Chimeric antigen receptors for treatment of myeloid malignancies

Abstract

Compositions and methods for treating acute myelogenous leukemia (AML) in a subject are disclosed. In particular, chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell metastasis to treat AML are disclosed. Immune effector cells, such as T cells or natural killer (NK) cells, engineered to express these CARs are also disclosed. Accordingly, also disclosed are methods of treating AML in a subject comprising adoptively transferring the disclosed immune effector cells, which are engineered to express the disclosed CARs.

Description

[0001] This application is a divisional application of patent application with the application date of August 14, 2020, the priority date of August 16, 2019, the application number of 202080071851.9, and the invention title of “Chimeric antigen receptors for treating myeloid malignancies”.

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of U.S. provisional application number 62 / 888,072, filed August 16, 2019, which is hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format as the file named “320803-2410_ST25” created on August 12, 2020. The content of the sequence listing is incorporated herein by reference in its entirety. BACKGROUND

[0006] Acute myeloid leukemia (AML) is a blood cancer in which the bone marrow produces abnormal myeloblasts. AML accounts for nearly one-third of all new leukemia cases each year. The American Cancer Society estimates that 21,380 patients will develop AML and 10,590 AML patients will die in 2017.

[0007] The standard of care for AML treatment has changed little in the past four decades. Hematopoietic stem cell transplantation after intensive chemotherapy 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 relapsed / refractory disease patients. Therefore, the 5-year overall survival rate is 27%, and the 5-year overall survival rate for patients over 60 years old is less than 10%. About 40-60% of hematopoietic stem cell transplant recipients develop graft-versus-host disease (GVHD). 30% of GVHD cases result in death.

[0008] According to the longitudinal data of the Center for International Blood and Marrow Transplant Research (CIBMTR), more than 1000 patients received allo-HCT for high-risk AML each year (Gupta, V. et al., Blood 117:2307-2318 (2011)). Even if patients can tolerate the myeloablative conditioning regimen, the relapse-free survival rate is limited to 67.8%, and 47.3% after reduced-intensity conditioning (Scott B.L. et al., J Clin Oncol 35:1154-1161 (2017)). Therefore, there is an urgent need for strategies to prevent AML relapse. SUMMARY

[0009] Disclosed are chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to treat myeloid malignancies. The disclosed CAR polypeptides comprise an anti-CD83 binding agent in an extracellular domain that can bind to CD83-expressing cells. Also disclosed are immune effector cells genetically modified to express the disclosed CAR polypeptides. Also disclosed are methods of treating a myeloid malignancy in a subject, the method involving administering to the subject an effective amount of immune effector cells genetically modified with the disclosed CD83-specific CAR.

[0010] Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells. They arise from genetic and epigenetic alterations that interfere with key processes such as self-renewal, proliferation, and differentiation. They include chronic phases, such as myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia (CMML), as well as acute phases, i.e., acute myeloid leukemia (AML). In some embodiments, the subject has AML. In some embodiments, the subject has Hodgkin lymphoma.

[0011] Although relapse remains an important cause of post-transplant failure and death, allo-HCT is often necessary to treat high-risk AML. Unlike the classic GVL mediated by HLA, CD83 CAR T cells selectively destroy malignant cells that express CD83. Thus, the disclosed CD83 CAR T cells can have efficacy in treating myeloid malignancies without reliance on allo-HCT. In some embodiments, the subject has been treated with hematopoietic stem cell transplantation. In other embodiments, the subject has not been treated with hematopoietic stem cell transplantation. In some embodiments, the subject is ineligible for allo-HCT.

[0012] In some embodiments, the anti-CD83 binding agent is an antibody fragment that specifically binds CD83. For example, the antigen binding domain can be a Fab or a single-chain variable fragment (scFv) of an antibody that specifically binds CD83. In some embodiments, the anti-CD83 binding agent is an aptamer that specifically binds CD83. For example, the anti-CD83 binding agent can be a peptide aptamer selected from a library of random sequences for its ability to bind CD83. The anti-CD83 binding agent can also be a natural ligand of CD83, or a variant and / or fragment thereof that is capable of binding CD83.

[0013] In some embodiments, the anti-CD83 scFv can comprise a variable heavy (V H ) domain having CDR1, CDR2, and CDR3 sequences and a variable light (V L ) domain having CDR1, CDR2, and CDR3 sequences.

[0014] For example, in some embodiments, V H the CDR1 sequence of the domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 1), SDGIS (SEQ ID NO: 7), or SNAMI (SEQ ID NO: 13);V H the CDR2 sequence of the domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 2), IISSGGNTYYASWAKG (SEQ ID NO: 8), or AMDSNSRTYYATWAKG (SEQ ID NO: 14);V H the CDR3 sequence of the domain comprises 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 the domain comprises 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 comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 5), YASTLAS (SEQ ID NO: 11), or QASSLAS (SEQ ID NO: 17); andV L the CDR3 sequence of the domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO: 6), TGTYGNSAWYEDA (SEQ ID NO: 12), or LGEYSISADNH (SEQ ID NO: 18).

[0015] For example, in some embodiments, V H the CDR1 sequence of the domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 1),V H the CDR2 sequence of the domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 2),V H the CDR3 sequence of the domain comprises the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 3),V L the CDR1 sequence of the domain comprises the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 4),V L the CDR2 sequence of the domain comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 5), andV LThe CDR3 sequence of the domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO: 6).

[0016] For example, in some embodiments, the V H The CDR1 sequence of the domain comprises the amino acid sequence SDGIS (SEQ ID NO: 7), the V H The CDR2 sequence of the domain comprises the amino acid sequence IISSGGNTYYASWAKG (SEQ ID NO: 8), and the V H The CDR3 sequence of the domain comprises the amino acid sequence VVGGTYSI (SEQ ID NO: 9), the V L The CDR1 sequence of the domain comprises the amino acid sequence QSSQS VYNNDFLS (SEQ ID NO: 10), the V L The CDR2 sequence of the domain comprises the amino acid sequence YASTLAS (SEQ ID NO: 11), and the V L The CDR3 sequence of the domain comprises the amino acid sequence TGTYGNSAWYEDA (SEQ ID NO: 12).

[0017] For example, in some embodiments, the V H The CDR1 sequence of the domain comprises the amino acid sequence SNAMI (SEQ ID NO: 13), the V H The CDR2 sequence of the domain comprises the amino acid sequence AMDSNSRTYYATWAKG (SEQ ID NO: 14), the V H The CDR3 sequence of the domain comprises the amino acid sequence GDGGSSDYTEM (SEQ ID NO: 15), the V L The CDR1 sequence of the domain comprises the amino acid sequence QSSQSVYGNNELS (SEQ ID NO: 16), the V L The CDR2 sequence of the domain comprises the amino acid sequence QASSLAS (SEQ ID NO: 17), and the V L The CDR3 sequence of the domain comprises the amino acid sequence LGEYSISADNH (SEQ ID NO: 18).

[0018] In some embodiments, the anti-CD83 scFv V HThe domain comprises the following amino acid sequence: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 19, VH-GBM00).

[0019] In some embodiments, the anti-CD83 scFv V L The domain comprises the following amino acid sequence: QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 20, VL-GBM00).

[0020] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSNNAINWVRQAPGKGLEWIGYIWSGGLTYYANWAEGRFTISKTSTTVDLKMTSPTIEDTATYFCARGINNSALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 21, 20D04).

[0021] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFTISDYDLSWVRQAPGEGLKYIGFIAIDGNPYYATWAKGRFTISKTSTTVDLKITAPTTEDTATYFCARGAGDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 23, 11G05).

[0022] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFTISDYDLSWVRQAPGEGLKYIGFIAIDGNPYYATWAKGRFTISKTSTTVDLKITAPTTEDTATYFCARGAGDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 23, 11G05).

[0023] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVHCQSVEESGGRLVTPGTPLTLTCTASGFSRSSYDMSWVRQAPGKGLEWVGVISTAYNSHYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGGSWLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 25, 14C12).

[0024] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVHCQSVEESGGRLVTPGTPLTLTCTASGFSRSSYDMSWVRQAPGKGLEWVGVISTAYNSHYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGGSWLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 25, 14C12).

[0025] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: MDXRAPTQLLGLLLLWLPGARCALVMTQTPASVSAAVGGTVTINCQSSQSVYDNDELSWYQQKPGQPPKLLIYALASKLASGVPSRFKGSGSGTQFALTISGVQCDDAATYYCQATHYSSDWYLTFGGGTEVVVKGFPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGTENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 26, 14C12).

[0026] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYDMTWVRQAPGKGLEWIGIIYASGTTYYANWAKGRFTISKTSTTVDLKVTSPTIGDTATYFCAREGAGVSMTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 27, 020B08).

[0027] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVSPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTSTTVDLEVTSLTTEDTATYFCSREHAGYSGDTGHLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVGIGPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 29, 006G05).

[0028] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVSPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTSTTVDLEVTSLTTEDTATYFCSREHAGYSGDTGHLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVGIGPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 29, 006G05).

[0029] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSDGISWVRQAPGKGLEWIGIISSGGNTYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARVVGGTYSIWGQGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 31, 96G08).

[0030] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSDGISWVRQAPGKGLEWIGIISSGGNTYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARVVGGTYSIWGQGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 31, 96G08).

[0031] In some embodiments, the anti-CD83 scFv V LDomain comprises the following amino acid sequence: MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAPVGGTVTINCQSSQSVYNNDFLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCTGTYGNSAWYEDAFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC (SEQ ID NO: 32, 96G08)

[0032] In some embodiments, the anti-CD83 scFv V H Domain comprises the following amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSNAMIWVRQAPREGLEWIGAMDSNSRTYYATWAKGRFTISRTSSITVDLKITSPTTEDTATYFCARGDGGSSDYTEMWGPGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 33, 95F04).

[0033] In some embodiments, the anti-CD83 scFv V LThe domain comprises the following amino acid sequence: MDTRAPTQLLGLLLLWLPGATFAQAVVTQTTSPVSAPVGGTVTINCQSSQSVYGNNELSWYQQKPGQPPKLLIYQASSLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCLGEYSISADNHFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC (SEQ ID NO: 34, 95F04)

[0034] In some embodiments, the anti-CD83 scFv V H The domain comprises the following amino acid sequence: QVQLVQSGGAVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAAVSYDGSNKYYADFVKGRFTISRDNPKNTLYLQMNSLRADDTAVYYCARRGGLDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA (SEQ ID NO: 35).

[0035] In some embodiments, the anti-CD83 scFv V L The domain comprises the following amino acid sequence: LTQPPPASGTPGQQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYYGNDQRPSGVPDRFSASKSGTSASLAISGLQSEDEAHYYCAAWDGSLNGGVIFGGGTKVTLG (SEQ ID NO: 36).

[0036] In some embodiments, the anti-CD83 scFv V L The domain comprises the following amino acid sequence: VTQPPSASGTPGQRVTISCSGSSSNIGTNPVNWYQQLPGTAPKLLIYTTDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLSGLYVFGTGTKVTVLG (SEQ ID NO: 37).

[0037] In some embodiments, the anti-CD83 scFv V L comprises the following amino acid sequence: MTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQRPGQSPQPLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVQAEDVGVYYCMQSLQLWTFGQGTKVEIKR (SEQ ID NO: 38).

