Chimeric antigen receptor (CAR) constructs having an NK receptor signaling domain

Abstract

Chimeric antigen receptor (CAR) polypeptides that can be used in conjunction with adoptive cell transfer to target and kill cancer are disclosed herein. Similar to other CARs, the disclosed CAR polypeptides include an ectodomain that includes a binding domain, a hinge domain, a transmembrane (TM) domain, and an endodomain that includes an endodomain containing the intracellular domain of an NK cell receptor and does not require a signaling domain from CD3 zeta (CD3ζ). Also disclosed are immune effector cells such as T cells or natural killer (NK) cells that have been modified to express these CARs. Accordingly, a method of providing anti-tumor immunity in a subject having a tumor-associated antigen-expressing cancer, involving adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CAR, is also disclosed.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,085, filed May 3, 2023; U.S. Provisional Patent Application No. 63 / 364,760, filed May 16, 2023; and U.S. Provisional Patent Application No. 63 / 365,291, filed May 25, 2022, which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a Sequence Listing submitted in ST.26 format entitled "320803_2660_Sequence_Listing," created on May 2, 2023. The contents of the Sequence Listing are hereby incorporated by reference in their entirety.

Background Art

[0003] Surgery, radiation therapy, and chemotherapy are standardly accepted techniques for the treatment of cancer, including leukemia, solid tumors, and metastases. Immunotherapy (also sometimes called biological therapy, biotherapy, or biological response modifier therapy), which uses the body's immune system directly or indirectly to shrink or eliminate cancer, has been studied for many years as an adjunct to conventional cancer treatment. The human immune system is an untapped resource for cancer treatment, and it is thought that effective treatments can be developed if the components of the immune system are properly utilized.

[0004] The main advancement in adoptive T cell therapy is the chimeric antigen receptor (CAR), a single-chain variable fragment (scFv) derived from an antibody fused to the signaling domain of the T cell receptor (TCR) (Davila, M.L., et al., Oncoimmunology, 2012.1(9):1577-1583). The intracellular domain of the first-generation CAR contains only CD3ζ, and the second-generation CAR also includes a co-stimulatory domain such as CD28 or 41BB. These second-generation CAR domains supported highly effective tumor killing in mice and led to the clinical evaluation of CAR T cell therapy in patients. The potential of CD19-targeted CAR T cells was confirmed by reports of a 90% complete remission rate in patients with B cell acute lymphoblastic leukemia (B-ALL) (Davila, M.L., et al., Sci Transl Med, 2014.6(224):224ra25, Maude, S.L., et al., N Engl J Med, 2014.371(16):1507-17). However, decreased persistence of CAR T cells and excessive T cell activation contribute to relapse and severe toxicity, respectively, suggesting an important need to understand CAR T cell biology (Gangadhar, T.C. and R.H. Vonderheide, Nat Rev Clin Oncol, 2014.11(2):91-9). Furthermore, relapse and toxicity are seen in all second-generation CARs, suggesting that the addition of co-stimulatory domains to the CAR improves efficacy at the expense of biological complications. SUMMARY OF THE INVENTION

[0005] Disclosed herein are chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to target and kill cancer. Similar to other CARs, the disclosed CAR polypeptides include an ectodomain that includes a binding domain, a hinge domain, a transmembrane (TM) domain, and an endodomain that includes a signaling region. However, unlike other CARs, the endodomain of the disclosed CAR polypeptides includes the intracellular domain of an NK cell receptor and does not require a signaling domain from CD3 zeta (CD3ζ).

[0006] Thus, the disclosed CAR polypeptides utilize a completely different signaling pathway (NKG2D / DAP10) from current CAR-T cells (such as CD3, OX40, ICOS, etc.). Since the signaling pathway does not depend on CD3ζ or ZAP70 signaling, it may be insensitive to PD-1-mediated inhibition. Furthermore, studies have shown that in T cells, NKG2D / DAP10 can bring about target killing and memory formation (Perez C, et al. J Immunother Cancer 2019), can replace the need for CD4 help (Zloza A, et al. Nat Med 2012 18:422-8), mediate resistance to immunosuppression by TGF-β, and enhance migratory properties.

[0007] Thus, a CAR polypeptide having an endodomain comprising a ligand-binding domain, a hinge domain, a transmembrane domain, and an intracellular domain of an NK cell receptor, or a fragment thereof capable of activating the killing of NK cells, is disclosed herein. In some embodiments, the CAR polypeptide further comprises an extracellular domain of an NK cell receptor, or a fragment thereof.

