid sequence SDGIS (SEQ ID NO: 7), V H The CDR2 sequence of the domain contains the amino acid sequence IISSGGNTYYASWAKG (SEQ ID NO: 8), V H The CDR3 sequence of the domain contains the amino acid sequence VVGGTYSI (SEQ ID NO: 9), V LThe CDR1 sequence of V contains the amino acid sequence QSSQS VYNNDFLS (SEQ ID NO: 10), and V L The CDR2 sequence of the V domain contains the amino acid sequence YASTLAS (SEQ ID NO: 11), and V L The CDR3 sequence of the V domain contains the amino acid sequence TGTYGNSAWYEDA (SEQ ID NO: 12).

[0014] For example, in some embodiments, V H The CDR1 sequence of the V domain contains the amino acid sequence SNAMI (SEQ ID NO: 13), and V H The CDR2 sequence of the V domain contains the amino acid sequence AMDSNSRTYYATWAKG (SEQ ID NO: 14), and V H The CDR3 sequence of the V domain contains the amino acid sequence GDGGSSDYTEM (SEQ ID NO: 15), and V L The CDR1 sequence of V contains the amino acid sequence QSSQSVYGNNELS (SEQ ID NO: 16), and V L The CDR2 sequence of the V domain contains the amino acid sequence QASSLAS (SEQ ID NO: 17), and V L The CDR3 sequence of the V domain contains the amino acid sequence LGEYSISADNH (SEQ ID NO: 18).

[0015] In some embodiments, the anti-CD83 scFv V H domain has the amino acid sequence:

Chemical formula

[0016] In some embodiments, the anti-CD83 scFv V L domain has the amino acid sequence: QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 20, VL-GBM00).

[0017] In some embodiments, the anti-CD83 scFv V HThe domain has the amino acid sequence:

Chem.

[0018] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence:

Chem.

[0019] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence:

Chem.

[0020] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence:

Chem.

[0021] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence:

Chem.

[0022] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence:

Chem.

[0023] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence: [Chemical formula] includes.

[0024] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence: [Chemical formula] includes.

[0025] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence: [Chemical formula] includes.

[0026] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence: [Chemical formula] includes.

[0027] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence: [Chemical formula] includes.

[0028] In some embodiments, the anti-CD83scFv V L domain has the amino acid sequence: [Chemical formula] includes.

[0029] In some embodiments, the anti-CD83scFv V H domain has the amino acid sequence: [Chemical formula] includes.

[0030] In some embodiments, the anti-CD83 scFv V L domain has the amino acid sequence:

Chemical formula

[0031] In some embodiments, the anti-CD83 scFv V H domain has the amino acid sequence:

Chemical formula

[0032] In some embodiments, the anti-CD83 scFv V L domain has the amino acid sequence: LTQPPPASGTPGQQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYYGNDQRPSGVPDRFSASKSGTSASLAISGLQSEDEAHYYCAAWDGSLNGGVIFGGGTKVTLG (SEQ ID NO: 36).

[0033] In some embodiments, the anti-CD83 scFv V L domain has the amino acid sequence: VTQPPSASGTPGQRVTISCSGSSSNIGTNPVNWYQQLPGTAPKLLIYTTDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLSGLYVFGTGTKVTVLG (SEQ ID NO: 37).

[0034] In some embodiments, the anti-CD83 scFv VL domain has the amino acid sequence: MTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQRPGQSPQPLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVQAEDVGVYYCMQSLQLWTFGQGTKVEIKR (SEQ ID NO: 38).

[0035] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLIHSDGNTYLDWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLRISRVEAEDIGVYYCMQATHWPRTFGQGTKVEIKR (SEQ ID NO: 39).

[0036] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVDSAGNTFLHWFHQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 40).

[0037] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: LTQSPLSLPVTLGQPASISCKSSQSLVDSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 41).

[0038] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVHSDGNMYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQATQPTWTFGQGTKLEIKR (SEQ ID NO: 42).

[0039] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSATYYCQQTYQGTKLEIKR (SEQ ID NO: 43).

[0040] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQSPSSLSASVGHPVTITCRASQSLISYLNWYHQKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLQPENFASYYCQHTDSFPRTFGHGTKVEIKR (SEQ ID NO: 44).

[0041] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: LTQPPSASGTPGQGVTISCRGSTSNIGNNVVNWYQHVPGSAPKLLIWSNIQRPSGIPDRFSGSKSGTSASLAISGLQSEDQAVYYCAVWDDGLAGWVFGGGTTVTVLS (SEQ ID NO: 45).

[0042] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: MTQAPVVSVALEQTVRITCQGDSLAIYYDFWYQHKPGQAPVLVIYGKNNRPSGIPHRFSGSSSNTDSLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTNLTVLG (SEQ ID NO: 46).

[0043] In some embodiments, the anti-CD83scFv V L domain comprises the amino acid sequence: LTQSPLSLPVTLGQPASISCKSNQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYCMQGTQWPRTFGGQGTKLDIKR (SEQ ID NO: 47).

[0044] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical Formula

[0045] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical formula

[0046] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical formula

[0047] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical formula

[0048] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical formula

[0049] In some embodiments, the anti-CD83scFv V H domain is humanized and has the amino acid sequence:

Chemical formula

[0050] In some embodiments, the anti-CD83scFv V LThe domain is humanized and contains the amino acid sequence: QLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 54, VL-GBM01).

[0051] In some embodiments, the anti-CD83 scFv V L The domain is humanized and contains the amino acid sequence: LPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 55, VL-GBM02).

[0052] The heavy and light chains are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker contains the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56).

[0053] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chemical Structure

[0054] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chemical Structure

[0055] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chemical Structure

[0056] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0057] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0058] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0059] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0060] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0061] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0062] In some embodiments, the anti-CD83 scFv has the amino acid sequence: [Chemical formula] and includes.

[0063] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chem.

[0064] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chem.

[0065] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chem.

[0066] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chem.

[0067] In some embodiments, the anti-CD83 scFv has the amino acid sequence:

Chem.

[0068] When using other CARs, the disclosed polypeptides may also contain a transmembrane domain and an intracellular domain capable of activating immune effector cells. For example, the intracellular domain may contain a signaling domain and one or more co-stimulatory signaling regions.

[0069] In some embodiments, the intracellular signaling domain is the CD3 zeta (CD3ζ) signaling domain. In some embodiments, the co-stimulatory signaling region comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof. In some examples, the co-stimulatory signaling region contains the cytoplasmic domain of 1, 2, 3, or 4 of one or more intracellular signaling and / or co-stimulatory molecules. In some embodiments, the co-stimulatory signaling region contains one or more mutations in the cytoplasmic domain of CD28 and / or 4-1BB that enhance signaling.

[0070] In some embodiments, the CAR polypeptide contains an incomplete internal domain. For example, the CAR polypeptide may contain only an intracellular signaling domain or a co-stimulatory domain, but not both. In these embodiments, the immune effector cell is not activated unless both its CAR polypeptide and a second CAR polypeptide (or endogenous T cell receptor) containing the missing domain both bind to their respective antigens. Thus, in some embodiments, the CAR polypeptide contains the CD3 zeta (CD3ζ) signaling domain but does not contain a co-stimulatory signaling region (CSR). In other embodiments, the CAR polypeptide contains the cytoplasmic domain of CD28, 4-1BB, or a combination thereof, but does not contain the CD3 zeta (CD3ζ) signaling domain (SD).

[0071] Also disclosed are isolated nucleic acid sequences encoding the disclosed CAR polypeptides, vectors containing these isolated nucleic acids, and cells containing these vectors. For example, the cell can be an immune effector cell selected from the group consisting of alpha-beta T cells, gamma-delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, and regulatory T cells.

[0072] Details of one or more embodiments of the present invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will be apparent from the description and drawings and from the claims.

Brief Description of the Drawings

[0073]

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Modes for Carrying Out the Invention

[0074] Before describing the present disclosure in further detail, it is to be understood that the present disclosure is not limited to the specific embodiments described, and as a matter of course, can itself vary. Since the scope of the present disclosure is limited only by the appended claims, it is also to be understood that the terms used herein are for the purpose of describing specific embodiments only and are not limiting.

[0075] When a range of values is provided, unless otherwise clearly indicated from the context, each value intermediate between the upper and lower limits of that range, to one tenth of the unit of the lower limit, and any other stated value or intermediate value in the stated range is to be understood as being included in the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the present disclosure, subject to any specifically excluded limitations in the stated range. The stated range includes one or both of the limits, and ranges excluding either or both of these included limits are also included in the present disclosure.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

[0077] All publications and patents cited in this application are hereby incorporated by reference herein, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and to disclose and describe the methods and / or materials related to the subject matter for which the publication is cited. Any citation of a publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present disclosure has the right to antedate such publication by virtue of prior disclosure. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently confirmed.

[0078] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and characteristics that can be readily separated from or combined with any of the characteristics of some other embodiments without departing from the scope or spirit of the disclosure. Any of the recited methods can be performed in the order of the recited events or any other order that is theoretically possible.

[0079] Embodiments of the disclosure use techniques such as chemistry, biology, which are within the skill in the art, unless otherwise indicated.

[0080] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to carry out the method and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, temperature is in °C, and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0081] Before describing embodiments of the disclosure in detail, it is to be understood that the disclosure is not limited to specific materials, reagents, reaction materials, manufacturing processes, etc., unless otherwise indicated, and can vary per se. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not limiting. In the disclosure, it is also possible for steps to be performed in different orders that are theoretically possible.

[0082] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0083] Chimeric antigen receptors (CARs) targeting CD83 in antigen-presenting cells are disclosed herein. Also disclosed are immune effector cells such as T cells or natural killer (NK) cells that are modified to express these CARs. CAR T cells expressing these CARs can suppress alloreactive donor cells such as T cells. Accordingly, disclosed is a method for preventing GVHD in a subject, the method involving adoptive transfer of the disclosed immune effector cells that are modified to express the disclosed CD83-specific CAR.