[0038] In some embodiments, the anti-CD83 scFv V L comprises the following amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLIHSDGNTYLDWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLRISRVEAEDIGVYYCMQATHWPRTFGQGTKVEIKR (SEQ ID NO: 39).

[0039] In some embodiments, the anti-CD83 scFv V L comprises the following amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVDSAGNTFLHWFHQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 40).

[0040] In some embodiments, the anti-CD83 scFv V L comprises the following amino acid sequence: LTQSPLSLPVTLGQPASISCKSSQSLVDSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 41).

[0041] In some embodiments, the anti-CD83 scFv V L comprises the following amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVHSDGNMYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQATQPTWTFGQGTKLEIKR (SEQ ID NO: 42).

[0042] In some embodiments, the anti-CD83 scFv V L domain comprises the following amino acid sequence: MTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSATYYCQQTYQGTKLEIKR (SEQ ID NO: 43).

[0043] In some embodiments, the anti-CD83 scFv V L domain comprises the following amino acid sequence: MTQSPSSLSASVGHPVTITCRASQSLISYLNWYHQKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLQPENFASYYCQHTDSFPRTFGHGTKVEIKR (SEQ ID NO: 44).

[0044] In some embodiments, the anti-CD83 scFv V L domain comprises the following amino acid sequence: LTQPPSASGTPGQGVTISCRGSTSNIGNNVVNWYQHVPGSAPKLLIWSNIQRPSGIPDRFSGSKSGTSASLAISGLQSEDQAVYYCAVWDDGLAGWVFGGGTTVTVLS (SEQ ID NO: 45).

[0045] In some embodiments, the anti-CD83 scFv V L domain comprises the following amino acid sequence: MTQAPVVSVALEQTVRITCQGDSLAIYYDFWYQHKPGQAPVLVIYGKNNRPSGIPHRFSGSSSNTDSLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTNLTVLG (SEQ ID NO: 46).

[0046] In some embodiments, the anti-CD83 scFv V L domain comprises the following amino acid sequence: LTQSPLSLPVTLGQPASISCKSNQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYCMQGTQWPRTFGGQGTKLDIKR (SEQ ID NO: 47).

[0047] In some embodiments, the anti-CD83 scFv V HThe domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 48, VH-GBM01).

[0048] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 49, VH-GBM02).

[0049] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 50, VH-GBM03).

[0050] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 51, VH-GBM04).

[0051] In some embodiments, the anti-CD83 scFv V HThe domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 52, VH-GBM05).

[0052] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the following amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 53, VH-GBM06).

[0053] In some embodiments, the anti-CD83 scFv V L The domain has been humanized and comprises the following amino acid sequence: QLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 54, VL-GBM01).

[0054] In some embodiments, the anti-CD83 scFv V L The domain has been humanized and comprises the following amino acid sequence: LPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 55, VL-GBM02).

[0055] The heavy chain and the light chain are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 56).

[0056] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAASSGGGGSGGGGSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAA (SEQ ID NO: 57).

[0057] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTV (SEQ ID NO: 58).

[0058] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 59).

[0059] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 60).

[0060] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 61).

[0061] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 62).

[0062] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 63).

[0063] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 64).

[0064] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 65).

[0065] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 66).

[0066] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 67).

[0067] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 67).

[0068] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 69).

[0069] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 70).

[0070] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 71).

[0071] As with other CARs, the disclosed polypeptides can also comprise a transmembrane domain and an intracellular domain capable of activating an immune effector cell. For example, the intracellular domain can comprise a signaling domain and one or more costimulatory signaling regions.

[0072] In some embodiments, the intracellular signaling domain is a CD3 zeta (CD3 zeta) signaling domain. In some embodiments, the costimulatory signaling region comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof. In some cases, the costimulatory signaling region comprises 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. In some embodiments, the costimulatory signaling region comprises one or more mutations in the cytoplasmic domain of CD28 and / or 4-1BB that enhance signaling.

[0073] In some embodiments, the CAR polypeptide comprises an incomplete intracellular domain. For example, the CAR polypeptide can comprise only an intracellular signaling domain or a costimulatory domain, but not both. In these embodiments, the immune effector cell is not activated unless both the immune effector cell and the second CAR polypeptide comprising a missing domain (or an endogenous T cell receptor) bind their respective antigens. Thus, in some embodiments, the CAR polypeptide comprises a CD3 zeta (CD3 zeta) signaling domain, but does not comprise a costimulatory signaling region (CSR). In other embodiments, the CAR polypeptide comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof, but does not comprise a CD3 zeta (CD3 zeta) signaling domain (SD).

[0074] Also disclosed are isolated nucleic acid sequences encoding the disclosed CAR polypeptides, vectors comprising these isolated nucleic acids, and cells comprising these vectors. For example, the cell can be an immune effector cell selected from the group consisting of an alpha-beta T cell, a gamma-delta T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine-induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine-activated killer (LAK) cell, and a regulatory T cell.

[0075] The details of one or more embodiments of the application are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figures 1A-1G Human CD83-targeting CAR T constructs and functional characteristics are shown. Figure 1AThe anti-CD83 single chain variable fragment is shown followed by a CD8 hinge and transmembrane domain, and a 41BB costimulatory domain and a CD3s activating domain. The CAR is tagged at the 3' end with a fluorescent reporter. The CAR reporter gene was cloned into an SFG retroviral vector. Figure 1B is a bar graph showing the amount of T cells (mean ± SEM) that produced the eGFP reporter after transduction in mock transduced T cells (eGFP negative) or CD83 CAR T cells (eGFP positive). Figure 1C is a bar graph showing the relative amount of CD4 or CD8 expression (mean ± SEM) in mock transduced T cells or CD83 CAR T cells, Sidak's test. Figure 1D and Figure 1E shows the amount of IFNy and IL-2 released by mock transduced T cells or CD83 CAR T cells after stimulation with CD83+ DCs. Figure 1F shows CD83 CAR T cells or mock transduced T cells were co-cultured with CD83+ DCs and cytotoxicity was measured on a real-time cell analysis system. Data are presented as mean normalized cell index of replicate wells over time. Normalized cell index was calculated as cell index at a given time point divided by cell index at the normalization time point (i.e. day 1 after addition of T cells). One of 2 representative experiments is shown, Dunnett's test. Figure 1G shows CD83+ DCs were stimulated with CD83 CAR T cells or mock transduced T cells and the absolute number of T cells was calculated every week over a 14 day period. One of 2 representative experiments is shown, Sidak's test. **P =.001-.01, ***P =.0001-.001, and ****P <.0001.

[0077] Figure 2Human CD83 chimeric antigen receptor T cells were shown to reduce alloreactivity. Human T cells were cultured with allogeneic cytokine-matured monocyte-derived dendritic cells (moDCs) at a DC:T cell ratio of 1:30 (i.e., 100,000 T cells and 3,333 moDCs). CD83 CART cells (autologous to the cultured T cells) were added to the moDCs at specific ratios (3:1 to 1:10, with a minimum of 333 CART cells added). T cell proliferation was measured by Ki-67 expression on day +5. CAR T cells were gated for GFP expression. Controls included T cells alone (i.e., no proliferation), mock-transduced T cells, and CD19 CART cells. These mock-transduced T cells did not express the chimeric antigen receptor but were treated identically to the CD83-transduced cells. The CD19 CART cells used the 41BB costimulatory domain and target an irrelevant antigen in this system. One representative experiment is shown.

[0078] Figures 3A-3D Figure 3: Differential expression of CD83 on activated human conventional CD4+ T cells (Tcon) compared to regulatory T cells (Treg). Human T cells were stimulated with allogeneic moDC (DC:T cell ratio 1:30) or CD3 / CD28 beads (bead:T cell ratio 1:30). CD83 expression on activated Tconv (CD4+, CD127+, CD25+) or Treg (CD4+, CD127-, CD25+, Foxp3+) was measured at baseline, 4 hours, 8 hours, 24 hours, and 48 hours after stimulation. The bar graph shows the expression of CD83 on allogeneic DC ( Figure 3A ) or CD3 / CD28 beads ( Figure 3B ) after stimulation with human CD83+ Tconv or Treg (mean ± SEM). N = 5 independent experiments, Sidak test. Human CD83 CAR or mock T cells were cultured with DC allogeneic stimulated PBMCs at a 1:10 ratio for 48 hours. Representative contour plots show the frequency of CD83+, CD3-, and CD3+ target cells over time ( Figure 3C ) and the expression of CD83 in eGFP+CART cells ( Figure 3D ). One representative experiment of two is shown. ****P < .0001.

[0079] Figures 4A-4J Human CD83 CART cells were shown to prevent xenogeneic GVHD. Figure 4A Shows acceptance of 25 x 10 6 Individual PBMCs were seeded at a low dose (1 x 10 6) or high dose (10 x 10 6 ) CD83 CAR or (1-10 x 10 6 ) mock transduced T cells. CARs were autologous to the PBMC donor. Another control group of mice received PBMCs alone. Figure 4A and Figure 4B Survival rates ( Figure 4A ) and GVHD ( Figure 4B ) clinical scores are shown. Clinical scores included a composite assessment of activity, coat and skin condition, weight loss, and posture. Data from 3 independent experiments were pooled, with a maximum of 9 mice per experimental group. Log-rank test. In separate experiments, recipient mice were humanely euthanized at +21 days and tissue GVHD severity was assessed by an expert, blinded pathologist. Heterologous GVHD pathway scores for recipient lungs ( Figures 4C-4F ) and livers ( Figures 4G-4J ), representative H&E images, amounts of Ki-67+, CD3+ T cells / HPF, and representative IHC images (CD3 = red, Ki-67 = brown) are shown. Data from 2 independent experiments were pooled, with a maximum of 6 mice per experimental group. Dunnett test (group comparison) or Mann-Whitney. **P =.001-.01 and ***P =.0001-.001.

[0080] Figures 5A-5D Human CD83-targeting CAR T cells significantly reduced CD83+ DCs are shown. NSG mice received 25 x 10 6 individual PBMCs plus 1 x 10 6 CD83 CAR or mock transduced T cells. Mice were humanely euthanized at +21 days and spleens were harvested. Figure 5A contains representative contour plots showing the frequency of human CD83+, CDlc+ DCs in the spleens of mice at +21 days. Figure 5B is a bar graph showing the absolute numbers (mean ± SEM) of human CD83+, CDlc+ DCs in the spleens of mice at +21 days, Dunn test. Figure 5C contains representative contour plots showing the percentage of MHC II+, CDlc+ DCs in the recipient spleens at +21 days. Figure 5D is a bar graph depicting the absolute numbers (mean ± SEM) of these cells, Dunn test. Data from 2 independent experiments were pooled, with a maximum of 6 mice per experimental group. **P =.001-.01.