[0008] The disclosed polypeptides can further contain, in some embodiments, one or more DAP10 or additional motifs of the endodomain of other signaling motifs, such as OX40, ICOS, CD3, etc. However, in some embodiments, the CAR polypeptide lacks any other intracellular signaling domain or co-stimulatory domain, such as the CD3ζ, 41BB, and / or CD28 domains.

[0009] NK receptor NKG2D In some embodiments, the NK cell receptor is hNKG2D. In some embodiments, the intracellular (IC) domain of the NK cell receptor comprises the amino acid sequence MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS (SEQ ID NO: 1), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1. In some embodiments, the TM has the amino acid sequence PFFFCCFIAVAMGIRFIIMVTIW (SEQ ID NO: 2), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 2. Thus, in some embodiments, the CAR comprises the amino acid sequence PFFFCCFIAVAMGIRFIIMVTIWMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS (SEQ ID NO: 3), or is 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 3, and comprises the hNKG2D TM / IC domain that can be incorporated into the plasma membrane when cross-linked and can bind to DAP10. In some embodiments, the extracellular domain has the amino acid sequence MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS (SEQ ID NO: 37), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 37.In some embodiments, hNKG2D has the amino acid sequence MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 38), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 38.

[0010] CD16 In some embodiments, the NK cell receptor is hCD16 (FCGR3A). Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence KTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 4), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 4. In some embodiments, the TM has the amino acid sequence VSFCLVMVLLFAVDTGLYFSV (SEQ ID NO: 5), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 5. Thus, in some embodiments, the CAR comprises the amino acid sequence VSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 6), or is 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 6 and comprises the hCD16 TM / IC domain, which can be incorporated into the plasma membrane when cross-linked and can bind to FcεR1γ. In some embodiments, the extracellular domain has the amino acid sequence MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQ (SEQ ID NO: 39), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 39.In some embodiments, hNKG2D has the amino acid sequence MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 40), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 40.

[0011] NKp30 In some embodiments, the NK cell receptor is hNKp30 (NCR3). Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence GSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPG (SEQ ID NO: 7), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 7. In some embodiments, the TM has the amino acid sequence AGTVLLLRAGFYAVSFLSVAV (SEQ ID NO: 8), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 8. Thus, in some embodiments, the CAR comprises the amino acid sequence AGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPG (SEQ ID NO: 9), or is 55, 56, 57, 58, 59, 60, 61, 61, 62, 64, or 65 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 9, the hNKp30 TM / IC which, when cross-linked, is incorporated into the plasma membrane and can bind to FcεR1γ. In some embodiments, the extracellular domain has the amino acid sequence MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLG (SEQ ID NO: 41), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 41.In some embodiments, hNKG2D has the amino acid sequence MAWMLLLILIMVHPGSCALWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPGG (SEQ ID NO: 42), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 42.

[0012] NKG2C In some embodiments, the NK cell receptor is hNKG2C. Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence IPFLEQNNSSPNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLASILPSSWIGVFRNSSHHPWVTINGLAFKHKIKDSDNAELNCAVLQVNRLKSAQCGSSMIYHCKHKL (SEQ ID NO: 10), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 10. In some embodiments, the TM has the amino acid sequence LTAEVLGIICIVLMATVLKTIVL (SEQ ID NO: 11), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 11. Thus, in some embodiments, the CAR comprises the amino acid sequence LTAEVLGIICIVLMATVLKTIVLIPFLEQNNSSPNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLASILPSSWIGVFRNSSHHPWVTINGLAFKHKIKDSDNAELNCAVLQVNRLKSAQCGSSMIYHCKHKL (SEQ ID NO: 12), or is 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 amino acids in length and comprises an hNKG2C TM / IC domain having a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 12, which is incorporated into the plasma membrane when crosslinked and can bind to DAP12. In some embodiments, the extracellular domain has the amino acid sequence MSKQRGTFSEVSLAQDPKRQQRKPKGNKSSISGTEQEIFQVELNLQNPSLNHQGIDKIYDCQGLLPPPEK (SEQ ID NO: 43), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 43.In some embodiments, hNKG2D has the amino acid sequence MSKQRGTFSEVSLAQDPKRQQRKPKGNKSSISGTEQEIFQVELNLQNPSLNHQGIDKIYDCQGLLPPPEKLTAEVLGIICIVLMATVLKTIVLIPFLEQNNSSPNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLASILPSSWIGVFRNSSHHPWVTINGLAFKHKIKDSDNAELNCAVLQVNRLKSAQCGSSMIYHCKHKL (SEQ ID NO: 44), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 44.