[0084] CD83-specific chimeric antigen receptor (CAR) CARs generally incorporate an antigen recognition domain from a single-chain variable fragment (scFv) of a monoclonal antibody (mAb) that has a transmembrane signaling motif involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45). Disclosed herein is a CD83-specific chimeric antigen receptor (CAR) that can be expressed in immune effector cells to suppress alloreactive donor cells.

[0085] The disclosed CARs generally consist of three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains a CD83-binding region and is involved in antigen recognition. Optionally, this also contains a signal peptide (SP), allowing the CAR to be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain (TD), as its name indicates, links the extracellular domain to the intracellular domain and is present within the cell membrane when expressed by the cell. The intracellular domain is the decisive part of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. For example, the intracellular domain may contain an intracellular signaling domain (ISD) and optionally a co-stimulatory signaling region (CSR).

[0086] The "signal transduction domain (SD)" generally contains an immunoreceptor tyrosine-based activation motif (ITAM) that activates a signal transduction cascade when phosphorylated. The term "costimulatory signal transduction region (CSR)" refers to the intracellular signal transduction domains from costimulatory protein receptors such as CD28, 41BB, and ICOS that can promote T cell activation by the T cell receptor.

[0087] In some embodiments, the internal domain contains either an SD or a CSR, but not both. In these embodiments, the immune effector cells containing the disclosed CAR are only activated when another CAR (or T cell receptor) containing the missing domain also binds to its individual antigen.

[0088] In some embodiments, the disclosed CAR has the formula: SP-CD83-HG-TM-CSR-SD; or SP-CD83-HG-TM-SD-CSR (wherein "SP" represents an optional signal peptide, "CD83" represents a CD83 binding region, "HG" represents an optional hinge domain, "TM" represents a transmembrane domain, "CSR" represents one or more costimulatory signal transduction regions, "SD" represents a signal transduction domain, "-" represents a peptide bond or linker) and is defined by.

[0089] Further CAR constructs are described, for example, in Fresnak AD, et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81, which is incorporated by reference in its entirety with respect to the teachings of these CAR models.

[0090] For example, the CAR can be a TRUCK, a universal CAR, a self-driving CAR, an armored CAR, a self-destructing CAR, a conditional CAR, a markt CAR, a TenCAR, a dual CAR, or an sCAR.

[0091] CAR T cells modified to be resistant to immunosuppression (armored CARs) can be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)) by an immune checkpoint switch receptor, or can be administered with monoclonal antibodies that block immune checkpoint signaling.

[0092] Self-destructing CARs can be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of T cells can be achieved based on the binding of ganciclovir to thymidine kinase in genetically modified lymphocytes or by more recently described systems of activation of human caspase 9 by small molecule dimerizers.

[0093] Conditional CAR T cells are non-responsive by default or are switched "off" until the addition of a small molecule that allows for complete transduction of both signal 1 and signal 2, thereby completing the circuit that activates the CAR T cells. Alternatively, the T cells can be modified to express an adapter-specific receptor that is affinity for a secondary antibody directed against the target antigen, which is administered subsequently.

[0094] Tandem CAR (TanCAR) T cells express a single CAR consisting of two linked single-chain variable fragments (scFvs) with different affinities that are fused to an intracellular co-stimulatory domain and a CD3ζ domain. TanCAR T cell activation is achieved only when the target cell co-expresses both targets.

[0095] Dual CAR T cells express two individual CARs with different ligand-binding targets; one CAR contains only the CD3ζ domain and the other CAR contains only the co-stimulatory domain. Dual CAR T cell activation requires co-expression of both targets.

[0096] Safety CAR (sCAR) consists of an extracellular scFv fused to a cytoplasmic inhibitory domain. sCAR T cells co-expressing a standard CAR will only be activated when they encounter target cells that retain the standard CAR target but lack the sCAR target.

[0097] The antigen recognition domain of the disclosed CARs is typically an scFv. However, there are many alternatives. Native T cell receptor (TCR) alpha and beta single chains have been described as having more novel recognition components such as simple external domains (e.g., the CD4 external domain for recognizing HIV-infected cells) and linked cytokines (leading to recognition of cells having cytokine receptors). In fact, almost anything that binds to a certain target with high affinity can be used as the antigen recognition region.

[0098] The internal domain is a crucial part of the CAR that transmits signals to immune effector cells after antigen recognition and activates at least one of the normal effector functions of immune effector cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity including cytokine secretion. Thus, the internal domain can include the "intracellular signaling domains" of the T cell receptor (TCR) and optionally co-receptors. Usually, the entire intracellular signaling domain can be used, while in many cases, it is not necessary to use the entire chain. To the extent that shortened portions of the intracellular signaling domain are used, such shortened portions can be used in place of the intact chain as long as they transmit effector function signals.

[0099] The cytoplasmic signaling sequences that regulate the primary activation of the TCR complex acting in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (FcγRIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0100] In certain embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3ζ) (TCR zeta, GenBank accno. BAG36664.1). The T cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as the T cell receptor T3 zeta chain or CD247 (surface antigen classification 247), is a protein encoded by the CD247 gene in humans.

[0101] First-generation CARs generally had an intracellular domain from the CD3ζ chain, which is the primary transducer of signals from the endogenous TCR. Second-generation CARs add intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the internal domain of the CAR to provide additional signals to the T cells. More recently, third-generation CARs combine multiple signaling domains to further enhance potency. T cells transplanted with these CARs have shown improved proliferation, activation, persistence, and tumor eradication effects independent of co-stimulatory receptor / ligand interactions (Imai C, et al. Leukemia 2004 18:676 - 84; Maher J, et al. Nat Biotechnol 2002 20:70 - 5).

[0102] For example, the intracellular domain of a CAR can be designed to include a CD3ζ signaling domain, either alone or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of a CAR can include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a part of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD123, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Thus, while a CAR is exemplified mainly with CD28 as a co-stimulatory signaling element, other co-stimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0103] In some embodiments, the CAR includes a hinge sequence. A hinge sequence is a short sequence of amino acids that promotes the flexibility of an antibody (see, for example, Woof et al., Nat. Rev. Immunol., 4(2):89-99 (2004)). The hinge sequence can be located between the antigen recognition portion (e.g., anti-CD83 scFv) and the transmembrane domain. The hinge sequence can be from any suitable molecule or any suitable sequence derived therefrom. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0104] The transmembrane domain can be derived from either natural or synthetic origin. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be from a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR and the alpha, beta or zeta chains of PAG / Cbp (i.e., may include at least its transmembrane region). Alternatively, the transmembrane domain can be synthesized, in which case it mainly contains hydrophobic residues such as leucine and valine. In some examples, a triplet of phenylalanine, tryptophan and valine can be seen at each end of the synthetic transmembrane domain. Short oligos or polypeptide linkers, such as 2-10 amino acids in length, can form a bond between the transmembrane domain and the endoplasmic domain of the CAR.

[0105] In some embodiments, the CAR has two or more transmembrane domains, which can be repeats of the same transmembrane domain or different transmembrane domains.

[0106] In some embodiments, the CAR is a multi-chain CAR as described in International Publication No. WO 2015 / 039523, which is incorporated herein by reference. The multi-chain CAR can include individual extracellular ligand-binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble at a juxtamembrane position and form a flexible structure closer to the native receptor that confers optimal signaling. For example, the multi-chain CAR can include a portion of the FCERI alpha chain and a portion of the FCERI beta chain such that the FCERI chains naturally dimerize together to form the CAR.

[0107] Tables 1, 2, and 3 below provide some example combinations of CD83 binding regions, costimulatory signaling regions, and intracellular signaling domains that can occur with the disclosed CARs.

[0108] [Table 1]

[0109] [Table 2]

[0110] [Table 3]

[0111] [Table 4]

[0112] [Table 5]

[0113]

Table 6

[0114]

Table 7

[0115]

Table 8

[0116]

Table 9

[0117]

Table 10

[0118]

Table 11

[0119]

Table 12

[0120]

Table 13

[0121]

Table 14

[0122]

Table 15

[0123]

Table 16

[0124]

Table 17

[0125]

Table 18

[0126]

Table 19

[0127]

Table 20

[0128]

Table 21

[0129]

Table 22

[0130]

Table 23

[0131]

Table 24

[0132]

Table 25

[0133]

Table 26

[0134]

Table 27

[0135]

Table 28

[0136]

Table 29

[0137]

Table 30

[0138]

Table 31

[0139]

Table 32

[0140]

Table 33

[0141]

Table 34

[0142]

Table 35

[0143]

Table 36

[0144]

Table 37

[0145]

Table 38

[0146]

Table 39

[0147]

Table 40

[0148]

Table 41

[0149]

Table 42

[0150]

Table 43

[0151]

Table 44

[0152]

Table 45

[0153]

Table 46

[0154]

Table 47

[0155]

Table 48

[0156]

Table 49

[0157]

Table 50

[0158]

Table 51

[0159]

Table 52

[0160]

Table 53

[0161]

Table 54

[0162]

Table 55

[0163]

Table 56

[0164]

Table 57

[0165]

Table 58

[0166]

Table 59

[0167]

Table 60

[0168]

Table 61

[0169]

Table 62

[0170]

Table 63

[0171]

Table 64

[0172]

Table 65

[0173]

Table 66

[0174]

Table 67

[0175]

Table 68

[0176]

Table 69

[0177]

Table 70

[0178]

Table 71

[0179]

Table 72

[0180]

Table 73

[0181]

Table 74

[0182]

Table 75

[0183]

Table 76

[0184]

Table 77

[0185]

Table 78

[0186]

Table 79

[0187]

Table 80

[0188]

Table 81

[0189]

Table 82

[0190]

Table 83

[0191]

Table 84

[0192]

Table 85

[0193]

Table 86

[0194]

Table 87

[0195]

Table 88

[0196]

Table 89

[0197]

Table 90

[0198]

Table 91

[0199]

Table 92

[0200] In some embodiments, the anti-CD83 binding substance is a single-chain variable fragment (scFv) antibody. The affinity / specificity of the anti-CD83 scFv is largely determined by specific sequences within the complementarity-determining regions (CDRs) in the heavy (V H ) and light (V L ) chains. Each V H and V L sequence has three CDRs (CDR1, CDR2, CDR3).