[0081] Figure 6: Human CD83-targeting CAR T cells significantly reduce CD4+, CD83+ T cells in vivo while increasing the Treg: activated Tconv ratio. NSG mice received 25 x 106human PBMC plus 1 x 10 6 Figure 6: Human CD83-targeting CAR T cells significantly reduce CD4+, CD83+ T cells in vivo while increasing the Treg: activated Tconv ratio. NSG mice received 25 x 106human PBMC plus 1 x 10

[0082] Figure 7: Human CD83 CAR T cells kill acute myeloid leukemia cell lines. Histograms show CD83 expression in proliferating (A) K562 and (B) Thp-1 cells, MFI noted in lower right corner. Human CD83 CAR or mock-transduced T cells were co-cultured with fresh K562 or Thp-1 cells at an E / T ratio of 10: 1. Target cell killing was monitored using the xCELLigence RTCA system, Dunnett’s test. Representative experiments are shown for each. **** P <.0001.

[0083] Figure 8: Human CD83 CART cells exhibit negligible on-target, off-tumor toxicity. CD34+ cells isolated from normal human bone marrow were co-incubated with CART cells, mock T cells, or culture medium alone at an effector to target ratio of 10: 1 for 4 hours. The cells were plated in duplicate in Methocult medium and cultured for 14 days, and then colonies were counted. The bar graph shows the amount of the following items: A) total colonies, B) colony-forming units (CFU)-granulocytes / macrophages (GM), C) CFU-granulocytes / erythrocytes / monocytes / megakaryocytes (GEMM) and D) erythroblast-forming units (BFU). The results represent 3 independent experiments, Dunnett test. NS = not significant.

[0084] Figure 9: Human CD83 CAR T cells can still kill and proliferate in response to CD83+ target cells when exposed to tacrolimus. A) Human CD83 CAR T cells or untransduced T cells from the same donor were cultured with allogeneic CD83+ cytokine-matured moDCs at varying T cell to DC ratios for 24 hours. Cultures were exposed to clinically relevant doses of tacrolimus (10 ng / ml) or DMSO control (< 0.01%). The bar graph shows DC lysis at 24 hours per colorimetric LDH assay. B) Human CD83 CAR T cells or untransduced T cells from the same donor were cultured with allogeneic CD83+ cytokine-matured moDCs at a 1:30 T:DC ratio. Tacrolimus or DMSO control was added once on day 0, and proliferation was assessed by colorimetric assay 3 days later. One representative experiment of two is shown for each, Sidak test. ***P = 0.0001-.001 and ****P < .0001.

[0085] Figure 10 :Human CD83 CART cells reduce the expansion of donor cells in vivo. NSG mice were transplanted with 25 x 10 6 Personal PBMC plus 1 x 10 6 CD83 CAR or mock-transduced T cells were administered to recipients of the CAR-T cells. Control groups consisted of mice that received no PBMCs (negative control) and mice that received PBMCs harboring unmodified T cells (secondary positive control). Recipient mice were humanely euthanized on day +21, and their spleens were removed for gross evaluation. Representative images show reduced spleen size in mice that received PBMCs and CD83 CAR T cells, supporting inhibition of donor T cell expansion in vivo. One representative experiment out of two.

[0086] Figure 11: Human CD83 CAR T cells eliminate CD83+ targets at +21 days. NSG mice received 25 x 106human PBMC plus 1 x 106CD83 CAR or mock transduced T cells. At +21 days, recipient mice were humanely euthanized and the amount of eGFP+ CAR, CD83+, CDlc+ DC and CD83+, CD4+ T cells were analyzed by flow cytometry. A) Bar graph showing the amount of eGFP+ CAR T cells in the recipient spleen at +21 days, and the percent reduction of CD83+ targets in the spleen (normalized to mice injected with mock T cells). B, C) Graphs showing linear regression of CD83+ targets according to the amount of eGFP+ CAR T cells recovered at +21 days (dashed line). Spearman's rank correlation coefficient is shown. Data from 2 independent experiments were pooled, with a maximum of 6 mice per experimental group. 6 Individual PBMC plus 1 x 10 6 CD83 CAR or mock transduced T cells. At +21 days, recipient mice were humanely euthanized and the amount of eGFP+ CAR, CD83+, CDlc+ DC and CD83+, CD4+ T cells were analyzed by flow cytometry. A) Bar graph showing the amount of eGFP+ CAR T cells in the recipient spleen at +21 days, and the percent reduction of CD83+ targets in the spleen (normalized to mice injected with mock T cells). B, C) Graphs showing linear regression of CD83+ targets according to the amount of eGFP+ CAR T cells recovered at +21 days (dashed line). Spearman's rank correlation coefficient is shown. Data from 2 independent experiments were pooled, with a maximum of 6 mice per experimental group.

[0087] Figure 12: DC depletion does not prevent xenogeneic GVHD mediated by human T cells. NSG mice received 7.5 x 106purified human T cells alone or together with 1.87 x 106autologous dendritic cells. Dendritic cells were isolated by magnetic bead purification (Miltenyi) and included plasmacytoid DC, CDlc+ type 1 myeloid DC and CDlc-, CD141 6 2 type 2 myeloid DC. Survival rate (A) and GVHD clinical score (B) are shown. Representative experiments are shown, with 4 mice per experimental group. 5 2 type 2 myeloid DC. Survival rate (A) and GVHD clinical score (B) are shown. Representative experiments are shown, with 4 mice per experimental group. 亮 2 type 2 myeloid DC. Survival rate (A) and GVHD clinical score (B) are shown. Representative experiments are shown, with 4 mice per experimental group.

[0088] Figure 13: Human CD83 CAR T cells do not reduce the amount of donor Thl 7 cells. NSG mice received 25 x 106human PBMC plus l x 106CD83 CAR or mock transduced T cells as described. At +21 days, mice were humanely euthanized and spleens harvested. A) Representative contour plot showing the frequency of human CD4+, IL-17+ Thl 7 cells in the mouse spleen at +21 days. B) Bar graph showing the absolute number of human Thl 7 cells in the mouse spleen at +21 days (mean ± SEM). Data from 2 independent experiments were pooled, with a maximum of 6 mice per experimental group.

[0089] Figure 14 Figure 14: Human CD83 CAR T cells persist at +100 days. NSG mice received 25 x 10 6 PBMC plus 1-10 x 10 6 CD83 CAR or 10 x 10 6Figure 14: CD83 CAR T cells reduce the amount of donor CD8+ T cells in vivo. NSG mice received 25 x 106human PBMCs plus 1 x 106CD83 CAR or mock transduced T cells as described. A) The amount of donor human CD8+ T cells was counted at +21 days, Dunn’s test. Data from 2 representative experiments were pooled, up to 6 mice per experimental group.

[0090] Figure 15: CD83 expression on U937 and MOLM-13 cells. Histograms show CD83 expression in proliferating A) U937 and B) MOLM-13 cells, MFI noted in lower right corner.

[0091] Figure 16 Figure 16: CD83 CAR T cells reduce the amount of donor CD8+ T cells in vivo. NSG mice received 25 x 106human PBMCs plus 1 x 106CD83 CAR or mock transduced T cells as described. A) The amount of donor human CD8+ T cells was counted at +21 days, Dunn’s test. Data from 2 representative experiments were pooled, up to 6 mice per experimental group. 6 Figure 17: CD83 CAR T cells reduce the amount of donor CD8+ T cells in vivo. NSG mice received 25 x 106human PBMCs plus 1 x 106CD83 CAR or mock transduced T cells as described. A) The amount of donor human CD8+ T cells was counted at +21 days, Dunn’s test. Data from 2 representative experiments were pooled, up to 6 mice per experimental group. DETAILED DESCRIPTION

[0092] Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to the particular implementations described and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0093] Where a series of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower magnitude, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 now described.

[0095] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0096] As will be apparent to one of ordinary skill in the art in light of the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any method recited can be carried out in the order of events recited or in any other order that is logically possible.

[0097] Unless otherwise indicated, the embodiments of the present disclosure will employ, unless otherwise indicated, chemistry, biological techniques, and the like within the skill of the art.

[0098] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc. ) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure is defined as 20 °C and 1 atmosphere.

[0099] Before describing embodiments of the disclosure in detail, it is to be understood that the disclosure is not limited in its application to the particulars of the specific embodiments described, as these may vary. Furthermore, it is to be understood that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present disclosure. It is also possible in the present disclosure that steps can be carried out in a different order than described, where logically possible.

[0100] It must be noted that as used herein and in the appended claims, the singular form "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc. ) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure is defined as 20 °C and 1 atmosphere.

[0101] Disclosed herein are chimeric antigen receptors (CARs) that target CD83 on antigen presenting cells. Also disclosed are immune effector cells, such as T cells or natural killer (NK) cells, engineered to express these CARs. CAR T cells expressing these CARs can suppress alloreactive donor cells, such as T cells. Thus, also disclosed are methods for preventing GVHD in a subject involving adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CD83-specific CARs.

[0102] CD83-specific chimeric antigen receptors (CARs)

[0103] CARs typically comprise 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 are CD83-specific chimeric antigen receptors (CARs) that can be expressed in immune effector cells to suppress alloreactive donor cells.

[0104] The disclosed CARs typically consist of three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises the CD83 binding region and is responsible for antigen recognition. It also optionally comprises a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of an immune effector cell. The transmembrane domain (TD), as the name suggests, links the extracellular domain to the intracellular domain and resides within the cell membrane when expressed by a cell. The intracellular domain is the business end of the CAR, transmitting an activation signal to the immune effector cell upon antigen recognition. For example, the intracellular domain can comprise an intracellular signaling domain (ISD) and an optional costimulatory signaling region (CSR).

[0105] A “signaling domain (SD)” typically comprises immunoreceptor tyrosine-based activation motifs (ITAMs) that activate a signaling cascade when the ITAMs are phosphorylated. The term “costimulatory signaling region (CSR)” refers to intracellular signaling domains from costimulatory protein receptors, such as CD28, 41BB, and ICOS, that are able to enhance T cell receptor activation of T cells.

[0106] In some embodiments, the intracellular domain comprises either a SD or a CSR, but not both. In these embodiments, the immune effector cells containing the disclosed CARs are only activated when the other CAR (or T cell receptor) containing the missing domain also binds its respective antigen.

[0107] In some embodiments, the disclosed CARs are defined by the formula:

[0108] SP-CD83-HG-TM-CSR-SD; or

[0109] SP-CD83-HG-TM-SD-CSR;

[0110] wherein "SP" represents an optional signal peptide,

[0111] wherein "CD83" represents a CD83 binding region,

[0112] wherein "HG" represents an optional hinge domain,

[0113] wherein "TM" represents a transmembrane domain,

[0114] wherein "CSR" represents one or more costimulatory signaling regions,

[0115] wherein "SD" represents a signaling domain, and

[0116] wherein "-" represents a peptide bond or linker.

[0117] Additional 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, the teachings directed to these CAR models are incorporated by reference in their entirety.

[0118] For example, the CAR can be a TRUCK, a universal CAR, a self-driving CAR, an armored CAR, a self-destruct CAR, a conditional CAR, a tagged CAR, a TenCAR, a biCAR, or a sCAR.

[0119] CAR T cells engineered to resist immune suppression (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)), have an immune checkpoint switch receptor, or can be administered with monoclonal antibodies that block immune checkpoint signaling.

[0120] Self-destruct CARs can be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of T cells can be achieved based on genetic modification of the lymphocytes or the recent description of ganciclovir binding to thymidine kinase in a system that activates human caspase 9 by small molecule dimerizers.