[0013] 2B4 In some embodiments, the NK cell receptor is h2B4. Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 13), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 13. In some embodiments, the TM has the amino acid sequence FLVIIVILSALFLGTLACFCV (SEQ ID NO: 14), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 14. Thus, in some embodiments, the CAR comprises the h2B4 TM / IC domain having the amino acid sequence FLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 15), or a variant / fragment thereof that is 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 amino acids in length and has at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 15, which is capable of signaling when cross-linked.In some embodiments, the extracellular domain has the amino acid sequence MLGQVVTLILLLLLKVYQGKGCQGSADHVVSISGVPLQLQPNSIQTKVDSIAWKKLLPSQNGFHHILKWENGSLPSNTSNDRFSFIVKNLSLLIKAAQQQDSGLYCLEVTSISGKVQTATFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWYRGSKLIQTAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQEFRFWP (SEQ ID NO: 45), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 45. In some embodiments, hNKG2D has the amino acid sequence MLGQVVTLILLLLLKVYQGKGCQGSADHVVSISGVPLQLQPNSIQTKVDSIAWKKLLPSQNGFHHILKWENGSLPSNTSNDRFSFIVKNLSLLIKAAQQQDSGLYCLEVTSISGKVQTATFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWYRGSKLIQTAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQEFRFWPFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 46), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 46.

[0014] DNAM-1 In some embodiments, the NK cell receptor is hDNAM-1. Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 16), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 16. In some embodiments, the TM has the amino acid sequence GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 17), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 17. Thus, in some embodiments, the CAR comprises the amino acid sequence GGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 18), or is 30, 31, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, or 82 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 18, the hDNAM TM / IC domain, which is capable of signaling when cross-linked. In some embodiments, the extracellular domain has the amino acid sequence MTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVA (SEQ ID NO: 47), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 47.In some embodiments, hNKG2D has the amino acid sequence MTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVAGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 48), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 48.

[0015] CD137 In some embodiments, the NK cell receptor is hCD137 (TNFRSF9). Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 19), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 19. In some embodiments, the TM has the amino acid sequence IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 20), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 20. Thus, in some embodiments, the CAR comprises the amino acid sequence IISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 21), or is 59, 60, 61, 61, 62, 64, 65, 66, 67, 68, or 69 amino acids in length and comprises an hCD137 TM / IC domain having a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 21, which is capable of signaling when crosslinked. In some embodiments, the extracellular domain has the amino acid sequence MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ (SEQ ID NO: 49), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 49.In some embodiments, hNKG2D has the amino acid sequence MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 50), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 50.

[0016] OX-40 In some embodiments, the NK cell receptor is hOX-40. Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 22), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 22. In some embodiments, the TM has the amino acid sequence LLLVASVIQGLGLLLCFTYICLHFSAL (SEQ ID NO: 23), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 23. Thus, in some embodiments, the CAR comprises the amino acid sequence LLLVASVIQGLGLLLCFTYICLHFSALVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 24), or is 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 amino acids in length and comprises an hOX-40 TM / IC domain having a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 24, which can signal when crosslinked. In some embodiments, the extracellular domain has the amino acid sequence MERVQPLEENVGNAARPRFERNK (SEQ ID NO: 51), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 51.In some embodiments, hNKG2D has the amino acid sequence MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 52), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 52.

[0017] CD27 In some embodiments, the NK cell receptor is hCD27. Thus, in some embodiments, the intracellular domain of the NK cell receptor comprises the amino acid sequence QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 25), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 25. In some embodiments, the TM has the amino acid sequence ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 26), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 26. Thus, in some embodiments, the CAR comprises the amino acid sequence ILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 27), or is 59, 60, 61, 61, 62, 64, 65, 66, 67, 68, or 69 amino acids in length and comprises an hCD27 TM / IC domain having a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 27, which can be incorporated into the plasma membrane when crosslinked and can bind to TRAF. In some embodiments, the extracellular domain has the amino acid sequence MNAVIISPSSLQFQKLRPVYTRIAGFKVAPLNKCSLARHFLEPGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO: 53), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 53.In some embodiments, hNKG2D has the amino acid sequence MNAVIISPSSLQFQKLRPVYTRIAGFKVAPLNKCSLARHFLEPGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 54), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 54.