[0201] In some embodiments, the anti-CD83 binding substance is derived from a natural antibody such as a monoclonal antibody. In some examples, the antibody is human. In some examples, the antibody has been modified to reduce its immunogenicity when administered to a human. For example, the modification includes one or more techniques selected from the group consisting of humanization, chimerization, CDR grafting, immunogenicity reduction, and mutagenesis of framework amino acids to correspond to the closest human germline sequence.

[0202] Bispecific CARs targeting CD83 and at least one additional antigen are also disclosed. CARs designed to function only in combination with another CAR that binds a different antigen are also disclosed. For example, in these embodiments, the intracellular domain of the disclosed CARs may contain only a signaling domain (SD) or a costimulatory signaling region (CSR), but not both. The second CAR (or endogenous T cell) provides the missing signal when activated. For example, if the disclosed CAR contains an SD but not a CSR, the immune effector cell containing this CAR is activated only when another CAR (or T cell) containing a CSR binds its respective antigen. Similarly, if the disclosed CAR contains a CSR but not an SD, the immune effector cell containing this CAR is activated only when another CAR (or T cell) containing an SD binds its respective antigen.

[0203] Nucleic acids and vectors Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed CD83 - specific CARs that enable the expression of the CD83 - specific CARs in the disclosed immune effector cells.

[0204] The nucleic acid sequences and regions encoding the disclosed CARs can be obtained using recombinant methods known in the art, such as, for example, screening a library from cells that express the gene, deriving the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be made synthetically rather than by cloning.

[0205] Expression of the nucleic acid encoding the CAR is generally achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for the control of expression of the desired nucleic acid sequence.

[0206] The disclosed nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0207] Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vector is a lentiviral or retroviral vector.

[0208] For gene transfer into mammalian cells, many virus-based systems have been developed. For example, retroviruses provide a convenient platform for gene delivery systems. The gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the target cells either in vivo or ex vivo.

[0209] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to human gene promoters such as the simian virus 40 (SV40) early promoter, the MND (myeloproliferative sarcoma virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include but are not limited to the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0210] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Generally, these are located 30 to 110 bp upstream of the start site region, but many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, and promoter function is maintained when the elements are inverted or moved relative to each other.

[0211] To evaluate the expression of a CAR polypeptide or a part thereof, the expression vector to be introduced into cells may contain either or both of a selectable marker gene and / or a reporter gene to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected through a viral vector. In other embodiments, the selectable marker may be done as an individual piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be adjacent to appropriate control sequences to enable their expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0212] Reporter genes are used to identify cells that may have been transfected and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue and whose expression is manifested by some easily detectable property, such as enzyme activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with a minimal 5' flanking region that shows the maximum level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate substances for their ability to drive promoter-mediated transcription.

[0213] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be transfected into the host cell by physical, chemical, or biological means.

[0214] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0215] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors and especially retroviral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells.

[0216] Chemical means for introducing polynucleotides into host cells include lipid-based systems, such as colloidal dispersion systems, for example, macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. A representative colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0217] When a non-viral delivery system is utilized, a representative delivery vehicle is a liposome. In another aspect, a nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, linked to the liposome via a linking molecule that associates with both the liposome and the oligonucleotide, encapsulated within the liposome, complexed with the liposome, dispersed within a lipid-containing solution, mixed with the lipid, combined with the lipid, contained as a suspension within the lipid, contained with micelles or micelle-complexed, or otherwise associated with the lipid. The lipid, lipid / DNA or lipid / expression vector association compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure with a micelle or with a "collapsed" structure. They can simply form aggregates that are optionally non-uniform in size or shape and are also dispersed in solution. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include classes of compounds containing fat droplets that occur naturally in the cytoplasm as well as long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar lipids, Inc., (Birmingham, Ala.).

[0218] Immune effector cells Also disclosed are immune effector cells (also referred to herein as "CAR-T cells") that are modified to express the disclosed CARs. These cells are preferably obtained from the subject to be treated (i.e., autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. In other embodiments, the immune effector cells are not HLA compatible. Immune effector cells can be obtained from many sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusions, spleen tissue, and tumors. Immune effector cells can be obtained from blood recovered from a subject using any of a number of techniques known to those of skill in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. Specific subpopulations of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies to surface markers specific for positive selection cells, for example, by incubating with antibody-conjugated beads for a time sufficient for positive selection of the desired immune effector cells. Alternatively, negative selection can be used to enrich the immune effector cell population using a combination of antibodies to surface markers specific for negative selection cells.

[0219] In some embodiments, immune effector cells include any white blood cells involved in defending the body from infectious diseases and foreign substances. For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells can include T lymphocytes.

[0220] T cells, or T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer (NK) cells, by the presence of T cell receptors (TCRs) on their cell surface. Since they mature in the thymus, they are called T cells (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0221] Helper T cells (T H cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). When activated, they rapidly divide and secrete small proteins called cytokines that regulate or promote an active immune response. These cells can differentiate into one of several subtypes, including T H 1, T H 2, T H 3, T H 17, T H 9 or T FH and secrete different cytokines to promote different types of immune responses.

[0222] Cytotoxic T cells (T C cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8 + T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into anergic states, thereby preventing autoimmune diseases.

[0223] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has resolved. Upon re-exposure to their cognate antigen, they rapidly expand into large numbers of effector T cells, thus providing an immune system with "memory" of past infections. Memory cells can be either CD4 + or CD8 + . Memory T cells generally express the cell surface protein CD45RO.

[0224] Regulatory T cells (T reg cells), also previously known as suppressor T cells, are important for maintaining immune tolerance. Their major roles are to dampen T cell-mediated immunity towards the end of an immune response and to suppress self-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4 + T reg cells - natural T reg cells and adaptive T reg cells are described.

[0225] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive and innate immune systems. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0226] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some instances, the T comprises cytotoxic CD8 + T lymphocytes. In some embodiments, the T cells comprise γδ T cells, which have a distinct T cell receptor (TCR) with one γ chain and one δ chain instead of the α and β chains.

[0227] Natural killer (NK) cells can constitute an important subset of cells of the innate immune system that kill virus-infected and transformed cells and are CD56 + CD3 ーThey are large granular lymphocytes (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Cytotoxic CD8 + Unlike cytotoxic CD8 T lymphocytes, NK cells can initiate cytotoxicity against tumor cells without prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and off-target, off-tumor effects.

[0228] Therapy The immune effector cells expressing the disclosed CARs suppress alloreactive donor cells, such as T cells, and prevent GVHD. Thus, the disclosed CARs can be administered to any subject at risk of GVHD. In some embodiments, the subject undergoes a bone marrow transplant, and the disclosed CAR-modified immune effector cells suppress the alloreactivity of donor T cells or dendritic cells.

[0229] The disclosed CAR-modified immune effector cells can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-15, or other cytokines or cell populations.

[0230] In some embodiments, the disclosed CAR-modified immune effector cells are administered in combination with ER stress blockade (the IRE-1 / XBP-1 pathway (e.g., compounds for targeting B-I09)). In some embodiments, the disclosed CAR-modified immune effector cells are administered in combination with a JAK2 inhibitor, a STAT3 inhibitor, an aurora kinase inhibitor, an mTOR inhibitor, or any combination thereof.

[0231] Briefly stated, the pharmaceutical composition may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are, in some embodiments, formulated for intravenous administration. The pharmaceutical composition can be administered in any manner appropriate for the treatment of MM. Although appropriate dosages can be determined by clinical trials, the amount and frequency of administration are determined by factors such as the condition of the patient and the severity of the patient's disease.

[0232] When a "therapeutically effective amount" is indicated, the exact amount of the composition of the invention to be administered can be determined by the physician taking into account individual differences in age, body weight, extent of transplantation and condition of the patient (subject). The pharmaceutical composition comprising T cells described herein can be administered at a dosage of from 10 4 to 10 9 cells / kg body weight, for example from 10 5 to 10 6 cells / kg body weight (including all integer values within these ranges). The T cell composition can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment schedule for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and appropriately adjusting the treatment.

[0233] In certain embodiments, it may be desirable to administer activated T cells to a subject and then subsequently re-collect blood (or perform apheresis), activate T cells therefrom according to the disclosed methods, and re-infuse these activated and expanded T cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw / multiple re-infusion protocol can serve to select a particular population of T cells.

[0234] Administration of the disclosed compositions can be performed in any convenient manner, including by injection, transfusion, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intra-articularly, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions can also be injected directly into the site of transplantation.

[0235] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) any of a number of suitable therapeutic modalities including, but not limited to, thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immune-depleting agents such as CAM PATH, anti-CD3 antibodies or other antibody therapeutic agents, cytokines, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. In some embodiments, CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) any of bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), T cell depletion therapy using any of cyclophosphamide or antibodies such as OKT3 or Campath (CAMPATH). In another embodiment, the cell composition of the invention is administered after B cell depletion therapy with an agent that reacts with CD20, such as Rituxan. For example, in some embodiments, a subject may receive standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In further embodiments, cells expanded before or after surgery are administered.