[0121] By default, the conditional CAR T cells are unresponsive or the switch is“off” until a small molecule is added to complete the circuit, thus enabling full transduction of signal 1 and signal 2, thereby activating the CAR T cells. Alternatively, the T cells can be engineered to express an adaptor-specific receptor with affinity for a secondary antibody against the target antigen that is subsequently administered.

[0122] Tandem CAR (TanCAR) T cells express a single CAR composed of two linked single-chain variable fragments (scFvs) with different affinities fused to one or more intracellular costimulatory domains and a CD3 zeta domain. TanCAR T cell activation is achieved only when the target cell expresses both targets simultaneously.

[0123] Dual CAR T cells express two separate CARs with different ligand-binding targets; one CAR contains only a CD3 zeta domain, while the other CAR contains only the one or more costimulatory domains. Dual CAR T cell activation requires co-expression of both targets.

[0124] Safety CARs (sCARs) are composed of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR will only be activated when they encounter a target cell with the standard CAR target but lacking the sCAR target.

[0125] The antigen recognition domain of the disclosed CARs is typically an scFv. However, there are many alternatives. Antigen recognition domains from native T cell receptor (TCR) alpha and beta single chains have been described, as have simple extracellular domains (e.g., the CD4 extracellular domain that recognizes HIV-infected cells) and more exotic recognition components, such as linked cytokines that result in recognition of cells bearing the cytokine receptor. In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition region.

[0126] The intracellular domain is the business end of the CAR, transmitting a signal to the immune effector cell after antigen recognition, activating at least one of the immune effector cell’s normal effector functions. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the intracellular domain can comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co-receptors. While it is generally possible to use the entire intracellular signaling domain, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the full chain so long as it transduces the effector function signal.

[0127] Cytosolic signaling sequences that modulate primary activation of the TCR complex in a stimulatory manner can include signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of cytoplasmic signaling sequences containing ITAMs include those derived from CD8, CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD32 (FcgammaRIIa), DAP10, DAP12, CD79a, CD79b, FcgammaRI gamma, FcgammaRIII gamma, Fc epsilon RI beta (FCERIB), and Fc epsilon RI gamma (FCERIG).

[0128] In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3 zeta) (TCR zeta, GenBank acc no. BAG36664.1). The T cell surface glycoprotein CD3 zeta (CD3 zeta) chain, also known as T cell receptor T3 zeta chain or CD247 (cluster of differentiation 247), is a protein that in humans is encoded by the CD247 gene.

[0129] First generation CARs typically have an intracellular domain from the CD3 zeta chain, which is the primary transmitter of endogenous TCR signals. Second generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the intracellular domain of the CAR to provide additional signals to the T cell. More recently, third generation CARs combine multiple signaling domains to further enhance potency. T cells transplanted with these CARs have demonstrated enhanced expansion, activation, persistence, and tumor eradication efficiency independent of costimulatory receptor / ligand interactions (Imai C, et al. Leukemia 2004 18:676-84; Maher J, et al. Nat Biotechnol 2002 20:70-5).

[0130] For example, the intracellular domain of a CAR can be designed to include a CD3 zeta signaling domain by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of a CAR of the application. For example, the cytoplasmic domain of a CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to the portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand required for the 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 with CD123, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Thus, while a CAR is primarily exemplified with CD28 as the costimulatory signaling element, other costimulatory elements can be used alone or in combination with other costimulatory signaling elements.

[0131] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., 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 any suitable sequence derived from or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0132] The transmembrane domain can be derived from a natural source or a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be derived 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 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, 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 alpha, beta, or zeta chain of PAG / Cbp (i.e., comprising at least one or more transmembrane regions thereof). Alternatively, the transmembrane domain can be synthetic, in which case it will comprise primarily hydrophobic residues, such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan, and valine will occur at each end of the synthetic transmembrane domain. A short oligonucleotide or polypeptide linker, such as between 2 and 10 amino acids in length, can form the junction between the transmembrane domain and the endoplasmic domain within the CAR.

[0133] In some embodiments, the CAR has more than one transmembrane domain, which can be repeats of the same transmembrane domain, or can be different transmembrane domains.

[0134] In some embodiments, the CAR is a multi-chain CAR, as described in WO 2015 / 039523, which is incorporated by reference for the present disclosure. Multi-chain CARs can contain separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble at a juxtamembrane location, forming a flexible structure closer to the native receptor, providing optimal signal transduction. For example, a multi-chain CAR can contain a portion of the FCERI alpha chain and a portion of the FCERI beta chain, such that the FCERI chains spontaneously dimerize together to form the CAR.

[0135] Tables 1, 2, and 3 below provide some example combinations of CD83 binding regions, costimulatory signaling regions, and intracellular signaling domains that can occur in the disclosed CARs.

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

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[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

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[0214]

[0215]

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[0220]

[0221]

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[0223]

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[0226]

[0227]

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[0229]

[0230]

[0231]

[0232]

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[0240]

[0241]

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[0250]

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[0256]

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[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

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[0269]

[0270]

[0271]

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[0278]

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[0280]

[0281]

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[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] In some embodiments, the anti-CD83 binding agent is a single chain variable fragment (scFv) antibody. The affinity / specificity of the anti-CD83 scFv is largely determined by the heavy (V H ) and light (V L ) chains within the complementarity determining regions (CDRs). Each V H and V L The sequence will have three CDRs (CDR1, CDR2, CDR3).

[0296] In some embodiments, the anti-CD83 binding agent is derived from a natural antibody, such as a monoclonal antibody. In some cases, the antibody is human. In some cases, the antibody is altered to reduce its immunogenicity when administered to humans. For example, the alteration comprises one or more techniques selected from the group consisting of chimerization, humanization, CDR grafting, deimmunization, and framework amino acid mutations to correspond to the closest human germline sequence.

[0297] Also disclosed are bispecific CARs that target CD83 and at least one additional antigen. Also disclosed are CARs designed to work in conjunction with another CAR that binds a different antigen. For example, in these embodiments, the disclosed CARs can contain only a signaling domain (SD) or a costimulatory signaling region (CSR), but not both. If the second CAR (or endogenous T cell) is activated, it provides the missing signal. For example, if the disclosed CAR contains a SD but not a CSR, the immune effector cell containing that CAR is only activated when the other CAR (or T cell) containing a CSR binds its respective antigen. Likewise, if the disclosed CAR contains a CSR but not a SD, the immune effector cell containing that CAR is only activated when the other CAR (or T cell) containing a SD binds its respective antigen.

[0298] Nucleic acids and vectors

[0299] Also disclosed are polynucleotides and polynucleotide vectors that encode the disclosed CD83-specific CARs, which allow for expression of the CD83-specific CARs in the disclosed immune effector cells.

[0300] Nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, a gene of interest can be produced synthetically, rather than cloned.

[0301] Expression of nucleic acids encoding CARs is typically 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 regulation of the expression of the desired nucleic acid sequence.

[0302] The disclosed nucleic acids can be cloned into a variety of types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0303] Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication 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 lentivirus or retrovirus vector.

[0304] A number of viral-based systems have been developed for the transfer of genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or in vivo and delivered to the cells of a subject.

[0305] One 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 levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the MND (myelocytomatosis virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Alternatively, the promoter can 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.

[0306] Additional promoter elements, such as enhancers, modulate the frequency of transcription initiation. Typically, these promoters are located in the region 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, so that the promoter function is preserved when the elements are inverted or moved relative to each other.

[0307] To assess expression of a CAR polypeptide or portion thereof, the expression vector to be introduced into a cell can also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked with appropriate regulatory sequences to enable their expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0308] Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene that is not present or not expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is evidenced by some readily detectable property (e.g., enzymatic activity). Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes can 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. Typically, the construct with the smallest 5' flanking region that shows the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to assess the ability of an agent to modulate promoter-driven transcription.

[0309] Methods of introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vector can be readily introduced into host cells, e.g., mammalian, bacterial, yeast, or insect cells, by any of a number of methods known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0310] Physical methods of introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0311] Biological methods of introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0312] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle).

[0313] In the case of use of a non-viral delivery system, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome by a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained in a lipid as a suspension, included or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector related compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, micellar form, or form with "collapsed" structures. They can also simply be interspersed in a solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include the fat droplets naturally occurring in cytoplasm and the class of compounds containing 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, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories, Plainview, NY; cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Birmingham, AL.

[0314] Immune effector cells

[0315] Also disclosed are immune effector cells engineered to express the disclosed CARs (also referred to herein as "CAR-T cells"). These cells are preferably obtained from the subject to be treated (i.e., are autologous). However, in some embodiments, an immune effector cell line or donor effector cell is used (allogeneic). However, in other embodiments, the immune effector cell is not HLA matched. Immune effector cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using 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 PERCOLL™ gradient centrifugation or by counterflow centrifugal elutriation. Particular 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 against surface markers unique to the cells for positive selection, for example, by incubation with antibody-conjugated beads for a sufficient time to positively select for the desired immune effector cells. Alternatively, enrichment of a population of immune effector cells can be accomplished by negative selection using a combination of antibodies against surface markers unique to the cells for negative selection.

[0316] In some embodiments, the immune effector cells comprise any white blood cell involved in protecting the body against infectious disease and foreign substances. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, the immune effector cells can comprise T lymphocytes.

[0317] T cells or T lymphocytes are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a different function.

[0318] T helper cells (T HHelper T cells assist other white blood cells in the immune process, 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 called CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in active immune responses. 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 , which secrete different cytokines to promote different types of immune responses.

[0319] Cytotoxic T cells (T C These cells are also called 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, which are present on the surface of all nucleated cells. Through the secretion of IL-10, adenosine, and other molecules by regulatory T cells, CD8+ cells can be inactivated to an anergic state, thereby preventing autoimmune diseases.

[0320] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has resolved. When re-exposed to the same antigen, they rapidly expand into a large population of effector T cells, providing the immune system with a "memory" of past infections. Memory cells can be CD4 + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0321] Regulatory T cells (T reg CD4 T cells, formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus. Two major classes of CD4 T cells have been described: + T reg Cells—naturally occurring T reg cells and adaptive T reg cell.

[0322] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) connect the adaptive immune system and the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0323] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subpopulations based on cell surface expression. For example, in some cases, the T comprise cytotoxic CD8 + T lymphocytes. In some embodiments, the T cells comprise gamma delta T cells, which have a unique T cell receptor (TCR) with one gamma chain and one delta chain instead of alpha and beta chains.

[0324] Natural killer (NK) cells are CD56 + CD3 - Large granular lymphocytes, which can kill virus-infected and transformed cells and constitute a key cell subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8 + T lymphocytes, NK cells are cytotoxic to 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 potential deadly complications of cytokine storm (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 depleting effects.

[0325] Therapeutic methods

[0326] 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 for GVHD. In some embodiments, the subject receives a bone marrow transplant and the disclosed CAR-modified immune effector cells suppress alloreactivity of donor T cells or dendritic cells.

[0327] The disclosed CAR-modified immune effector cells can be administered alone or in combination with a diluent and / or other components (such as IL-2, IL-15 or other cytokines) or cell populations as a pharmaceutical composition.

[0328] In some embodiments, the disclosed CAR-modified immune effector cells are administered in combination with ER stress blockade (compounds targeting the IRE-1 / XBP-1 pathway (e.g., 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.