[0018] 2DS5 In some embodiments, the NK cell receptor is killer cell immunoglobulin-like receptor 2DS5 (KIR2DS5). In some embodiments, the intracellular (IC) domain of the NK cell receptor comprises the amino acid sequence LLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVSYA (SEQ ID NO: 28), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 28. In some embodiments, the TM has the amino acid sequence VLIGTSVVKLPFTILLFFL (SEQ ID NO: 29), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 29. Thus, in some embodiments, the CAR comprises the amino acid sequence VLIGTSVVKLPFTILLFFLLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVSYA (SEQ ID NO: 30), or is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 30, the hNKG2D TM / IC domain, which is incorporated into the plasma membrane when cross-linked and can bind to DAP12. In some embodiments, the extracellular domain has the amino acid sequence MSLMVISMACVAFFLLQGAWPHEGFRRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGTFNHTLRLIGEHIDGVSKGNFSIGRMTQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNRTFQADFPLDPATHGGTYRCFGSFRDSPYEWSKSSDPLLVSVTGNSSNSWPSPTEPSSETGNPRHLH (SEQ ID NO: 55), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 55.In some embodiments, hNKG2D has the amino acid sequence MSLMVISMACVAFFLLQGAWPHEGFRRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGTFNHTLRLIGEHIDGVSKGNFSIGRMTQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNRTFQADFPLDPATHGGTYRCFGSFRDSPYEWSKSSDPLLVSVTGNSSNSWPSPTEPSSETGNPRHLHVLIGTSVVKLPFTILLFFLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVSYA (SEQ ID NO: 56), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 56.

[0019] KIR3DS1 In some embodiments, the NK cell receptor is KIR3DS1. In some embodiments, the intracellular (IC) domain of the NK cell receptor comprises the amino acid sequence HRWCSNKKKCCCNGPRACREQK (SEQ ID NO: 31), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 31. In some embodiments, the TM has the amino acid sequence ILIGTSVVKIPFTILLFFLL (SEQ ID NO: 32), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 32. Thus, in some embodiments, the CAR has the hNKG2D TM / IC domain having the amino acid sequence ILIGTSVVKIPFTILLFFLLHRWCSNKKKCCCNGPRACREQK (SEQ ID NO: 33), or a variant / fragment thereof, having a length of 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 amino acids and having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 33, which is incorporated into the plasma membrane when crosslinked and can bind to DAP12. In some embodiments, the extracellular domain has the amino acid sequence MLLMVVSMACVGLFLVQRAGPHMGGQDKPFLSAWPSAVVPRGGHVTLRCHYRHRFNNFMLYKEDRIHVPIFHGRIFQEGFNMSPVTTAHAGNYTCRGSHPHSPTGWSAPSNPMVIMVTGNHRKPSLLAHPGPLVKSGERVILQCWSDIMFEHFFLHREWISKDPSRLVGQIHDGVSKANFSIGSMMRALAGTYRCYGSVTHTPYQLSAPSDPLDIVVTGLYEKPSLSAQPGPKVQAGESVTLSCSSRSSYDMYHLSREGGAHERRLPAVRKVNRTFQADFPLGPATHGGTYRCFGSFRHSPYEWSDPSDPLLVSVTGNPSSSWPSPTEPSSKSGNLRHLH (SEQ ID NO: 57), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 57.In some embodiments, hNKG2D has the amino acid sequence MLLMVVSMACVGLFLVQRAGPHMGGQDKPFLSAWPSAVVPRGGHVTLRCHYRHRFNNFMLYKEDRIHVPIFHGRIFQEGFNMSPVTTAHAGNYTCRGSHPHSPTGWSAPSNPMVIMVTGNHRKPSLLAHPGPLVKSGERVILQCWSDIMFEHFFLHREWISKDPSRLVGQIHDGVSKANFSIGSMMRALAGTYRCYGSVTHTPYQLSAPSDPLDIVVTGLYEKPSLSAQPGPKVQAGESVTLSCSSRSSYDMYHLSREGGAHERRLPAVRKVNRTFQADFPLGPATHGGTYRCFGSFRHSPYEWSDPSDPLLVSVTGNPSSSWPSPTEPSSKSGNLRHLHILIGTSVVKIPFTILLFFLLHRWCSNKKKCCCNGPRACREQK (SEQ ID NO: 58), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 58.