[0236] A major concern associated with CAR-T cells as a form of "biological therapy drugs" is their operability in vivo and the potential for their immune-stimulating side effects. To better control CAR-T therapy and prevent unwanted side effects, various properties have been modified, including off-switches, safety mechanisms, and conditional control mechanisms. Both self-destruct and mark / tagged CAR-T cells are modified, for example, to have an "off-switch" that promotes the clearance of CAR-expressing T cells. Self-destruct CAR-T contains the CAR but is also modified to express an apoptosis-promoting suicide gene or "exclusion gene" inducibility upon administration of an exogenous molecule. Various suicide genes can be used for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC TAG, and truncated EGFR (endothelial growth factor receptor). HSK, for example, converts the prodrug ganciclovir (GCV) to GCV-triphosphate, which incorporates itself into replicating DNA and ultimately leads to cell death. iCasp9 is a chimeric protein containing a component of the FK506-binding protein that binds to the small molecule AP1903, leading to caspase 9 dimerization and apoptosis. However, mark / tagged CAR-T cells retain the CAR but are also modified to express a selectable marker. Administration of an mAb against this selectable marker promotes the clearance of CAR-T cells. Truncated EGFR is one such targetable antigen by an anti-EGFR mAb, and administration of cetuximab plays a role in promoting the elimination of CAR-T cells. CARs engineered to have these properties are also called sCAR for "switchable CAR" and RCAR for "regulatable CAR". "Safety CAR", also known as "inhibitory CAR" (iCAR), is modified to express two antigen-binding domains. One of these extracellular domains is for the first antigen and binds to the intracellular co-stimulatory and stimulatory domains. However, the second extracellular antigen-binding domain is specific for normal tissue and binds to an intracellular checkpoint domain such as CTLA4, PD1, or CD45.It is also possible to incorporate multiple intracellular inhibitory domains into the iCAR. Some inhibitory molecules that can provide these inhibitory domains include B7-H1, B7-1, CD160, PIH, 2B4, CEACAM (CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1 and TGFβ-R. In the presence of normal tissue, stimulation of this second antigen-binding domain serves to inhibit the CAR. It should be noted that due to this dual antigen specificity, the iCAR is also in the form of a bispecific CAR-T cell. The safety CAR-T modification promotes the specificity of CAR-T cells for tissues and is advantageous in situations where certain normal tissues may express very low levels of antigens that lead to off-target effects with standard CARs (Morgan 2010). Conditional CAR-T cells express an extracellular antigen-binding domain linked to an intracellular co-stimulatory domain and an individual intracellular co-stimulatory molecule. Upon administration of an exogenous molecule, the resulting proteins come together intracellularly to modify the co-stimulation and stimulation domain sequences to complete the CAR circuit. In this way, CAR-T activation can be regulated and, in some cases, "fine-tuned" or individualized for a particular patient. Similar to the dual CAR design, in the case of a conditional CAR that is inactive, the stimulation and co-stimulation domains are physically separated; for this reason, they are also referred to as "split CARs".

[0237] Generally, CAR-T cells are produced using α-β T cells, but γ-δ T cells can also be used. In some embodiments, the described CAR constructs, domains, and modified properties used to produce CAR-T cells can be similarly used in the production of other types of CAR-expressing immune cells, including NK (natural killer) cells, B cells, mast cells, bone marrow-derived macrophages, and NKT cells. Alternatively, CAR-expressing cells can be produced to have the properties of both T cells and NK cells. In further embodiments, transduction with the CAR can be autologous or allogeneic.

[0238] Several different methods for CAR expression can be used, including retroviral transduction (including γ-retroviruses), lentiviral transduction, transposons / transposases (Sleeping Beauty and PiggyBac systems), and messenger RNA-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) has similarly been increasing in importance with respect to the potential for modification of CAR-T cells. The CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator-like effector nuclease) systems are three potential ways by which CAR-T cells can be generated.

[0239] Definitions The term "amino acid sequence" refers to a list of abbreviations, letters, words, or phrases representing amino acid residues. As used herein, "amino acid abbreviation" is the conventional one-letter code for amino acids and is represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0240] The term "antibody" refers to immunoglobulins, their derivatives that maintain specific binding ability, and proteins having binding domains that are homologous or nearly homologous to immunoglobulin binding domains. These proteins can be derived from natural sources or can be produced, in part or completely, synthetically. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In representative embodiments, the antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of these immunoglobulin molecules and human or humanized versions of immunoglobulin molecules that selectively bind to a target antigen.

[0241] The term "antibody fragment" refers to any derivative of an antibody that is less than full length. In representative embodiments, an antibody fragment retains at least a substantial portion of the specific binding ability of the full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. Antibody fragments can be made by any means. For example, an antibody fragment can be made enzymatically or chemically by fragmentation of an intact antibody, recombinantly from a gene encoding a partial antibody sequence, or made synthetically, either wholly or in part. An antibody fragment can optionally be a single-chain antibody fragment. Alternatively, the fragment can include multiple chains linked together, for example, by disulfide bonds. The fragment can optionally also be a complex of multiple molecules. Functional antibody fragments generally include at least about 50 amino acids, more generally at least about 200 amino acids.

[0242] The term "antigen-binding site" refers to the region of an antibody that specifically binds to an epitope on an antigen.

[0243] The term "aptamer" refers to an oligonucleic acid or peptide molecule that binds to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamer can adapt to a unique three-dimensional structure and recognize the target molecule with high affinity and specificity. A "nucleic acid aptamer" is a DNA or RNA oligonucleic acid that binds to a target molecule through its structure, thereby inhibiting or suppressing the function of such a molecule. The nucleic acid aptamer can be composed of DNA, RNA, or a combination thereof. A "peptide aptamer" is a combinatorial protein molecule in which a variable peptide sequence is inserted into a constant scaffold protein. The identification of peptide aptamers is generally performed under stringent yeast two-hybrid conditions, which increases the likelihood that the selected peptide aptamer will be stably expressed and correctly folded in the intracellular context.

[0244] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other property of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and any harmful side effects in the subject.

[0245] The term "chimeric molecule" refers to a single molecule created by linking two or more molecules that exist individually in their native states. A single chimeric molecule has all of the desired functionality of its constituent molecules. One type of chimeric molecule is a fusion protein.

[0246] The term "modified antibody" refers to a recombinant molecule that includes at least one antibody fragment that contains an antigen-binding site derived from the variable domain of the heavy and / or light chain of an antibody, and optionally includes all or part of the variable and / or constant domain of an antibody from any Ig class (e.g., IgA, IgD, IgE, IgG, IgM, and IgY).

[0247] The term "epitope" refers to the region of an antigen to which an antibody preferentially and specifically binds. A monoclonal antibody preferentially binds to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0248] The term "fusion protein" refers to a polypeptide formed by the ligation of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. A fusion protein can be formed by chemical coupling of the constituent peptides, or it can be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single-chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Conventional techniques in molecular biology are used to ligate two genes in-frame into a single nucleic acid, and then the nucleic acid is expressed in a suitable host cell under conditions in which the fusion protein is produced, to prepare the fusion protein.

[0249] The term "Fab fragment" refers to a fragment of an antibody containing an antigen-binding site that results from cleavage of an antibody with the enzyme papain, which cleaves at the N-terminus of the hinge region with respect to the inter-H-chain disulfide bond, generating two Fab fragments from one antibody molecule.

[0250] The term "F(ab’)2 fragment" refers to a fragment of an antibody containing two antigen-binding sites that results from cleavage of an antibody molecule with the enzyme pepsin, which cleaves at the C-terminus of the hinge region with respect to the inter-H-chain disulfide bond.

[0251] The term "Fc fragment" refers to a fragment of an antibody that includes the constant domain of its heavy chain.

[0252] The term "Fv fragment" refers to a fragment of an antibody that includes the variable domains of its heavy and light chains.

[0253] A "gene construct" refers to a nucleic acid, such as a vector, plasmid, viral genome, etc. that contains a "coding sequence" for a polypeptide or is otherwise transcribable into a biologically active RNA (such as antisense, decoy, ribozyme, etc.), which can be transfected into a cell, for example, a mammalian cell in certain embodiments, and can cause the expression of the coding sequence in the transfected cell. The gene construct may include one or more regulatory elements operably linked to the coding sequence as well as intron sequences, polyadenylation sites, origins of replication, marker genes, and the like.

[0254] The term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the sequences being compared is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of nucleotides that are identical or match at positions shared by those nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate identity between two sequences, including, for example, FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to a specific polypeptide described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When using BLASTP, percent similarity is based on the BLASTP positives score and percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" indicates the number and percentage of total residues in sequence pairs showing high scores of identity; BLASTP "Positives" indicates the number and percentage of residues that have positive alignment scores and are similar to each other. Amino acid sequences having these degrees of identity or similarity, or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein, are contemplated and encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides can be deduced using the genetic code and obtained by conventional means, particularly by reverse translating their amino acid sequences using the genetic code.

[0255] The term "linker" is recognized in the art and refers to a molecule or group of molecules that connects two compounds such as two polypeptides. A linker can be composed of a single linking molecule or can include linking molecules and spacer molecules that are intended to separate the linking molecule and the compound by a specific distance.

[0256] The term "multivalent antibody" refers to an antibody or modified antibody that contains multiple antigen recognition sites. For example, a "bivalent" antibody has two antigen recognition sites, while a "tetravalent" antibody has four antigen recognition sites. The terms "monospecific", "bispecific", "trispecific", "tetraspecific", etc. refer to the number of different antigen recognition site specificities present in a multivalent antibody (as opposed to the number of antigen recognition sites). For example, all of the antigen recognition sites of a "monospecific" antibody bind to the same epitope. A "bispecific" antibody has at least one antigen recognition site that binds to a first epitope and at least one antigen recognition site that binds to a second epitope different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind to the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, some of which bind to a first epitope and some of which bind to a second epitope different from the first epitope.

[0257] The term "nucleic acid" refers to a natural or synthetic molecule that includes a single nucleotide or two or more nucleotides linked by a phosphate group at the 3' end of one nucleotide to the 5' end of another nucleotide. Nucleic acids are not limited by length and thus can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0258] The term "operably linked" refers to the functional relationship of nucleic acids with another nucleic acid sequence. Promoters, enhancers, transcription and translation stop sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, an operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0259] The terms "peptide", "protein", and "polypeptide" are used interchangeably to refer to natural or synthetic molecules containing two or more amino acids in which the carboxyl group of one amino acid is linked to the alpha amino group of another amino acid.