[0329] Briefly, a pharmaceutical composition may comprise a target cell population as described herein, along with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer (e.g., neutral buffered saline, phosphate buffered saline, etc.); a carbohydrate (e.g., glucose, mannose, sucrose, or dextran, mannitol); a protein; a polypeptide or amino acid (e.g., glycine); an antioxidant; a chelating agent (e.g., EDTA or glutathione); an adjuvant (e.g., aluminum hydroxide); and a preservative. In some embodiments, the compositions used in the disclosed methods are formulated for intravenous administration. The pharmaceutical composition may be administered in any manner suitable for treating MM. The amount and frequency of administration will be determined by factors such as the patient's condition and the severity of their disease, although appropriate dosages can be determined through clinical trials.

[0330] When a "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, degree of engraftment, and condition of the patient (subject). In general, it can be said that the pharmaceutical composition comprising the T cells described herein can be 10 4 to 10 9 cells / kg body weight, such as 10 5 to 10 6 The T cell compositions can be administered at a dose of 10 cells / kg body weight, including all integer values ​​within those ranges. These doses can also be administered multiple times. The cells can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). One skilled in the art of medicine can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0331] In certain embodiments, it can be desirable to administer activated T cells to a subject, then to draw 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 10 cc to 400 cc of apheresis. In certain embodiments, T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc of apheresis. This multiple apheresis / multiple reinfusion protocol can be used to select certain T cell populations.

[0332] Administration of the disclosed compositions can be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intranodally, 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 intravenous injection. The compositions can also be injected directly into a graft site.

[0333] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., prior to, concurrently with, or following) any number of related therapeutic modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells can be combined with chemotherapy, radiation, immunosuppressive agents (such as cyclosporin, azathioprine, methotrexate, mycophenolic acid, and FK506), antibodies, or other immunolytic agents (such as CAM PATH, anti-CD3 antibodies, or other antibody therapies), cytotoxins, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., prior to, concurrently with, or following) a bone marrow transplant, T cell ablative therapy using any one of a number of chemotherapeutic agents (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (such as OKT3 or CAMPATH). In another embodiment, the cell compositions of the application are administered following B cell ablative therapy (such as an agent reactive with CD20, e.g., Rituxan). For example, in some embodiments, a subject can receive standard therapy of high dose chemotherapy followed by peripheral blood stem cell transplant. In certain embodiments, following the transplant, the subject receives an infusion of the expanded immune cells of the application. In another embodiment, the expanded cells are administered prior to or following surgery.

[0334] A major issue with CAR-T cells as a form of “living therapeutic agent” is their maneuverability in vivo and their potential immunostimulatory side effects. To better control CAR-T therapy and prevent unwanted side effects, a variety of features have been engineered, including off switches, safety mechanisms, and conditionally controlled mechanisms. For example, suicide and tagged / tagged CAR-T cells have been engineered to have an “off switch” that promotes the elimination of CAR-expressing T cells. Suicide CAR-Ts contain a CAR but are also engineered to express a pro-apoptotic suicide gene or “elimination gene” that can be induced upon administration of an exogenous molecule. A variety of 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). For example, HSK converts the prodrug ganciclovir (GCV) into GCV-triphosphate, which incorporates itself into replicating DNA, ultimately leading to cell death. iCasp9 is a chimeric protein containing a component of FK506 binding protein that can bind the small molecule AP1903, leading to dimerization of Caspase 9 and apoptosis. However, tagged / tagged CAR-T cells are cells that have a CAR but are also engineered to express a selection marker. Administration of a mAb against this selection marker will promote the elimination of CAR-T cells. Truncated EGFR is a such a targetable antigen for anti-EGFR mAbs, administration of cetuximab can promote elimination of CAR-T cells. CARs built with these features are also called sCARs for ‘switchable CARs’, and RCARs for ‘regulatable CARs’. “Safety CARs”, also called “inhibitory CARs” (iCARs), are engineered to express two antigen binding domains. One of these extracellular domains is directed against a first antigen and is coupled to an intracellular costimulatory and stimulatory domain. However, the second extracellular antigen binding domain has specificity for normal tissue and is coupled to an intracellular checkpoint domain such as CTLA4, PD1, or CD45. It is also possible to incorporate multiple intracellular inhibitory domains into iCARs. Some of the 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, the stimulation of this second antigen binding domain will act to inhibit the CAR. It is important to note that due to this dual antigen specificity, iCARs are also a form of bispecific CAR-T cells.SafeCAR-T engineering enhances CAR-T cell tissue specificity and is advantageous in situations where normal tissues may express very low levels of antigen, which would result in off-target effects with standard CARs (Morgan 2010). Conditional CAR-T cells express an extracellular antigen-binding domain linked to an intracellular costimulatory domain and a separate intracellular costimulator. The costimulatory and stimulatory domain sequences are engineered in such a way that, upon administration of the exogenous molecule, the resulting proteins will assemble together intracellularly to complete the CAR circuit. In this way, CAR-T activation can be modulated and potentially even "fine-tuned" or personalized for specific patients. Similar to dual-CAR designs, the stimulatory and costimulatory domains are physically separated when inactive in conditional CARs; for this reason, these are also referred to as "split-CARs."

[0335] Typically, CAR-T cells are established using α-β T cells, but γ-δ T cells can also be used. In some embodiments, the CAR constructs, domains, and engineering features used to generate CAR-T cells can be similarly used to generate other types of CAR-expressing immune cells, including NK (natural killer) cells, B cells, mast cells, myeloid phagocytes, and NKT cells. Alternatively, CAR-expressing cells can be established to have the characteristics of T cells and NK cells. In another embodiment, cells transduced with CAR can be autologous or allogeneic.

[0336] Several different CAR expression methods are available, including retroviral transduction (including gamma-retrovirus), lentiviral transduction, transposon / transposase (Sleeping Beauty and PiggyBac systems), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) is also becoming increasingly important for the potential of engineering CAR-T cells. CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator-like effector nuclease) systems are three potential approaches for generating CAR-T cells.

[0337] definition

[0338] The term "amino acid sequence" refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are the conventional one-letter code for amino acids, which are represented as follows: A, alanine; B, asparagine or aspartate; C, cysteine; D aspartate; E, glutamine, glutamate; 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 glutamate.

[0339] The term "antibody" refers to an immunoglobulin, a derivative thereof that retains the ability to specifically bind, and a protein having a binding domain that is homologous or largely homologous to a binding domain of an immunoglobulin. These proteins can be derived from natural sources, or produced in part or in whole synthetically. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class from any species, including any human class: IgG, IgM, IgA, IgD, and IgE. In exemplary 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 those immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules that selectively bind a target antigen.

[0340] The term "antibody fragment" refers to any derivative of less than full-length antibody. In exemplary embodiments, an antibody fragment retains at least a significant portion of the full-length antibody's ability to specifically bind. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabodies, Fc, and Fd fragments. An antibody fragment can be produced by any means. For example, an antibody fragment can be produced enzymatically or chemically from fragmentation of an intact antibody, it can be produced recombinantly from a gene encoding a partial antibody sequence, or it can be produced wholly or partially synthetically. An antibody fragment can optionally be a single chain antibody fragment. Alternatively, the fragment can comprise multiple chains linked together, e.g., by disulfide bonds. The fragment can also optionally be a multi-molecular complex. A functional antibody fragment typically comprises at least about 50 amino acids, and more typically at least about 200 amino acids.

[0341] The term "antigen binding site" refers to the region of an antibody that specifically binds an epitope of an antigen.

[0342] The term "aptamer" refers to an oligonucleic acid or a peptide molecule that binds to a specific target molecule. These molecules are typically selected from a library of random sequences. Selected aptamers are able to adapt a unique tertiary structure and recognize target molecules with high affinity and specificity. A "nucleic acid aptamer" is a DNA or RNA oligonucleic acid that binds to a target molecule through its conformation, thereby inhibiting the function of that molecule. Nucleic acid aptamers can be composed of DNA, RNA, or a combination thereof. A "peptide aptamer" is a combinatorial protein molecule with a variable peptide sequence inserted into a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast two-hybrid conditions, which increases the likelihood that the selected peptide aptamer is stably expressed and correctly folded in the cellular environment.

[0343] The term "carrier" refers to a compound, composition, material, or structure that aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of a compound or composition for its intended use or purpose when combined with the compound or composition. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in a subject.

[0344] The term "chimeric molecule" refers to a single molecule created by joining two or more molecules that exist separately in their natural state. The single chimeric molecule has the functions required of all of its constituent molecules. One type of chimeric molecule is a fusion protein.

[0345] The term "engineered antibody" refers to a recombinant molecule comprising at least an antibody fragment comprising an antigen binding site derived from an antibody heavy and / or light chain variable domain, and can optionally comprise all or a portion of the variable and / or constant domains from an antibody of any Ig class (e.g., IgA, IgD, IgE, IgG, IgM, and IgY).

[0346] The term "epitope" refers to the region of an antigen to which an antibody preferentially and specifically binds. A monoclonal antibody preferentially binds a single, specific epitope of a molecule that can be defined molecularly. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0347] The term "fusion protein" refers to a polypeptide formed by the linkage of two or more polypeptides via a peptide bond formed between the amino terminus of one polypeptide and the carboxy terminus of another polypeptide. A fusion protein can be formed by chemical coupling of the constituent polypeptides, or it can be expressed as a single polypeptide from a nucleic acid sequence encoding a single continuous fusion protein. A single chain fusion protein is a fusion protein having a single continuous polypeptide backbone. Fusion proteins can be prepared using routine techniques in molecular biology to join two in-frame genes into a single nucleic acid, which is then expressed in a suitable host cell under conditions that yield the fusion protein.

[0348] The term "Fab fragment" refers to an antibody fragment that contains the antigen binding site produced by cleavage of an antibody with papain, which cleaves at the hinge region N-terminal to the inter-heavy chain disulfide bond and produces two Fab fragments from one antibody molecule.

[0349] The term "F(ab')2 fragment" refers to an antibody fragment that contains two antigen binding sites, produced by cleavage of an antibody molecule with pepsin, which cleaves at the hinge region C-terminal to the inter-heavy chain disulfide bond.

[0350] The term "Fc fragment" refers to an antibody fragment that contains its heavy chain constant domains.

[0351] The term "Fv fragment" refers to an antibody fragment that contains its heavy and light chain variable domains.

[0352] A "gene construct" refers to a nucleic acid, such as a vector, plasmid, viral genome, etc., that includes a "coding sequence" for a polypeptide or is otherwise transcribable into a biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), that can be transfected into a cell, e.g., in certain embodiments, a mammalian cell, and that can cause the coding sequence to be expressed in the cell transfected with the construct. A gene construct can include one or more regulatory elements operably linked to the coding sequence as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0353] The term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between a nucleic acid or amino acid sequence is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin) 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 particular polypeptide described herein and preferably exhibiting essentially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Unless otherwise indicated, similarity scores will be based on the use of BLOSUM62. When using BLASTP, the percent similarity is based on the BLASTP positive score, while the percent sequence identity is based on the BLASTP identity score. BLASTP "identity" shows the number and fraction of total residues in high-scoring sequence pairs that are identical; and BLASTP "positive" shows the number and fraction of residues that have a positive value of alignment score and are similar to each other. The present disclosure contemplates and encompasses amino acid sequences having these degrees of identity or similarity, or any intermediate degree of identity or similarity, to the amino acid sequences disclosed herein. Polynucleotide sequences of similar polypeptides are inferred using the genetic code, and can be obtained by routine methods, particularly by reverse translation of their amino acid sequences using the genetic code.