[0020] NKp80 / KLRF1 In some embodiments, the NK cell receptor is NKp80 / KLRF1. In some embodiments, the intracellular (IC) domain of the NK cell receptor comprises the amino acid sequence MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYK (SEQ ID NO: 34), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 34. In some embodiments, the TM has the amino acid sequence ILLGISGTVNGILTLTLISLI (SEQ ID NO: 35), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 35. Thus, in some embodiments, the CAR comprises the amino acid sequence MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLI (SEQ ID NO: 36), or is 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 amino acids in length and has a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 36 and comprising the hNKG2D TM / IC domain that can incorporate signaling when cross-linked. In some embodiments, the extracellular domain has the amino acid sequence LLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY (SEQ ID NO: 59), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 59.In some embodiments, hNKG2D has the amino acid sequence MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLILLVSQGVLLKCQKGSCSNATQYEDTGDLKVNNGTRRNISNKDLCASRSADQTVLCQSEWLKYQGKCYWFSNEMKSWSDSYVYCLERKSHLLIIHDQLEMAFIQKNLRQLNYVWIGLNFTSLKMTWTWVDGSPIDSKIFFIKGPAKENSCAAIKESKIFSETCSSVFKWICQY (SEQ ID NO: 60), or a variant / fragment thereof having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 60.

[0021] Binding domain In some embodiments, the binding domain is an antibody fragment that specifically binds to a target molecule on a cell, such as a tumor-associated antigen (TAA). For example, the antigen-binding domain can be a Fab or single-chain variable fragment (scFv) of an antibody that specifically binds to the target molecule. In some embodiments, the binding domain is an aptamer that specifically binds to the tar target molecule. For example, the binding domain can be a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule. The binding domain can also be the natural ligand of the target molecule, or a variant and / or fragment thereof that can bind to the target molecule.

[0022] CD33 For example, an scFv that selectively binds to CD33 is described in US2020 / 0223920, which is incorporated by reference in its entirety for the description of these antibodies and their sequences. For example, in some embodiments, the anti-CD33 region of the disclosed antibody or CAR is derived from hybridoma 27A3, 33G3, 36C2, 6A11, 35D5, 38G5, or a combination thereof. In some embodiments, the anti-CD33 region (e.g., scFv) has a variable heavy (V H) domain, and a variable light (V) having a CDR1 sequence, a CDR2 sequence and a CDR3 sequence L ) domain can be included.

[0023] For example, in some embodiments, the CDR1 sequence of the V H ) domain includes the amino acid sequence GFTFSNYG (SEQ ID NO: 61), GYTFTSYW (SEQ ID NO: 62), or GFSLSRYS (SEQ ID NO: 63), and the CDR2 sequence of the V H ) domain includes the amino acid sequence ISSGGGDT (SEQ ID NO: 64), IHPSDSET (SEQ ID NO: 65), or IWGGGYT (SEQ ID NO: 66), and the CDR3 sequence of the V H ) domain includes the amino acid sequence ARDYGGTWDYFDY (SEQ ID NO: 67), AREEGQLGHGGAMDY (SEQ ID NO: 68), or ARYIDSSGYDY (SEQ ID NO: 69), and the CDR1 sequence of the V L ) includes the amino acid sequence QDISKY (SEQ ID NO: 70), QTVNDD (SEQ ID NO: 71), SSVSY (SEQ ID NO: 72), or ENIYSY (SEQ ID NO: 73), and the CDR2 sequence of the V L ) domain includes the amino acid sequence YTS, YVS, DTS, or NAK, and the CDR3 sequence of the V L ) domain includes the amino acid sequence QQGDTFPWT (SEQ ID NO: 78), QQDYSSPYT (SEQ ID NO: 79), QQWSSNPLT (SEQ ID NO: 80), or QHHYGTPYT (SEQ ID NO: 81), or any combination thereof.

[0024] Thus, in some embodiments, the anti-CD33 scFv V HThe domain comprises the amino acid sequence EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQTSDKRLEWVASISSGGGDTYYPDNVKGRFTISRENAKNTLYLQMSSLNSEDTALYYCARDYGGTWDYFDYWGQGTTLTVSS (SEQ ID NO: 82), QVQLQQPGAELVRPGVSVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKAILTVDKSSSTAYMQLSSPTSEDSAVYYCAREEGQLGHGGAMDYWGQGTSVTVSS (SEQ ID NO: 83), or QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSS (SEQ ID NO: 84).

[0025] In some embodiments, the anti-CD33 scFv V L The domain comprises the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK (SEQ ID NO: 85), SIVMTQTPKFLLVSAGDRVTITCKASQTVNDDVAWYQQKPGQSPKLLIYYVSNRHTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK (SEQ ID NO: 86), QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 87), or DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIK (SEQ ID NO: 88).

[0026] The heavy and light chains are preferably separated by a linker. Linkers suitable for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 89).

[0027] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence: EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQTSDKRLEWVASISSGGGDTYYPDNVKGRFTISRENAKNTLYLQMSSLNSEDTALYYCARDYGGTWDYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK (SEQ ID NO: 90, 6A11HC1_LC).