[0260] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0261] The term "polypeptide fragment" or "fragment", when used with respect to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but the remaining amino acid sequence is usually identical to the reference polypeptide. Such deletions can occur at the amino terminus or carboxy terminus of the reference polypeptide or alternatively at both. Fragments are generally at least about 5, 6, 8, or 10 amino acids in length, at least about 14 amino acids in length, at least about 20, 30, 40, or 50 amino acids in length, at least about 75 amino acids in length or at least about 100, 150, 200, 300, 500 or more amino acids in length. Fragments can retain one or more of the biological activities of the reference polypeptide. In various embodiments, the fragment can include the enzymatic activity and / or interaction sites of the reference polypeptide. In another embodiment, the fragment can have immunogenic properties.

[0262] The term "protein domain" refers to a portion of a protein, several portions of a protein, or the entire protein that exhibits structural integrity; this determination can be based on the amino acid composition of a portion of the protein, several portions of the protein, or the entire protein.

[0263] The term "single-chain variable fragment or scFv" refers to an Fv fragment in which the heavy-chain domain and the light-chain domain are linked. One or more scFv fragments can be linked to other antibody fragments (such as the constant domains of the heavy or light chains) to form an antibody construct having one or more antigen recognition sites.

[0264] "Spacer", as used herein, refers to a peptide that links proteins, including fusion proteins. Generally, a spacer has no specific biological activity other than linking proteins or preserving some minimal distance or other spatial relationship between them. However, the constituent amino acids of a spacer can be selected to affect some properties of the molecule, such as the folding of the molecule, the net charge, or the hydrophobicity.

[0265] The term "specifically binds", as used herein, when referring to a polypeptide (including an antibody) or a receptor, refers to a binding reaction that is a determining factor for the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological agents. Thus, if a specified ligand or antibody binds only a small amount to other proteins present in a sample or to other proteins that the ligand or antibody can contact in an organism, under the specified conditions (e.g., the immunassay conditions in the case of an antibody), this specified ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule binds to that second molecule with an affinity of about 10 5 M ー1 (e.g., 10 6 M ー1 、10 7 M ー1 、10 8 M ー1 、10 9 Mー1 and 10 10 M ー1 and 10 11 M ー1 and 10 12 M ー1 and have an affinity constant (Ka) greater than that of 10 M or more.

[0266] The term "specifically deliver" as used herein refers to the preferential association of a molecule with a cell or tissue bearing a specific target molecule or marker and does not refer to a cell or tissue lacking that target molecule. Of course, it is recognized that non-specific interactions between the molecule and non-target cells or tissues can occur to a certain degree. Nevertheless, specific delivery can be distinguished as being mediated through specific recognition of the target molecule. Generally, as a result of specific delivery, a much stronger association occurs between the delivery molecule and the cells bearing the target molecule than between the delivery molecule and the cells lacking the target molecule.

[0267] The term "subject" refers to any individual that is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, such as a physician.

[0268] The term "therapeutically effective" refers to the amount of a composition used that is sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement only requires alleviation or change and does not necessarily require elimination.

[0269] The terms "transformation" and "gene transfer" mean the introduction of a nucleic acid, such as an expression vector, into a recipient cell, including the introduction of the nucleic acid into the chromosomal DNA of the recipient cell.

[0270] The term "treatment" refers to the medical management of a patient with the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. This term includes active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition or disorder, and causal treatment, i.e., treatment directed to the elimination of the cause of an associated disease, pathological condition or disorder. Furthermore, this term includes symptomatic treatment, i.e., treatment planned for the relief of symptoms rather than the cure of a disease, pathological condition or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the progression of an associated disease, pathological condition or disorder; and supportive treatment, i.e., treatment used in addition to another specific treatment aimed at improving an associated disease, pathological condition or disorder.

[0271] The term "variant" refers to an amino acid or peptide sequence having a substitution within the wobble site of each codon (i.e., DNA and RNA) encoding a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence, which includes a conservative amino acid substitution, a non-conservative amino acid substitution (i.e., a degenerate variant), an amino acid added to the C-terminus of an amino acid or peptide, or a peptide.

[0272] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which a vector sequence is ligated. The term "expression vector" includes any vector (e.g., plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., ligated to transcriptional control elements).

[0273] Numerous embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Example

[0274] Example 1: CD83 chimeric antigen receptor T cells prevent GVHD and suppress myeloid leukemia Materials and Methods Study Plan: This is a preclinical trial for the design, production, and efficacy of human CD83 CAR T cells for GVHD prevention. The first part of this trial describes the in vitro activity of CD83 CAR T cells with respect to the CAR construct and phenotype, cytokine production, on-target killing, and proliferation in response to CD83+ targets. Next, the immunosuppressive effect of CD83 CAR T cells in vitro using a standard allo-MLR is clarified. Furthermore, CD83 expression was measured among human T cells showing different expression of CD83 on Tconv vs Treg cells. In a human T cell-mediated xenogeneic GVHD model (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)), the preclinical efficacy of CD83 CAR in GVHD prevention was revealed. This includes a thorough evaluation of in vivo target killing of CD83+ dendritic cells and Tconv. The effect of CD83 CAR T cells in various T cell subsets in vivo is also shown. CD83 was found to be expressed on human malignant myeloid cell lines, and these human malignant myeloid cell lines were effectively killed by CD83 CAR T cells using the xCELLigence RTCA (real-time cell analysis) system (Li G. et al., JCI Insight 3 (2018)). For the GVHD experiments, a human pre-mortem endpoint was used. Mice were monitored frequently for the GVHD clinical score. GVHD histopathology was evaluated and scored blindly by a specialized pathologist (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci USA., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). In vivo data in mice were pooled from at least two independent experiments with 6-9 mice per experimental group.

[0275] CD83 CAR T cell construct and production: The CD83 CAR was synthesized and cloned into an SFG retroviral construct by GENEWIZ (Li, G. et al., Methods Mol Biol 1514:111 - 118 (2017); Li G. et al., JCI Insight 3 (2018)). Next, the CD83 SFG cloning construct was transfected into H29 cells using calcium phosphate, and RD114 was transduced using the retroviral supernatant from the transfected H29 cells. The retroviral supernatant of RD114 cells was filtered through a 0.45 μm filter (MilliporeSigma) to purify the gammaretrovirus. Specifically, CD83 CAR T cells were generated by transducing human T cells as described (Li G. et al., JCI Insight 3 (2018)). Briefly, leukocytes obtained from apheresis from healthy human donors (All Cells) were isolated by density gradient centrifugation. T cells were isolated using magnetic beads (Stem Cells Inc.) and stimulated with human Dynabeads CD3 and CD28 (Thermo fisher) in RPMI with recombinant human IL - 2. CD83 gammaretrovirus was used to transduce activated T cells on RetroNectin (TaKaRa Bio Inc.) - coated plates. Seven to eight days after activation, the CD83 CAR T cells were bead - depleted. The transfection or transduction efficiency was estimated by GFP+ cells when detected by flow cytometry.

[0276] Monoclonal antibodies and flow cytometry: Fluorescent dye-conjugated mouse anti-human monoclonal antibodies included anti-CD3, CD4, CD8, CD25, CD83, CD1c, CD127, MHCII, Foxp3, Ki-67, IFN-γ, IL-17A, and IL-4 (BD Biosciences, San Jose, CA, USA; eBioscience, San Jose, CA, USA; Cell Signaling Technology, Boston, MA, USA). To determine viability, LIVE / DEAD Fixable Yellow or Aqua Dead Cell Stain (Life Technologies, Grand Island, NY) was used. Viable events were captured on a BD FACSCanto II or LSRII flow cytometer (FlowJo software, ver. 7.6.4; TreeStar, Ashland, OR, USA).

[0277] Cytokine immunoassay: CD83 CAR and mock-transduced T cells (1 × 10 5 cells) were co-cultured with CD83+ moDCs (1 × 10 4 cells) for 24 hours. Supernatants were collected and analyzed using a human luminex assay kit (R&D Systems) on a Luminex 100 system (Luminex) and a Simple Plex Assay Kit (Biotechne) on an Ella instrument (Biotechne) according to the manufacturer's instructions (Li G. et al., JCI Insight 3 (2018)).

[0278] Cytotoxicity and in vitro proliferation of human CD83 CAR T cells: In E-Plate 96, normalized CD83 CAR T cells (1 × 10 5Individual cells) were cultured in a 10:1 effector-to-target (ET) ratio in pairs with CD83+ moDC, K562, or Thp-1 cells. A cytotoxicity assay was performed on an xCELLigence RTCA (real-time cell analysis) instrument (ACEA Biosciences) according to the manufacturer's instructions. Similarly, human CD83 CAR T cells were co-cultured with moDC at a 1:1 ET ratio in a non-tissue culture-treated 6-well plate in triplicate. Cells were grown in human T cell complete medium supplemented with 60 IU / mL IL-2. Trypan blue staining was performed, and cell viability and total cell numbers in each well were measured on a cell counter (Bio-Rad) on days +1, +7, and +14.

[0279] In vitro allogeneic MLR: As described, human monocyte-derived dendritic cells (moDC) were generated, differentiated, and matured with cytokines (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)). T cell preparations (10 5 individuals) purified from leukocyte concentrates (OneBlood or Memorial Blood Center) were cultured with allogeneic moDC (T cell:DC ratio 30:1) (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci USA., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). CD83 CAR, CD19 CAR, or mock-transduced T cells (autologous to the T cell donor) were added to the allogeneic MLR at a range of CAR-to-DC ratios. T cell proliferation was measured by Ki-67 expression after 5 days.