[0354] The term "linker" is art-recognized and refers to a molecule or group of molecules that links two compounds, such as two polypeptides. The linker can consist of a single linking molecule or can comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and the compounds by a specific distance.

[0355] The term "multivalent antibody" refers to an antibody or engineered antibody comprising more than one antigen recognition site. 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," and the like 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, a "monospecific" antibody has antigen recognition sites that all bind the same epitope. A "bispecific" antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope different from the first epitope.

[0356] The term "nucleic acid" refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides joined by a phospho group at the 3' position of one nucleotide to the 5' end of another nucleotide. Nucleic acids are not limited by length, thus nucleic acids can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0357] The term "operably linked to" refers to the functional relationship of a nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to a 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.

[0358] The terms "peptide," "protein," and "polypeptide" are used interchangeably and refer to a natural or synthetic molecule comprising two or more amino acids joined by a carboxyl of one amino acid to an alpha amino of another amino acid.

[0359] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0360] The term "polypeptide fragment" or "fragment," when used in reference to a particular polypeptide, refers to a polypeptide having amino acid residues missing from the reference polypeptide itself, but with the remaining amino acid sequence generally identical to the reference polypeptide. Such deletions can occur at the amino- or carboxy-terminus of the reference polypeptide, or, alternatively, both. Fragments are generally at least about 5, 6, 8, or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40, or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500, or more amino acids long. Fragments can retain one or more biological activities of the reference polypeptide. In various embodiments, fragments can comprise enzymatic activities and / or interaction sites of the reference polypeptide. In another embodiment, fragments can be immunogenic.

[0361] The term "protein domain" refers to a portion of a protein, portions of a protein, or the entire protein that exhibits structural integrity; this determination can be based on the amino acid composition of the portion of the protein, portions of the protein, or the entire protein.

[0362] The term "single-chain variable fragment or scFv" refers to an Fv fragment in which the heavy and light chain domains are connected. One or more scFv fragments can be linked to other antibody fragments (such as constant domains of a heavy or light chain) to form an antibody construct with one or more antigen recognition sites.

[0363] As used herein, "spacer" refers to a peptide that links proteins, including fusion proteins. Typically, the spacer has no particular biological activity other than linking the proteins or maintaining some minimal distance or other spatial relationship between them. However, the constituent amino acids of the spacer can be selected to influence certain properties of the molecule, such as folding of the molecule, net charge, or hydrophobicity.

[0364] As used herein, the term "specifically binds," when referring to a polypeptide (including an antibody) or a receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biological materials. Thus, under designated conditions (e.g., the conditions of an immunoassay for an antibody), a particular ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins or other proteins that the ligand or antibody can contact in the sample or organism. Typically, a "specific binding" first molecule for a second molecule has a binding affinity of greater than about 10 5 M -1 (e.g., 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 109 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 or more) affinity constant (Ka).

[0365] As used herein, the term "specific delivery" refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker, as opposed to preferential binding with a cell or tissue lacking that target molecule. Of course, one recognizes that some degree of non-specific interaction between the molecule and non-target cells or tissues can occur. However, specific delivery can be distinguished as being mediated by specific recognition of the target molecule. Typically, specific delivery results in a much stronger association between the delivered molecule and a cell bearing the target molecule than between the delivered molecule and a cell lacking the target molecule.

[0366] The term "subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, e.g., a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject who is receiving treatment by a clinician, e.g., a physician.

[0367] 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. The improvement need only be a reduction or alteration, not necessarily elimination.

[0368] The terms "transformation" and "transfection" refer to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell, including the introduction of a nucleic acid into the chromosomal DNA of the cell.

[0369] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, the term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than curing the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment directed to supplementing another specific therapy directed for the improvement of the associated disease, pathological condition, or disorder.

[0370] The term "variant" refers to a peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., degenerate variants), substitutions within wobble positions of each codon encoding amino acids (i.e., DNA and RNA), amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0371] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which it is linked to a cell. The term "expression vector" includes any vector that contains a gene construct in a form suitable for expression by a cell (e.g., linked to a transcription control element) (e.g., a plasmid, cosmid or bacteriophage chromosome).

[0372] A number of embodiments of the application have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the application. Accordingly, other embodiments are within the scope of the following claims.

[0373] Examples

[0374] Example 1: CD83-targeted chimeric antigen receptor T cells prevent GVHD and kill myeloid leukemia

[0375] Materials and Methods

[0376] Study Design: This is a preclinical study on the design, production, and efficacy of human CD83 CAR T cells for GVHD prevention. Part one of this study describes the CAR construct and the in vitro activity of CD83 CAR T cells in response to CD83+ targets in phenotype, cytokine production, targeted killing, and proliferation. The immunosuppressive effect of CD83 CAR T cells was subsequently demonstrated in vitro using a standard alloMLR. In addition, CD83 expression was measured in human T cells, showing differential expression of CD83 on Tconv and Treg cells. Preclinical efficacy of CD83 CARs in GVHD prevention was demonstrated in a human T cell-mediated xenogeneic GVHD model (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017)). This included a comprehensive assessment of in vivo targeted killing of CD83+ dendritic cells and Tconv. The effect of CD83 CAR T cells on various T cell subsets in vivo was also shown. CD83 has been shown to be expressed on human malignant myeloid cell lines and 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 GVHD experiments, humane end points were used. Mice were frequently monitored for GVHD clinical scores. GVHD histopathology was evaluated and scored by a blinded expert pathologist (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci U S A. 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). Murine in vivo data were pooled from at least two independent experiments with 6-9 mice per experimental group.

[0377] CD83 CAR T cell constructs and generation: CD83 CARs were synthesized by GENEWIZ and cloned into SFG retroviral constructs (Li, G. et al., Methods Mol Biol 1514: 111-118 (2017); Li G. et al., JCI Insight 3 (2018)). CD83 SFG clone constructs were then transfected into H29 cells using calcium phosphate and RD114 was transduced using retroviral supernatant from transfected H29 cells. Retroviral supernatant from RD114 cells was filtered through a 0.45 μm filter (MilliporeSigma) to purify gamma retrovirus. 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 a healthy human donor (AllCells) 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. Activated T cells were transduced with CD83 gamma retrovirus on RetroNectin (TaKaRa Bio Inc.) coated plates. CD83 CAR T cells were debeaded 7-8 days after activation. Gene transfer or transduction efficiency was estimated by GFP+ cells as detected by flow cytometry.

[0378] Monoclonal antibodies and flow cytometry: Fluorochrome-conjugated mouse anti-human monoclonal antibodies included anti-CD3, CD4, CD8, CD25, CD83, CD1c, CD127, MHCII, Foxp3, Ki-67, IFN-g, IL-17A, and IL-4 (BD Biosciences, San Jose, CA, USA; eBioscience, San Jose, CA, USA; Cell Signaling Technology, Boston, MA, USA). Live / Dead fixable yellow or aqueous dead cell stain (Life Technologies, Grand Island, NY) was used to determine viability. Live events were acquired on a BD FACSCanto II or LSRII flow cytometer (FlowJo software, version 7.6.4; TreeStar, Ashland, OR, USA).

[0379] Cytokine immunoassay: CD83 CAR and mock transduced T cells (1 x 10 5 ) were co-cultured with CD83+ moDC (1 x 10 4 ) for 24 hours. Supernatants were harvested and analyzed on Luminex 100 system (Luminex) using human luminex assay kit (R&D Systems) and on Ella instrument (Biotechne) using Simple Plex assay kit (Biotechne) following the manufacturer’s instructions (Li G. et al., JCI Insight 3 (2018)).

[0380] Cytotoxicity and in vitro proliferation of human CD83 CAR T cells: Normalized CD83 CAR T cells (1 x 10 5 cells) were repeatedly cultured with CD83+ moDC, K562, or Thp-1 cells at an ET ratio of 10: 1 in E-Plate 96. Cytotoxicity assays were performed on 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 an ET ratio of 1: 1 in non-tissue culture treated 6-well plates in triplicates. Cells were grown in human T cell complete media supplemented with 60 IU / ml IL-2. Cell viability and total cell number in each well were measured with trypan blue staining on a cell counter (Bio-Rad) at +1 day, +7 days, and +14 days.

[0381] In vitro alloMLR: As described, human monocyte-derived dendritic cells (moDC) are cytokine producing, differentiated, and mature (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017)). T cells (10 5Allo-MLR: Purified human T cells were cultured (1 x 105cells / well) with allogeneic moDC (T cell : DC ratio 30 : 1) in 100 μΐ of complete RPMI supplemented with 10% heat-inactivated pooled human serum (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci U S A. 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 allo-MLR at a range of CAR to DC ratios. T cell proliferation was measured by Ki-67 expression after 5 days.

[0382] CD83 expression time course: Purified human T cells were stimulated with allogeneic moDC (T cell : DC ratio 30 : 1) or CD3 / CD28 beads (T cell : bead ratio 30 : 1). After 4, 8, 24, and 48 hours of culture, T cells were harvested from triplicate wells of a 96-well plate. T cells were stained for CD3, CD4, CD127, CD25, and CD83, then fixed. CD83 expression was assessed 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-). Where indicated, CD83 CAR or mock T cells were cultured with DC allogeneic stimulated PBMC and CD83 expression was assessed in CD3- and CD3+ target cells over 48 hours.

[0383] Colony forming units: CD34+ cells isolated from normal human bone marrow were purchased from AllCells. 10 3Cells were co-cultured with CD83 virus-transduced CAR T cells, mock T cells, or media alone. Cells were incubated at an E:T ratio of 10: 1 for 4 hours. Following incubation, cells were plated in MethoCult media (StemCell) in 6-well SmartDish plates (StemCell) and cultured for 14 days, according to the manufacturer’s instructions. At the end of the culture period, colonies were imaged, analyzed, and counted using STEMvision software.

[0384] 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 animal facility. Recipient mice received a single infusion of 25 x 10 6 fresh human PBMCs (OneBlood) on day 0 of transplant. As indicated, mice received either PBMCs alone, or PBMCs plus CD83 CAR T cells (low dose: 1 x 10 6 or high dose: 10 x 10 6 ), or PBMCs plus mock-transduced T cells (10 x 10 6 ). Each independent experiment was performed with a different human PBMC donor, with CAR T cells and mock-transduced T cells derived from the PBMC donor. Mice were monitored for GVHD clinical scores and moribundity. Where indicated, short-term experiments were completed at +21 days by humane euthanasia to assess blinded GVHD target organ pathology, tissue-resident lymphocytes, and human DC and T cell subpopulation content in murine spleen (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci U S A., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). Tissue samples were prepared, stained (Ventana Medical Systems), and imaged (Vista) to identify human Ki67+ T cells as previously described (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017)). These mice were transplanted with CD83 CAR (1 x 10 6 ) or mock-transduced T cells (1 x 106 ) of PBMCs (25 x 10 6 All vertebrate animal work was performed under AICUC-approved protocols.