[0028] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence: QVQLQQPGAELVRPGVSVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKAILTVDKSSSTAYMQLSSPTSEDSAVYYCAREEGQLGHGGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK (SEQ ID NO: 91, 6A11HC2_LC).

[0029] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence: QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQTVNDDVAWYQQKPGQSPKLLIYYVSNRHTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK (SEQ ID NO: 92, 27A3HC_LC1).

[0030] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence: QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 93, 27A3HC_LC2).

[0031] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence: QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIK (SEQ ID NO: 94, 27A3HC_LC3).

[0032] CD123 For example, scFvs that selectively bind to CD123 are described in 2020 / 0165348, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences. For example, in some embodiments, the anti-CD123 scFv is derived from hybridoma 3F5, 4E10, 12H5, 15A12, 17E7, 12H11, or combinations thereof. In some embodiments, the anti-CD123 scFv can include 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. For example, in some embodiments, the CDR1 sequence of the V H domain includes the amino acid sequence GYTFTDYN (SEQ ID NO: 95), the CDR2 sequence of the V H domain includes the amino acid sequence INPNNGGT (SEQ ID NO: 96), the CDR3 sequence of the V H domain includes the amino acid sequence ARKGYGGNYDYFDY (SEQ ID NO: 97), the CDR1 sequence of V L includes the amino acid sequence QSIGTS (SEQ ID NO: 98), the CDR2 sequence of the V L domain includes the amino acid sequence YASx (SEQ ID NO: 99), and the CDR3 sequence of the V L domain includes the amino acid sequence QQSNSWPYT (SEQ ID NO: 100).

[0033] In some embodiments, the CDR1 sequence of the V H domain includes the amino acid sequence GFNIKDTY (SEQ ID NO: 101) or GFSLSTYGMG (SEQ ID NO: 102), the CDR2 sequence of the V H domain includes the amino acid sequence IDPANGNT (SEQ ID NO: 103) or IYWDDDK (SEQ ID NO: 104), the CDR3 sequence of the V H domain includes the amino acid sequence ALYYYGGSLDY (SEQ ID NO: 105) or AQSLIYDGYYGFAY (SEQ ID NO: 106), and the CDR1 sequence of V L includes the amino acid sequence QSLLYSGNQKNY (SEQ ID NO: 107).L The CDR2 sequence of the domain contains the amino acid sequence WASx (SEQ ID NO: 108), and V L The CDR3 sequence of the domain contains the amino acid sequence QQYYSYPRT (SEQ ID NO: 109).

[0034] In some embodiments, V H The CDR1 sequence of the domain contains the amino acid sequence GYTFTYYG (SEQ ID NO: 110), and V H The CDR2 sequence of the domain contains the amino acid sequence INTYSGVP (SEQ ID NO: 111), and V H The CDR3 sequence of the domain contains the amino acid sequence ARWIYYSDLYGMDY (SEQ ID NO: 112), and V L The CDR1 sequence of contains the amino acid sequence QSIVHSNGDTY (SEQ ID NO: 113), and V L The CDR2 sequence of the domain contains the amino acid sequence KVSx (SEQ ID NO: 114), and V L The CDR3 sequence of the domain contains the amino acid sequence FQGSHVPWT (SEQ ID NO: 115).

[0035] In some embodiments, V H The CDR1 sequence of the domain contains the amino acid sequence GYTFSSYW (SEQ ID NO: 116) or GYTLTTYL (SEQ ID NO: 117), and V H The CDR2 sequence of the domain contains the amino acid sequence INPSSGYT (SEQ ID NO: 118) or INPNSGSS (SEQ ID NO: 119), and V H The CDR3 sequence of the domain contains the amino acid sequence ARDGNYDHWYFDV (SEQ ID NO: 120) or AIRHYGGSLFDY (SEQ ID NO: 121), and V L The CDR1 sequence of contains the amino acid sequence QDINSY (SEQ ID NO: 122) or QSLLNSRTRKNY (SEQ ID NO: 123), and V L The CDR2 sequence of the domain contains the amino acid sequence WAS or RAN, and V L The CDR3 sequence of the domain contains the amino acid sequence LQYDELLT (SEQ ID NO: 126) or EQSYNLFT (SEQ ID NO: 127).