[0280] Time-course changes in CD83 expression: Purified human T cells were stimulated with either allogeneic moDCs (T cell:DC ratio of 30:1) or CD3 / CD28 beads (T cell:bead ratio of 30:1). T cells were harvested from triplicate wells in 96-well plates at 4, 8, 24, and 48 hours of culture. T cells were stained for CD3, CD4, CD127, CD25, and CD83 and then fixed. CD83 expression was evaluated in activated Tconv (CD3+, CD4+, CD127+, CD25+) (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)), Treg (CD3+, CD4+, CD127-, CD25+) (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)), and CD8 T cells (CD3+, CD4-). As designated, CD83 CAR or mock T cells were cultured with PBMCs stimulated with allogeneic DCs, and CD83 expression was evaluated between CD3- and CD3+ target cells over 48 hours.

[0281] Colony-forming units: CD34+ cells isolated from normal human bone marrow were purchased from AllCells. 10 3 cells were co-cultured with either CAR T cells transduced with the CD83 virus, mock T cells, or medium alone. Cells were incubated at a 10:1 E:T ratio for 4 hours. After incubation, cells were seeded in MethoCult medium (StemCell) in 6-well SmartDish plates (StemCell) according to the manufacturer's instructions and cultured for 14 days. At the end of the culture period, colonies were imaged, analyzed, and counted using STEMvision software.

[0282] Xenogeneic GVHD model: NOD scid gamma (NSG) mice (male or female, 6 - 24 weeks old) were housed in an IACU-C approved colony maintained at the Moffitt / USF vivarium. On day 0 of transplantation, 25×10 6Fresh human PBMC (OneBlood) was administered once. As specified, mice were given either PBMC alone or PBMC + CD83 CAR T cells (low dose: 1×10 6 cells or high dose: 10×10 6 cells) or PBMC + mock-transduced T cells (10×10 6 cells). Each independent experiment was performed using different human PBMC donors, and the CAR T cells and mock-transduced T cells were derived from the PBMC donor. Mice were monitored for GVHD clinical score and pre-terminal status. When specified, short-term experiments were completed on day +21 via humane euthanasia to evaluate the content of blinded GVHD target organ pathology, tissue-resident lymphocytes, and human DC and T cell subsets in the mouse spleen (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci USA., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). As previously described, tissue samples were prepared, stained (Ventana Medical Systems), and imaged (Vista) to identify human Ki67+ T cells (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)). These mice were transplanted with PBMC (25×10 6 cells) with or without CD83 CAR (1×10 6 cells) or mock-transduced T cells (1×10 6 cells). All vertebrate experiments were conducted under an AICUC-approved protocol.

[0283] Statistical analysis: Data were reported as mean ± SEM. Correction was performed for multiple comparisons, and ANOVA was used for group comparisons, including Dunnett's or Sidak's post hoc tests. Mann-Whitney was used for all others. For comparison of survival curves, the log-rank test was used. Statistical analysis was performed using Prism software version 5.04 (GraphPad). Statistical significance was defined as two-sided P < 0.05 (two-sided).

[0284] Results Schematic of the human CD83 CAR construct: The anti-CD83 single-chain variable fragment (scFv) was paired against the CD8 hinge and transmembrane domain, followed by the intracellular 41BB co-stimulatory domain and the CD3ζ activation domain (Figure 1A). To facilitate tracking of CAR T cells, the construct contains an eGFP tag that can be used to identify CAR T cells among normal non-CAR T cells (Figure 1A). CD83-targeted CAR T cells were transduced with retrovirus and generated as described by the inventors (Figure 1A) (Li, G. et al., Methods Mol Biol 1514:111-118 (2017); Li G. et al., JCI Insight 3 (2018)).

[0285] Characterization of human CD83 CAR T cells: The CD83 CAR construct showed high transduction efficiency, with over 60% of T cells expressing eGFP (Figure 1B). CD4 expression was similar between both groups, while a significant decrease in CD8 expression was observed among CD83 CAR T cells compared to mock-transduced T cells (Figure 1C). However, CD83 CAR T cells showed robust IFNγ and IL-2 production when cultured with CD83+ target cells; cytokine-matured human monocyte-derived DCs (moDCs), etc. (Figure 1D, E). Furthermore, CD83 CAR T cells showed a potent killing effect on CD83+ moDCs and proliferation compared to mock-transduced T cells and in contrast to CD83+ moDCs (Figure 1F, 1G). The target moDCs in these experiments were syngeneic to the T cells, and thus, lysis and proliferation by mock-transduced T cells corresponded to baseline alloreactivity (Figure 1F, 1G).

[0286] Human CD83 CAR T cells reduce alloreactivity: To test whether human CD83 CAR T cells reduce alloreactivity in vitro, their inhibitory function in the mixed lymphocyte reaction (alloMLR) was examined. CD83 and mock-transduced CAR T cells were generated from healthy donor, human T cells. CD19 CAR T cells, which target B cells and inappropriate cell types in the alloMLR, were also used as an additional control. Furthermore, CD19 and CD83 CAR T cells were similar in that both received costimulation via 41BB. Autologous T cells (1×10 5 cells) and allogeneic cytokine-matured CD83 + moDCs (3.33×10 3 cells) were added to a 5-day alloMLR. The CAR T cell:moDC ratio ranged from 3:1 to 1:10. CD83 CAR T cells strongly reduced alloreactive T cell proliferation (Figure 2, upper panel). Conversely, mock-transduced and CD19-targeted CAR T cells had no inhibitory effect on alloreactive T cells (Figure 2, middle and lower panels).

[0287] CD83 is differentially expressed in activated human Tconv compared to Treg: CD83 is an established marker of human dendritic cell maturation and is also expressed on activated human B cells (Szabolcs P. et al., Blood 87:4520-4530 (1996); Krzyzak L. et al., J Immunol 196:3581-3594 (2016)). Using a CD83 reporter mouse system, it has previously been shown that activated mouse T cells also express CD83 (Lechmann, M. et al., Proc Natl Acad Sci USA 105:11887-11892 (2008)). It is known that CD83 is expressed on human T cells after stimulation and is detectable on circulating T cells from patients with acute GVHD (Ju X. et al., J Immunol 197:4613-4625 (2016)). However, the detailed expression of CD83 on CD4+ Treg vs CD4+ Tconv or CD8+ T cells is unknown. Experimental confirmation has shown that human T cell expression of CD83 occurs with stimulation including allogeneic dendritic cells or CD3 / CD28 beads (Figures 3A, 3B). Importantly, it has been revealed that CD83 is differentially expressed on human CD4+ Tconv (CD127+, CD25+) compared to immunosuppressive CD4+ Treg (CD127-, CD25+) or cytolytic CD8+ T cells in response to DC-allogeneic activation (Figure 3A). CD4+ Tconv expression of CD83 peaks at 4-8 hours of DC-allogeneic stimulation and declines to baseline levels by 48 hours, with minimal amounts observed on Treg or CD8+ T cells (Figure 3A). Expression of CD83 is greater with supra-physiological CD3 / CD28 bead stimulation, also resulting in a delayed increase in CD83 expression on Treg and CD8+ T cells by 48 hours from activation (Figure 3B). Considering that CD83 expression is shared between pro-inflammatory, mature DC and alloreactive Tconv, it was investigated whether CD83 CAR T cells could deplete any target cells in culture. Human CD83 CAR or mock T cells were cultured with autologous peripheral blood mononuclear cells (PBMC) stimulated by allogeneic moDC, and the amount of CD83+ target cells was evaluated at 4, 8, 24, and 48 hours of culture.The inventors observed similar spikes in CD83 expression by CD3- and CD3+ target cells over 8 hours (Figure 3C). However, CD83 CAR T cells essentially eliminated CD83+ target cells by 48 hours of culture, well below their baseline amounts by 8 hours after culture (Figure 3C). Furthermore, CD83-T cells remained present in all experimental groups (Figure 3C), confirming that T cells were not indiscriminately destroyed. Next, the expression of CD83 on eGFP+ CAR T cells over 48 hours was evaluated. CD83 expression on CAR T cells was moderate, and an increase in the percentage of eGFP+ CAR T cells was still observed by 48 hours of culture (Figure 3D), providing evidence that CD83 CAR T cells are clearly not subject to fratricide mediated by CD83. To parallel clinical practice, the functional ability of CD83 CAR T cells in the presence of clinically relevant doses of tacrolimus (5 - 10 ng / mL) was tested. Interestingly, CD83 CAR T cells can still kill and proliferate in response to CD83+ target cells despite exposure to tacrolimus (Figures 9A, 9B).

[0288] Human CD83-targeted CAR T cells prevent allo-GVHD: To evaluate the efficacy of human CD83 CAR T cells in vivo, an allo-GVHD model was used. Using an established NSG mouse model (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)), 25×10 6 human PBMC + 1 - 10×10 6All recipients were inoculated on day 0 with either individual autologous CD83 or mock-transduced CAR T cells. For clinical signs of allogeneic GVHD, transplanted mice were monitored daily until day +100. In NSG mice injected with CD83 or mock-transduced CAR T, there was no evidence of early GVHD or toxicity compared to PBMC alone (Figures 4A, 4B). However, CD83 CAR T cells significantly improved allogeneic GVHD survival after transplantation compared to PBMC alone or mock-transduced CAR T cells (Figure 4A). Furthermore, CD83-targeted CAR T cells reduced the clinical severity of allogeneic GVHD (Figure 4B). Notably, mice in both dose cohorts of CD83-targeted CAR T cells demonstrated survival of over 90% at 3 months (Figure 4A). In individual experiments, NSG mice that received transplants were given PBMC alone or with mock-transduced T cells (1×10 6 cells) or CD83-targeted CAR T cells (1×10 6 cells), euthanized humanely on day +21, and the severity of target organ GVHD was evaluated. A blinded expert pathologist determined the GVHD pathology score (Betts B.C. et al., Science translational medicine 9:eaai8269(2017); Betts B.C. et al., Proc Natl Acad Sci USA.,201712452(2018); Betts B.C. et al., Front Immunol 9:2887(2018)). CD83 CAR T cells eliminated allogeneic GVHD target organ tissue damage by human T cells in the recipient lungs (Figures 4C - 4E) and livers (Figures 4G - J) compared to PBMC alone or mock-transduced T cells. Furthermore, human T cells that directly infiltrated the mouse target organs were only slightly present, and these were not proliferative based on Ki-67 staining (Figures 4E, 4F, 4I, 4J).