[0385] Statistical Analysis: Data are reported as mean ± SEM. ANOVA was used for group comparisons, including Dunnett's or Sidak's post hoc test, corrected for multiple comparisons. Mann-Whitney was used for all other analyses. For comparison of survival curves, the log-rank test was used. Statistical analyses were performed using Prism software, version 5.04 (GraphPad). Statistical significance was defined as two-tailed P < 0.05 (two-tailed).

[0386] Results

[0387] Schematic diagram of the human CD83 CAR construct: an anti-CD83 single-chain variable fragment (scFv) paired with the CD8 hinge and transmembrane domains, followed by the intracellular 41BB costimulatory domain and the CD3ζ activation domain ( Figure 1A To facilitate tracking of CAR T cells, the construct contains an eGFP tag, which can be used to identify CAR T cells among normal non-CAR T cells ( Figure 1A As we have published, CD83-targeted CAR T cells were generated by retroviral transduction ( Figure 1A ) (Li, G. et al., MethodsMol Biol 1514:111-118 (2017); Li G. et al., JCI Insight 3 (2018)).

[0388] Characterization of human CD83 CAR T cells: The CD83 CAR construct demonstrated high transduction efficiency, with over 60% of T cells expressing eGFP ( Figure 1B While CD4 expression was similar in both groups, a significant decrease in CD8 expression was observed in CD83 CAR T cells compared with mock-transduced T cells ( Figure 1C However, when cultured with CD83+ target cells, CD83 CAR T cells displayed robust IFNγ and IL-2 production; as did cytokine-matured human monocyte-derived DCs (moDCs) ( Figure 1D , E). In addition, compared with mock-transduced T cells, CD83 CAR T cells showed effective killing and proliferation of CD83+ moDCs ( Figure 1F 、 1G The target moDCs in these experiments were allogeneic to T cells, so the lysis and proliferation of simulated transduced T cells represented baseline alloreactivity ( Figure 1F, 1G ).

[0389] Human CD83 CAR T cells reduce alloreactivity: To test whether human CD83 CAR T cells reduce alloreactivity in vitro, their suppressive function in an allogeneic mixed leukocyte reaction (alloMLR) was investigated. CD83 and mock transduced CAR T cells were generated from healthy donor human T cells. CD19 CAR T cells target B cells, a cell type that is not relevant in the alloMLR, and were used as another control. In addition, CD19 and CD83 CAR T cells are similar in that they both receive costimulation through 41BB. CAR T cells were added to 5-day alloMLRs composed of autologous T cells (1 x 10 5 ) and allogeneic cytokine-matured CD83+ moDCs (3.33 x 10 3 ). The CAR T cell : moDC ratio ranged from 3 : 1 to 1 : 10. CD83 CAR T cells effectively reduced allogeneic reactive T cell proliferation ( Figure 2 , top panel). In contrast, mock transduced T cells and CD19-targeting CAR T cells had no suppressive effect on allogeneic reactive T cells ( Figure 2 , middle and bottom panels).

[0390] CD83 is differentially expressed on activated human Tconv compared to Tregs: 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 was previously shown that activated murine T cells also express CD83 (Lechmann, M. et al., Proc Natl Acad Sci U S A 105:11887-11892 (2008)). CD83 is known to be expressed on human T cells after stimulation and can be detected on circulating T cells from patients with acute GVHD (Ju X. et al., J Immunol 197:4613-4625 (2016)). However, the precise expression of CD83 on CD4+ Tregs versus CD4+ Tconv or CD8+ T cells was not known. Experiments confirmed that human T cells express CD83 coincident with stimulation, including allogeneic dendritic cells or CD3 / CD28 beads ( Figure 3A , 3B). Importantly, CD83 was shown to be differentially expressed on human CD4+ Tconv (CD127+, CD25+) compared to immunosuppressive CD4+ Tregs (CD127-, CD25+) or cytolytic CD8+ T cells in response to DC allogeneic activation ( Figure 3A ). CD4+ Tconv expression of CD83 peaked at 4-8 hours of DC allogeneic stimulation and declined to baseline levels by 48 hours, with the least amount observed on Tregs or CD8+ T cells ( Figure 3A ). CD83 expression was more abundant under supraphysiologic CD3 / CD28 bead stimulation, which also resulted in a late increase in CD83 expression on Tregs and CD8+ T cells at 48 hours post-activation ( Figure 3B ). Given that CD83 expression is shared between pro-inflammatory mature DCs and allogeneic reactive Tconv, it was investigated whether CD83 CAR T cells could deplete either target cell in culture. Human CD83 CAR or mock T cells were cultured with autologous peripheral blood mononuclear cells (PBMCs) stimulated by allogeneic moDCs and the amount of CD83+ target cells was assessed at 4, 8, 24, and 48 hours of culture. We observed a similar peak in CD83 expression on CD3- and CD3+ target cells at 8 hours ( Figure 3C ). However, CD83+ target cells were largely depleted by CD83 CAR T cells at 48 hours of culture and were far below the baseline amount at 8 hours post-culture ( Figure 3C ). Furthermore, CD83- T cells remained in all experimental groups ( Figure 3C ), supporting that T cells were not indiscriminately destroyed. Next, the expression of CD83 on eGFP+ CAR T cells was assessed over 48 hours. CD83 expression on CAR T cells was modest and an increase in the proportion of eGFP+ CAR T cells was still observed at 48 hours of culture ( Figure 3D ), providing evidence that CD83 CAR T cells do not succumb significantly to CD83-mediated fratricide. To parallel clinical practice, the functional capacity of CD83 CAR T cells was tested in the presence of clinically relevant doses of tacrolimus (5-10 ng / ml). Interestingly, CD83 CAR T cells could still kill and proliferate in response to CD83+ target cells despite exposure to tacrolimus ( Figure 9A 、 9B ).

[0391] Human CD83-targeted CAR T cells prevent xenogeneic GVHD: The efficacy of human CD83 CAR T cells in vivo was assessed using a xenogeneic GVHD model. An established NSG mouse model (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017)) was used in which recipients were inoculated with 25 x 10 6 PBMC plus 1-10 x 10 6 autologous CD83 or mock-transduced CAR T cells on day 0. Transplanted mice were monitored daily for xenogeneic GVHD clinical symptoms until +100 days. NSG mice infused with CD83 or mock-transduced CAR T cells had no evidence of early GVHD or toxicity compared to PBMC alone ( Figure 4A 、 4B ). However, CD83 CAR T cells significantly improved xenogeneic GVHD survival after transplantation compared to PBMC alone or mock-transduced CAR T cells ( Figure 4A ). In addition, CD83-targeted CAR T cells reduced xenogeneic GVHD clinical severity ( Figure 4B ). Notably, mice in both dose groups of CD83-targeted CAR T cells exhibited 90% or greater 3-month survival ( Figure 4A ). In separate experiments, transplanted NSG mice received PBMC alone or with mock-transduced T cells (1 x 10 6 ) or CD83-targeted CAR T cells (1 x 10 6 ) and were humanely euthanized at +21 days to assess target organ GVHD severity. GVHD path score was determined by blinded expert pathologists (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci U S A., 201712452 (2018); Betts B.C. et al., Front Immunol 9:2887 (2018)). CD83 CAR T cells abrogated human T cell damage to xenogeneic GVHD target organ tissues in recipient lungs ( Figures 4C-4E ) and livers ( Figure 4G -J) compared to PBMC alone or mock-transduced T cells. In addition, few human T cells directly infiltrated murine target organs and they did not proliferate according to Ki-67 staining ( Figure 4E ,4F , 4I, 4J).

[0392] Human CD83-targeting CAR T cells significantly reduce CD83+ DCs in vivo: Mature CD83+ dendritic cells are associated with priming of alloreactive donor T cells. Therefore, the impact of CD83 CAR T cells on human CD1c+ DC immune recovery in engrafted mice was determined. NSG mice engrafted with human PBMCs plus CD83 CAR or mock-transduced T cells were euthanized at +21 days. After harvesting the recipient spleens, it was determined that CD83-targeting CAR T cells reduced donor cell expansion in vivo, as indicated by the much smaller spleens in this treatment group ( Figure 10 ). CD83-targeting CAR T cells significantly reduced the amount of human CD1c+, CD83+ DCs in recipient mice ( Figure 5A , 5B ). While the proportion of MHC class II-expressing CD1c+ DCs was similar across experimental groups, mice engrafted with CD83 CAR T cells overall exhibited significantly fewer total DCs ( Figure 5C , 5D ).

[0393] Human CD83-targeting CAR T cells significantly reduce CD4+, CD83+ T cells in vivo while increasing the Treg: activated Tconv ratio: An eGFP tag was used to confirm the presence of infused human CD83 CAR T cells that were detectable in murine spleens at +21 days ( Figure 6A ). The total amount of human CD4+ T cells in the spleens of mice treated with CD83-targeting CAR T cells was significantly reduced at +21 days ( Figure 6B , 6C ). Since a large number of CD83+ CD4+ Tconv were observed after DC allo-stimulation in vitro, experiments were performed to confirm that CD83+ Tconv were increased in mice treated with PBMCs alone or mock-transduced T cells at +21 days ( Figure 6D ). Furthermore, the amount of CD83+ Tconv was significantly reduced in recipients that received CD83 CAR T cells in vivo ( Figure 6D ). Overall, CD83 CAR T cells robustly depleted CD83+ target cells at +21 days compared to mock T cells ( Figure 11A ). While the higher number of circulating eGFP+ CAR T cells at +21 days was associated with a lower number of CD83+ DCs, the reduction in CD83+ T cells was consistent across the number of CAR T cells in vivo ( Figure 11B , 11C ).

[0394] In separate experiments, NSG mice were transplanted with human T cells alone or T cells plus dendritic cells. While the lack of dendritic cells slightly delayed the onset of GVHD, the median GVHD survival was similar between the two groups ( Figure 12A , 12B ). This is in agreement with the work of others showing that purified human T cells are sufficient to induce xenogeneic GVHD (Li W. et al., JCI Insight 1 (2016)).

[0395] It is hypothesized that CD83-targeted CAR T cells protect the recipient from GVHD by mainly eliminating alloreactive Tconv associated with GVHD while enhancing the ratio of Treg to alloreactive Tconv ( Figures 6E-6G ). At +21 days, the frequency of human Tregs in the spleen of all experimental groups of mice was similar ( Figure 6E ). Similar to the decrease in total CD4+ T cells, the absolute number of Tregs was significantly reduced in mice treated with CD83-targeted CAR T cells ( Figure 6F ). However, the ratio of Tregs (CD4+, CD127-, CD25+, Foxp3+) to activated Tconv (CD4+, CD127+, CD25+) (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017)) was significantly increased in mice receiving CD83-targeted CAR T cells ( Figure 6G ). Th1 cells contribute to the pathogenesis of GVHD. Importantly, mice treated with CD83 CAR T cells exhibited a significant reduction in human CD4+, IFNy+ Th1 cells ( Figure 6H , 6I ). Moreover, the amount of human Th2 cells (CD4+, IL-4+) residing in the spleen was also significantly reduced in mice injected with CD83 CAR T cells ( Figure 6H , 6J ). In contrast, CD83-targeted CAR T cells did not suppress the amount of human Th17 cells in the recipient spleen compared to PBMC alone or mock-transduced CAR T cells ( Figure 13A , 13B ). Interestingly, in long-term experiments, eGFP+ CD83 CAR T cells were also detected in the spleen of mice surviving to the +100 days endpoint ( Figure 14 ). More than 3 months after transplantation, at low (1 x 10 6 ) or high (10 x 10 6A dose-dependent reduction in circulating CD83+ target cells was observed in mice treated with 100 mg of CD83 CAR T cells ( Figure 14 ).