[0036] In some embodiments, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GYTFTDYN (SEQ ID NO: 128), GFNIKDTY (SEQ ID NO: 129), GFSLSTYGMG (SEQ ID NO: 130), GYTFTYYG (SEQ ID NO: 131), GYTFSSYW (SEQ ID NO: 132), or GYTLTTYL (SEQ ID NO: 133), and V H The CDR2 sequence of the domain comprises the amino acid sequence INPNNGGT (SEQ ID NO: 96), IDPANGNT (SEQ ID NO: 103), IYWDDDK (SEQ ID NO: 104), INTYSGVP (SEQ ID NO: 111), INPSSGYT (SEQ ID NO: 134), or INPNSGSS (SEQ ID NO: 135), and V H The CDR3 sequence of the domain comprises the amino acid sequence ARKGYGGNYDYFDY (SEQ ID NO: 97), ALYYYGGSLDY (SEQ ID NO: 105), AQSLIYDGYYGFAY (SEQ ID NO: 106), ARWIYYSDLYGMDY (SEQ ID NO: 112), ARDGNYDHWYFDV (SEQ ID NO: 136), or AIRHYGGSLFDY (SEQ ID NO: 137), and V L The CDR1 sequence of comprises the amino acid sequence QSIGTS (SEQ ID NO: 98), QSLLYSGNQKNY (SEQ ID NO: 107), QSIVHSNGDTY (SEQ ID NO: 113), QDINSY (SEQ ID NO: 122), or QSLLNSRTRKNY (SEQ ID NO: 138), and V L The CDR2 sequence of the domain comprises the amino acid sequence YASx, WASx, KVSx, or RAN, and V L The CDR3 sequence of the domain comprises the amino acid sequence QQSNSWPYT, QQYYSYPRT, FQGSHVPWT, LQYDELLT (SEQ ID NO: 140), or EQSYNLFT (SEQ ID NO: 141), or any combination thereof.

[0037] Thus, in some embodiments, the anti-CD123 scFv V HThe domain comprises the amino acid sequence: EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGTINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARKGYGGNYDYFDYWGQGTTLTVSS (SEQ ID NO: 142, 3F5HC1), EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTIYASKFQGKATITADTSSNTAYMQLSSLTSGDTAVYYCALYYYGGSLDYWGQGTTLTVSS (SEQ ID NO: 143, 12H1HC1), QVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSNNQVFLKITSVDTADTATYYCAQSLIYDGYYGFAYWGQGTLVTVSA (SEQ ID NO: 144, 12H1HC2), QIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLEWMGWINTYSGVPTYADDFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARWIYYSDLYGMDYWGQGTSVTVSS (SEQ ID NO: 145, 12H2HC1), QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSS (SEQ ID NO: 146, 15A12HC1), or QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSS (SEQ ID NO: 147, 15A12HC2).

[0038] In some embodiments, the anti-CD123 scFv V LThe domain includes the amino acid sequence: DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK (SEQ ID NO: 148, 3F5LC1), DIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK (SEQ ID NO: 149, 12H1LC1), DVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGDTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYHCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 150, 12H2LC), DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK (SEQ ID NO: 151, 15A12LC1), or DIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK (SEQ ID NO: 152, 15A12LC2).

[0039] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It includes EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGTINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARKGYGGNYDYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK (SEQ ID NO: 153, 3F5HC1_LC).

[0040] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It includes EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTIYASKFQGKATITADTSSNTAYMQLSSLTSGDTAVYYCALYYYGGSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK (SEQ ID NO: 154, 12H1HC1_LC1).

[0041] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It includes QVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSNNQVFLKITSVDTADTATYYCAQSLIYDGYYGFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK (SEQ ID NO: 155, 12H1HC2_LC1).

[0042] In some embodiments, the anti-CD123 scFv has the amino acid sequence: QIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLEWMGWINTYSGVPTYADDFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARWIYYSDLYGMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGDTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYHCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 156, 12H2HC1_LC1), and includes.

[0043] In some embodiments, the anti-CD123 scFv has the amino acid sequence: QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK (SEQ ID NO: 157, 15A12HC1_LC1), and includes.

[0044] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It contains QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK (SEQ ID NO: 158, 15A12HC1_LC2).

[0045] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It contains QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK (SEQ ID NO: 159, 15A12HC2_LC1).

[0046] In some embodiments, the anti-CD123 scFv has the amino acid sequence: It contains QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK (SEQ ID NO: 160, 15A12HC2_LC2).

[0047] CD99 For example, scFvs that selectively bind to CD99 are described in US20200397882, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences.

[0048] In some embodiments, the anti-CD99 region of the disclosed antibody or CAR is derived from hybridoma 1H3, 4C5, 9G12, 3C7, 2F11, 4D5, 4F4, 6A10, or combinations thereof. In some embodiments, the anti-CD99 region (e.g., scFv) can have 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.