[0289] Human CD83-targeted CAR T cells significantly reduce CD83+ DCs in vivo: Mature, CD83+ dendritic cells are associated with increased sensitivity of alloreactive donor T cells. As such, the effect of CD83 CAR T cells on the immune reconstitution of human CD1c+ DCs was investigated in transplanted mice. NSG mice transplanted with human PBMC+CD83 CAR or mock-transduced T cells were euthanized on day +21. It was determined that CD83-targeted CAR T cells attenuated donor cell proliferation in vivo, as indicated by the significantly smaller spleens in this treatment group at the time of recipient spleen excision (Figure 10). CD83-targeted CAR T cells significantly decreased the amount of human CD1c+, CD83+ DCs in recipient mice (Figures 5A, 5B). While the proportion of CD1c+ DCs expressing MHC class II was similar between experimental groups, mice transplanted with CD83 CAR T cells showed significantly fewer DCs overall (Figures 5C, 5D).

[0290] Human CD83-targeted CAR T cells significantly reduce CD4+, CD83+ T cells while increasing the in vivo Treg:activated Tconv ratio: To confirm that the injected human CD83 CAR T cells were detectable in the mouse spleen on day +21, an eGFP tag was used (Figure 6A). On day +21, the total amount of human CD4+ T cells was significantly decreased in the spleens of mice treated with CD83-targeted CAR T cells (Figures 6B, 6C). Since a substantial amount of CD83+CD4+ Tconv was observed in vitro after DC-allostimulation, experiments were performed on day +21 to confirm the increase in CD83+ Tconv between mice treated with PBMC alone or mock-transduced T cells (Figure 6D). Furthermore, the amount of CD83+ Tconv was significantly decreased in recipients of CD83 CAR T cells in vivo (Figure 6D). Overall, CD83 CAR T cells resulted in robust elimination of CD83+ target cells by day +21 compared to mock T cells (Figure 11A). The higher number of circulating eGFP+ CAR T cells was associated with a lower number of CD83+ DCs on day +21, while the decrease in CD83+ T cells was uniform across CAR T cell numbers in vivo (Figures 11B, 11C).

[0291] In individual experiments, human T cells alone or T cells + dendritic cells were transplanted into NSG mice. The absence of dendritic cells slightly delayed the onset of GVHD, while the median GVHD survival was similar between both groups (Figures 12A, 12B). This is consistent with other studies and indicates that purified human T cells are sufficient to induce xenogeneic GVHD (Li W. et al., JCI Insight 1(2016)).

[0292] CD83-targeted CAR T cells were hypothesized to protect recipients from GVHD mainly by improving the ratio of Tregs to alloreactive Tconvs while eliminating alloreactive Tconvs involved in GVHD (Figures 6E–6G). The frequency of human Tregs in mouse spleens was similar among all experimental groups on day +21 (Figure 6E). Similar to the decrease in total CD4+ T cells, the absolute number of Tregs was significantly decreased in mice treated with CD83-targeted CAR T cells (Figure 6F). However, the ratio of Tregs (CD4+, CD127−, CD25+, Foxp3+) to activated Tconvs (CD4+, CD127+, CD25+) (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017)) was significantly increased in mice administered CD83-targeted CAR T cells (Figure 6G). Th1 cells contribute to GVHD pathogenesis. Importantly, mice treated with CD83 CAR T cells showed a substantial decrease in human CD4+, IFNγ+ Th1 cells (Figures 6H, 6I). Furthermore, the amount of splenic resident human Th2 cells (CD4+, IL-4+) was also significantly decreased in mice injected with CD83 CAR T cells (Figures 6H, 6J). Conversely, CD83-targeted CAR T cells did not suppress the amount of human Th17 cells in the recipient spleen compared with PBMC alone or mock-transduced CAR T cells (Figures 13A, 13B). Interestingly, eGFP+ CD83 CAR T cells were also detected in the spleens of mice that survived to the day +100 endpoint in the long-term experiment (Figure 14). More than 3 months after transplantation, a dose-dependent decrease in circulating CD83+ target cells was observed between mice treated with low (1×10 6 cells) or high (10×10 6 cells) doses of CD83 CAR T cells (Figure 14).

[0293] Human CD83 CAR T cells kill acute myeloid leukemia cell lines: According to long-term data from the Center for International Blood and Marrow Transplant Research (CIBMTR), more than 1,000 patients undergo allogeneic HCT annually for high-risk AML (Gupta, V. et al., Blood 117:2307-2318 (2011)). Even when patients may be tolerant to myeloablative preconditioning therapy, the relapse-free survival rate was limited to 67.8%, compared with 47.3% after reduced-intensity pre-transplantation therapy (Scott B. L. et al., J Clin Oncol 35:1154-1161 (2017)). Therefore, there is a strong need for strategies to prevent AML relapse. Considering the potent lytic activity of CD83 CAR T cells in preventing allogeneic GVHD and its sufficient tolerance by transplanted mice, experiments were conducted to investigate whether human myeloid leukemia might express CD83. It was found that CD83 was actually expressed on malignant myeloid K562, Thp-1, U937, and MOLM-13 cell lines (Figure 7A, 7B, Figure 15A, 15B). Furthermore, using the xCELLigence platform, CD83 CAR T cells showed significant antitumor activity against K562 and Thp-1 cells (Figure 7C, 7D). Therefore, human CD83 CAR T cells have the ability to prevent GVHD and cause direct killing of AML.

[0294] Human CD83 CAR T cells exhibit negligible off-target, off-tumor toxicity: Human AML antigens are often shared by progenitor stem cells. While CD83 CAR T cells clearly kill AML targets, they were confirmed to enable the proliferation and differentiation of hematopoietic stem cells in colony-forming units (CFUs) (Figures 8A - 8D). Overall, the total number of colonies was similar among mock T cells, CD83 CAR T cells, and the medium treatment group. A decrease in granulocyte / macrophage CFUs was observed with CD83 CAR T cells, but this was not significantly different compared to medium alone (Figure 8B). Furthermore, colonies from granulocyte / erythrocyte / monocyte / megakaryocyte CFUs and erythroid burst-forming units were basically the same among the treatment groups (Figures 8C, 8D). These experiments provide evidence that human CD83 CAR T cells selectively kill AML while preserving normal hematopoiesis.

[0295] Discussion The use of CAR T cells as a cellular immunotherapy to prevent GVHD is an epoch-making strategy different from pharmacological immunosuppression or adoptive transfer of donor Tregs. Targeted cells expressing CD83 effectively deplete inflammation, mature DCs, and alloreactive CD4 + Tcovnv from transplant recipients. Donor CD8 + T cells can also be involved in GVHD (Okiyama N. et al., J Invest Dermatol 134:992 - 1000 (2014); Shindo T. et al., Blood 121:4617 - 4626 (2013)). Although there were only slightly more human CD8 + T cells expressing CD83, CD83 CAR T cells also significantly reduced the amount of donor CD8 + T cells (Figure 16). Mechanistically, dendritic cell depletion did not reduce allogeneic GVHD, so in vivo elimination of alloreactive T cells was presumed to drive the effectiveness of these CAR T cells. In vivo depletion of alloreactive T effectors by CD83 CAR T cells is also involved in a marked increase in the Treg:activated Tconv ratio, a clinically significant indicator in the control of GVHD (Koreth J. et al., N Engl J Med 365:2055 - 2066 (2011)).

[0296] CD83 CAR T cells significantly reduce pathogenic human Th1 and Th2 cells in vivo. Experiments using STAT4 and STAT6 knockout donor T cells have shown that Th1 and Th2 cells independently mediate lethal GVHD in mice (Nikolic, B. et al., J Clin Invest 105:1289-1298 (2000)). Furthermore, the combination of Th1 and Th2 cells in vivo synergistically exacerbates murine GVHD (Nikolic, B. et al., J Clin Invest 105:1289-1298 (2000)). In part, Th1 and Th2 cells cause tissue-specific damage to the intestine and lung, respectively (Yi T. et al., Blood 114:3101-3112 (2009)). Currently, strategies exist to target the donor Th1 response, which is mainly driven by p40 cytokine neutralization or inhibition of appropriate downstream receptor signaling (Betts B.C. et al., Science translational medicine 9:eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci USA., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018); Pidala J. et al., Haematologica 2017.171199 (2017); Yu Y. et al., Blood 118:5011-5020 (2011)). However, approaches that simultaneously target pathogenic Th1 and Th2 cells are only scarce. Therefore, human CD83 CAR T cells correspond to a cell product for simultaneously suppressing the donor Th1 / Th2 response after allogeneic HCT. Human Th17 cells were not significantly affected by CD83 CAR T cells, but the treated mice were clearly protected from GVHD.Donor Th17 cells have the potential to contribute to GVHD (Iclozan C. et al., Biol Blood Marrow Transplant 16:170 - 178 (2010)), while the lack of available Th1 cells seems to reduce the pathogenicity of surviving Th17 cells (Yu Y. et al., Blood 118:5011 - 5020 (2011)).