[0396] Human CD83 CAR T cells kill acute myeloid leukemia cell lines: According to longitudinal data from the Center for International Blood and Marrow Transplant Research (CIBMTR), over 1,000 patients undergo allo-HCT annually for high-risk AML (Gupta, V. et al., Blood 117:2307-2318 (2011)). Even when patients can tolerate a myeloablative preparative regimen, relapse-free survival is limited to 67.8%, and 47.3% after reduced-intensity conditioning (Scott BL et al., J Clin Oncol 35:1154-1161 (2017)). Therefore, strategies to prevent AML relapse are urgently needed. Given that CD83 CAR T cells have potent lytic activity in xenogeneic GVHD prevention and are well tolerated by transplanted mice, experiments were conducted to investigate whether human myeloid leukemias potentially express CD83. It was found that CD83 is indeed expressed on the malignant myeloid K562, Thp-1, U937 and MOLM-13 cell lines ( Figure 7A 、 7B , Figure 15A 、 15B In addition, using the xCELLigence platform, CD83 CAR T cells demonstrated significant anti-tumor activity against K562 and Thp-1 cells ( Figure 7C 、 7D ). Therefore, human CD83 CAR T cells have the ability to prevent GVHD and provide direct killing of AML.

[0397] Human CD83 CAR T cells exhibit negligible on-target and off-tumor toxicity: Human AML antigens are often shared with progenitor stem cells. Although CD83 CAR T cells clearly kill AML targets, they have been shown to allow the growth and differentiation of hematopoietic stem cells in colony-forming units (CFUs). Figures 8A-8D Overall, the total number of colonies was similar in the mock T cell, CD83 CAR T cell, and medium treatment groups. Although a decrease in granulocyte / macrophage CFU was observed for CD83 CAR T cells, it was not significantly different compared with medium alone ( Figure 8B In addition, the colonies from granulocyte / erythrocyte / monocyte / megakaryocyte CFU and erythroid burst-forming unit were essentially the same in the treatment groups ( Figure 8C 、 8D These experiments provide evidence that human CD83 CAR T cells selectively kill AML while preserving normal hematopoietic function.

[0398] Discussion

[0399] The use of CAR T cells as a cellular immunotherapy to prevent GVHD is an innovative strategy, distinct from pharmacologic immunosuppression or adoptive transfer of donor Tregs. Targeting cells expressing CD83 effectively depletes inflammatory mature DCs as well as alloreactive CD4+ Tconv in the transplant recipient. Donor CD8+ T cells can also mediate GVHD (Okiyama N. et al., J Invest Dermatol 134:992-1000 (2014); Shindo T. et al., Blood 121:4617-4626 (2013)). While few CD8+ T cells express CD83, CD83 CAR T cells also significantly reduced the amount of donor CD8+ T cells ( Figure 16 ). Mechanistically, it is hypothesized that the in vivo elimination of alloreactive T cells drives the efficacy of these CAR T cells, as depletion of dendritic cells did not reduce xenogeneic GVHD. The in vivo depletion of alloreactive T effectors by CD83 CAR T cells also mediated a significant increase in the Treg: activated Tconv ratio, a clinically relevant indicator of control of GVHD (Koreth J. et al., N Engl J Med 365:2055-2066 (2011)).

[0400] CD83 CAR T cells significantly reduced pathogenic human Th1 and Th2 cells in vivo. Experiments using STAT4 and STAT6 knockout donor T cells demonstrated that Th1 and Th2 cells independently mediated 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 worsened murine GVHD (Nikolic, B. et al., J Clin Invest 105: 1289-1298 (2000)). To some extent, Th1 and Th2 cells caused tissue-specific injury to the intestine and lung, respectively (Yi T. et al., Blood 114: 3101-3112 (2009)). There are currently strategies targeting donor Th1 responses, and are primarily driven by neutralization of p40 cytokines or inhibition of associated downstream receptor signaling (Betts B.C. et al., Science translational medicine 9: eaai8269 (2017); Betts B.C. et al., Proc Natl Acad Sci U S A. 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, few methods simultaneously target pathogenic Th1 and Th2 cells. Thus, human CD83 CAR T cells represent a cellular product that can simultaneously suppress donor Th1 / Th2 responses following allo-HCT. Human Th17 cells were largely unaffected by CD83 CAR T cells, despite the fact that treated mice were clearly protected from GVHD. While donor Th17 cells can contribute to GVHD (Iclozan C. et al., Biol Blood Marrow Transplant 16: 170-178 (2010)), the lack of available Th1 cells can mitigate the pathogenicity of surviving Th17 cells (Yu Y. et al., Blood 118: 5011-5020 (2011)).

[0401] The disclosed data support that human CD83 CAR T cells provide durable protection against activated Tconv and GVHD mortality. While CD83 is not significantly expressed on human Tregs, mice treated with human CD83 CAR T cells exhibited a reduction in the amount of Tregs. This can be due to limited availability of CD4+ T cell precursors for Treg differentiation, or to reduced IL-2 concentration due to overall reduction in circulating donor T cells. In rodents, CD83 is involved in the stability of Tregs in vivo, while mice carrying 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 Tconv was significantly increased in mice treated with CD83 CAR T cells compared to controls. An increased ratio of Tregs to Tconv is a clinically relevant immune indicator and even correlates with response to Treg-directed GVHD therapy, 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 were well-tolerated in vivo and eliminated immune-mediated organ damage. Thus, the role of CD83 can be different in murine and human Tregs.

[0402] CD83 is a unique immunomodulatory molecule. In mice, soluble CD83 mediates immunosuppressive effects by enhancing Treg responses through indoleamine 2,3-dioxygenase and TGFp mechanisms (Bock F. et al., J Immunol 191 : 1965-1975 (2013)). The extracellular domain of human CD83 also showed impairment of allogeneic reactive T cell proliferation in vitro (Lechmann M. et al., J Exp Med 194: 1813-1821 (2001)). In contrast, direct neutralization of CD83 with monoclonal antibody 3C12C significantly reduced human T cell-mediated xenogeneic GVHD in vivo (Wilson J. et al., J Exp Med 206: 387-398 (2009)). CD83 antibodies also preserved Treg and anti-viral responses of 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 preserving critical effector and Treg functions. Unlike monoclonal antibodies, CD83 CAR T cells alone cause robust target cell killing; NK cell-mediated antibody-dependent cellular cytotoxicity is not required (Seldon T. A. et al., Leukemia 30: 692-700 (2016)). This is an advantage when rapid, efficient elimination of allogeneic reactive T cells is needed to prevent GVHD. In fact, human CD83-targeting CAR T cells provide durable GVHD prevention, even after a single infusion, detectable in mice for +100 days.

[0403] In addition to abrogating alloreactive T cells in GVHD prevention, CD83 emerges as a promising candidate to target myeloid malignancies. CD83 expression was observed on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, CD83 CAR T cells efficiently killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Therefore, experiments were performed in human CFU assays to assess stem cell killing, demonstrating negligible on-target / off-tumor toxicity. Although relapse remains an important cause of post-transplant failure and death, allo-HCT is often necessary to treat high-risk AML. Unlike the classic GVL mediated by HLA, CD83 CAR T cells selectively destroy malignant cells expressing CD83. Moreover, CD83 was recently found to be expressed on Hodgkin’s lymphoma as well (Li Z. et al., Haematologica 103:655-665 (2018)). Thus, CD83 CAR T cells can have efficacy in treating AML or HL without reliance on allo-HCT. Given the clinical success of CD19 CAR T cells in ALL and diffuse large B-cell lymphoma, this is a powerful transformation (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)).

[0404] In summary, CD83 CAR T cells represent the first human programmed cytolytic effector cell designed to prevent GVHD. The transformative potential of CD83 CAR T cells has been demonstrated in GVHD prevention, although it is anticipated to have advantages in preventing rejection following transplantation of solid organs or vascularized composite allotransplants. Furthermore, CD83 CAR T cells retain their killing activity even when exposed to calcineurin inhibitors. CD83 CAR T cells can overcome barriers in HLA disparity among hematopoietic cells and donor selection for solid organs and greatly expand the application of curative transplant procedures in patients in need. Importantly, CD83 CAR T cells provide a platform to eliminate alloreactive T cells without the need for broad inhibitory, nonselective calcineurin inhibitors or glucocorticoids. Moreover, the ability of CD83 CAR T cells to kill myeloid leukemia cells further expands their clinical impact. Thus, CD83 CAR T cells have the potential to reduce transplant-related mortality and improve outcomes following allo-HCT.

[0405] 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 the disclosed invention belongs. The publications cited herein and materials incorporated by reference are specifically incorporated by reference.

[0406] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating a myeloid malignancy in a subject, the method comprising administering to the subject an effective amount of 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.

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

3. The method of claim 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 method of claim 3, wherein the anti-CD83 scFv comprises a variable heavy (VH) having CDR1, CDR2, and CDR3 sequences. H ) domain and a variable light (V) having CDR1, CDR2, and CDR3 sequences L ) domain, wherein the V H The CDR1 sequence of the 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 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 domain comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15; L The CDR1 sequence comprises the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16; the V 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 the V L The CDR3 sequence of the domain comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:12, or SEQ ID NO:

18.

5. The method of claim 4, wherein the anti-CD83 scFv V H The domain comprises the amino acid sequence of 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.

6. The method of claim 4 or 5, wherein the anti-CD83 scFv V L The domain comprises the amino acid sequence of SEQ ID NO:20, SEQ ID NO:54, or SEQ ID NO:

55.

7. The method of any one of claims 1 to 6, wherein the anti-CD83 scFv comprises the amino acid sequence of 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.

8. The method of any one of claims 1 to 7, 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.

9. The method of any one of claims 1 to 8, wherein the CAR polypeptide is defined by the formula: SP-CD83-HG-TM-CSR-ISD; or SP-CD83-HG-TM-ISD-CSR Where "SP" stands for signal peptide, "CD83" represents the CD83 binding region, wherein "HG" represents an optional hinge domain, Where "TM" stands for transmembrane domain, "CSR" stands for costimulatory signaling region. wherein "ISD" stands for intracellular signaling domain, and Wherein "-" represents a divalent linker.

10. The method of any one of claims 1 to 9, wherein the intracellular signaling domain comprises a CD3ζ (CD3 zeta) signaling domain.

11. The method of any one of claims 1 to 10, further comprising administering a checkpoint inhibitor to the subject.

12. The method of claim 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 of any one of claims 1 to 12, wherein the myeloid malignancy comprises acute myeloid leukemia (AML).

14. The method of any one of claims 1 to 12, wherein the myeloid malignancy comprises Hodgkin's lymphoma.

15. The method of any one of claims 1 to 14, wherein the subject has been treated with hematopoietic stem cell transplantation.

16. The method of any one of claims 1 to 14, wherein the subject has not been treated with hematopoietic stem cell transplantation.

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