[0297] From the disclosed data, it is confirmed that human CD83 CAR T cells continuously prevent activated Tconv and GVHD death. Despite the fact that CD83 is not significantly expressed on human Tregs, mice treated with human CD83 CAR T cells showed a decrease in the amount of Tregs. This may be due to the limited availability of T cell precursors or a decrease in the overall concentration of IL - 2 due to a decrease in circulating donor T cells. In rodents, CD83 contributes to Treg stability in vivo, and mice with CD83 - deficient Tregs are prone to autoimmune syndromes (Doebbeler M. et al., JCI Insight 3 (2018)). However, in xenotransplantation experiments, the ratio of human Tregs to activated Tconvs was significantly increased in mice treated with CD83 CAR T cells compared to the control. An increase in the ratio of Tregs to Tconvs is a clinically significant immune indicator and even correlates with the response to Treg - directed GVHD therapies such as low - dose IL - 2 (Koreth J. et al., N Engl J Med 365:2055 - 2066 (2011); Koreth J. et al., Blood 128:130 - 137 (2016)). Furthermore, human CD83 CAR T cells showed good tolerance and eliminated immune - mediated organ damage in vivo. Therefore, the role of CD83 may differ between mouse and human Tregs. +

[0298] ​CD83 is a unique immunomodulatory molecule. In mice, soluble CD83 mediates immunosuppressive effects by promoting Treg responses through indoleamine 2,3-dioxygenase- and TGFβ-mediated mechanisms (Bock F. et al., J Immunol 191:1965-1975 (2013)). The extracellular domain of human CD83 has also been shown to attenuate alloreactive T cell proliferation in vitro (Lechmann M. et al., J Exp Med 194:1813-1821 (2001)). Conversely, direct neutralization of CD83 with the monoclonal antibody, 3C12C, significantly reduces human T cell-mediated xenogeneic GVHD in vivo (Wilson J. et al., J Exp Med 206:387-398 (2009)). CD83 antibodies have also been shown to preserve Treg and antiviral responses by donor, human CD8+ T cells (Seldon T.A. et al., Leukemia 30:692-700 (2016)). This suggests that while soluble CD83 can have immunosuppressive properties, targeting cell surface expression of CD83 can prevent GVHD while retaining critically important effector and Treg functions. Unlike monoclonal antibodies, CD83 CAR T cells induce only robust target cell killing and do not require NK-cell-mediated antibody-dependent cellular cytotoxicity (Seldon T.A. et al., Leukemia 30:692-700 (2016)). This is an advantage when rapid and efficient elimination of alloreactive T cells is required to prevent GVHD. Indeed, human CD83-targeted CAR T cells provided sustained GVHD prophylaxis and were detectable in mice up to day +100 even after a single injection.

[0299] In addition to the elimination of alloreactive T cells in GVHD prevention, CD83 appears to be a promising candidate for targeting myeloid malignancies. CD83 expression was observed on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Furthermore, CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Therefore, experiments were conducted to evaluate stem cell killing in the human CFU assay, which revealed negligible on-target, off-tumor toxicity. Allogeneic HCT is often necessary to treat high-risk AML, but relapse remains an important cause of post-transplant failure and death. Unlike HLA-mediated classical GVL, CD83 CAR T cells selectively destroy malignant cells expressing CD83. Furthermore, CD83 has recently been found to be expressed in Hodgkin lymphoma (Li Z. et al., Haematologica 103:655-665 (2018)). Therefore, CD83 CAR T cells may be effective in the treatment of AML or HL independently of allogeneic HCT. Considering the clinical success of CD19 CAR T cells in ALL and diffuse large B-cell lymphoma, this is strong in a bridging point (Neelapu S.S. et al., N Engl J Med 377:2531-2544 (2017); Schuster S.J. et al., Engl J Med 380:45-56 (2019); Maude S.L. et al., N Engl J Med 378:439-448 (2018); Davila M.L. et al., Sci Transl Med 6:224ra225 (2014)).

[0300] In conclusion, CD83 CAR T cells correspond to the first human programmed cytolytic effector cells designed to prevent GVHD. The bridging ability of CD83 CAR T cells has shown tin GVHD prevention and is expected to have merits in preventing rejection even after transplantation of solid organ or vascularized composite tissue allografts. Furthermore, CD83 CAR T cells retain their killing activity even when exposed to calcineurin inhibitors. CD83 CAR T cells can overcome the HLA mismatch barrier in hematopoietic cell and solid organ donor selection and can greatly expand the application of therapeutic transplantation procedures to patients in need. Importantly, CD83 CAR T cells are broadly suppressive and provide a platform for eliminating alloreactive T cells without the need for non-selective calcineurin inhibitors or glucocorticoids. Furthermore, the clinical impact of CD83 CAR T cells is further expanded by their ability to kill myeloid leukemia cells. Therefore, CD83 CAR T cells are likely to reduce transplantation-related mortality and improve outcomes after allogeneic HCT.

[0301] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosed invention belongs. Publications and the materials cited therein are specifically incorporated by reference.

[0302] One of ordinary skill in the art will recognize many equivalents to the specific embodiments of the invention described herein or will be able to ascertain such equivalents using no more than routine experimentation. Such equivalents are intended to be encompassed by the following claims. Some aspects of the invention are described below. 1. A method of treating a myeloid malignancy in a subject, the method comprising administering to the subject an immunoeffector cell genetically modified to express a chimeric antigen receptor (CAR) polypeptide comprising an effective amount of a CD83 antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region. 2. The method according to item 1, wherein the immune effector cell is a regulatory T cell. 3. The method according to item 1 or 2, wherein the CD83 antigen-binding domain is a single-chain variable fragment (scFv) of an antibody that specifically binds to CD83. 4. The anti-CD83 scFv comprises a variable heavy chain (V H ) domain having CDR1, CDR2, and CDR3 sequences and a variable light chain (V L ) domain having CDR1, CDR2, and CDR3 sequences. The CDR1 sequence of the V H domain comprises amino acid sequence SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 13; the CDR2 sequence of the V H domain comprises amino acid sequence SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14; the CDR3 sequence of the V H domain comprises amino acid sequence SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15; the CDR1 sequence of the V L comprises amino acid sequence SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16; the CDR2 sequence of the V L domain comprises amino acid sequence SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17; and the CDR3 sequence of the V L domain comprises amino acid sequence SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18. The method according to item 3. 5. The V H domain of the anti-CD83 scFv comprises amino acid sequence SEQ ID NO: 19, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. The method according to item 4. 6. The V L domain of the anti-CD83 scFv comprises amino acid sequence SEQ ID NO: 20, SEQ ID NO: 54, or SEQ ID NO: 55. The method according to item 4 or 5. 7. The anti-CD83 scFv comprises amino acid sequence SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71. The method according to any one of items 1 to 6. 8. The method according to any one of items 1 to 7, wherein the co-stimulatory signal transduction region comprises the cytoplasmic domain of a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. 9. The CAR polypeptide is of the formula: SP-CD83-HG-TM-CSR-ISD; or SP-CD83-HG-TM-ISD-CSR (wherein, "SP" represents a signal peptide, "CD83" represents a CD83 binding region, "HG" represents an optional hinge domain, "TM" represents a transmembrane domain, "CSR" represents a co-stimulatory signal transduction region, "ISD" represents an intracellular signal transduction domain, "-" represents a bivalent linker) defined by the method according to any one of items 1 to 8. 10. The method according to any one of items 1 to 9, wherein the intracellular signal transduction domain comprises a CD3 zeta (CD3ζ) signal transduction domain. 11. The method according to any one of items 1 to 10, further comprising administering a checkpoint inhibitor to the subject. 12. The method according to item 11, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof. 13. The method according to any one of items 1 to 12, wherein the myeloid malignancy comprises acute myeloid leukemia (AML). 14. The method according to any one of items 1 to 12, wherein the myeloid malignancy comprises Hodgkin lymphoma. 15. The method according to any one of items 1 to 14, wherein the subject has been treated with hematopoietic stem cell transplantation. 16. The method according to any one of items 1 to 14, wherein the subject has not been treated with hematopoietic stem cell transplantation.

Claims

1. 1. A pharmaceutical composition for use in a method of treating a myeloid malignancy in a subject, comprising immune effector cells genetically modified to express a chimeric antigen receptor (CAR) polypeptide comprising a CD83 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory signaling region, said method comprising administering to said subject an effective amount of said immune effector cells; the CD83 antigen-binding domain is a single chain variable fragment (scFv) of an antibody that specifically binds to CD83; The anti-CD83 scFv comprises a variable heavy chain (VH) having CDR1, CDR2 and CDR3 sequences. Ｈ ) domain and a variable light chain (V) having CDR1, CDR2 and CDR3 sequences. Ｌ ) domain, The V Ｈ The CDR1 sequence of the V domain comprises the amino acid sequence SEQ ID NO:1, SEQ ID NO:7, or SEQ ID NO:13; Ｈ The CDR2 sequence of the V domain comprises the amino acid sequence SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:14; Ｈ The CDR3 sequence of the V domain comprises the amino acid sequence SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:15; Ｌ the CDR1 sequence of comprises the amino acid sequence SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:16; Ｌ The CDR2 sequence of the V domain comprises the amino acid sequence SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:17; and Ｌ The pharmaceutical composition, wherein the CDR3 sequence of the domain comprises the amino acid sequence SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:

1.

2. The pharmaceutical composition of claim 1 , wherein the immune effector cell is a regulatory T cell.

3. The V of the anti-CD83 scFv Ｌ The pharmaceutical composition of claim 1 or 2, wherein the domain comprises the amino acid sequence SEQ ID NO:20, SEQ ID NO:54 or SEQ ID NO:

55.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the anti-CD83 scFv comprises the amino acid sequence SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:

71.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the costimulatory signaling region comprises a cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

6. The CAR polypeptide has the formula: SP-CD83-HG-TM-CSR-ISD; or SP-CD83-HG-TM-ISD-CSR (wherein "SP" represents a signal peptide, "CD83" represents the CD83 binding domain; "HG" represents an optional hinge domain; "TM" stands for transmembrane domain; "CSR" stands for costimulatory signaling region; "ISD" stands for intracellular signaling domain; "-" represents a bivalent linker. The pharmaceutical composition according to any one of claims 1 to 5, wherein said pharmaceutical composition is defined by:

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the intracellular signaling domain comprises a CD3 zeta (CD3ζ) signaling domain.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the method further comprises administering a checkpoint inhibitor to the subject.

9. 9. The pharmaceutical composition of claim 8, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the myeloid malignant tumor comprises acute myeloid leukemia (AML).

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the myeloid malignancies include Hodgkin's lymphoma.

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