[0049] In some embodiments, the CDR1 sequence of the V H domain comprises the amino acid sequence GFDIKDTY (SEQ ID NO: 161), TYAMY (SEQ ID NO: 162), TFWM (SEQ ID NO: 163), or TFWMQ (SEQ ID NO: 164), the CDR2 sequence of the V H domain comprises the amino acid sequence IDPANGDT (SEQ ID NO: 165), RIRSKVNNYATYYADSVKDRFT (SEQ ID NO: 166), or TIYPGDDDTRYTQKFKGRAT (SEQ ID NO: 167), the CDR3 sequence of the V H domain comprises the amino acid sequence ARRGGLS (SEQ ID NO: 168), DPMDY (SEQ ID NO: 169), or SGYERGPYYFDS (SEQ ID NO: 170), or SGYERGPYYF (SEQ ID NO: 171), and the CDR1 sequence of the V L domain comprises the amino acid sequence GNIHNY (SEQ ID NO: 172), GSSKSLLHSNGNTYLY (SEQ ID NO: 173), KSSQSLLCRSNQKNYLA (SEQ ID NO: 174), or KSSQSLLYRSNQKNYLA (SEQ ID NO: 175), the CDR2 sequence of the V L domain comprises the amino acid sequence NAK, RVSNLAS (SEQ ID NO: 177), or WASTRES (SEQ ID NO: 178).L The CDR3 sequence of the domain comprises the amino acid sequence QHFWSTPWT (SEQ ID NO: 179), MQHLEYPYT (SEQ ID NO: 180), or QQYYSYPLT (SEQ ID NO: 181).

[0050] Thus, in some embodiments, the anti-CD99 V H domain comprises the amino acid sequence EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 182, 1H3H7).

[0051] Thus, in some embodiments, the anti-CD99 V H domain comprises the amino acid sequence EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 183, 4C5E2).

[0052] Thus, in some embodiments, the anti-CD99 V H domain comprises the amino acid sequence EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 184, 4C5H10).

[0053] Thus, in some embodiments, the anti-CD99 V H domain comprises the amino acid sequence It includes QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 185, 9G12C9).

[0054] Therefore, in some embodiments, the anti-CD99 V H domain has the amino acid sequence DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 186, 9G12G6 HB1), and includes it.

[0055] In some embodiments, the anti-CD99 V H domain has the amino acid sequence QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 187, 9G12G6 HB3), and includes it.

[0056] In some embodiments, the anti-CD99 V L domain has the amino acid sequence DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 188, 1H3H9), and includes it.

[0057] In some embodiments, the anti-CD99 V L domain is It includes GNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 189, 1H3H7 LC1).

[0058] In some embodiments, the anti-CD99 V L domain is It includes GNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 190, 1H3H7 LC2).

[0059] In some embodiments, the anti-CD99 V L domain is It includes DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 191, 4C5E2).

[0060] In some embodiments, the anti-CD99 V L domain is It includes DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 192, 4C5H10).

[0061] In some embodiments, the anti-CD99 V L domain is It contains DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 193, 9G12C9).

[0062] In some embodiments, the anti-CD99 V L domain contains the amino acid sequence DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 194, 9G12G6).

[0063] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 195).

[0064] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 196).

[0065] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 197).

[0066] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 198).

[0067] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 199).

[0068] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 200).

[0069] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 201).

[0070] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 202), and includes.

[0071] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 203), and includes.

[0072] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 204), and includes.

[0073] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 205), and includes.

[0074] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 206), and includes.

[0075] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 207).

[0076] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 208).

[0077] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 209).

[0078] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 210), and includes.

[0079] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 211), and includes.

[0080] In some embodiments, the anti-CD99 scFv has the amino acid sequence: EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 212), and includes.

[0081] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 213), and includes.

[0082] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 214), and includes.

[0083] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[SEQ ID NO:215]]: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK.

[0084] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[SEQ ID NO:216]]: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK.

[0085] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[SEQ ID NO:217]]: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0086] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 218), and includes.

[0087] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 219), and includes.

[0088] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 220), and includes.

[0089] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 221), and includes.

[0090] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 222), and includes.

[0091] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[ID=]], DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 223).

[0092] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[ID=]], DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 224).

[0093] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes [[ID=]], DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 225).

[0094] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 226), and includes.

[0095] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 227), and includes.

[0096] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 228), and includes.

[0097] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 229), and comprises.

[0098] In some embodiments, the anti-CD99 scFv has the amino acid sequence: QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 230), and comprises.

[0099] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 231).

[0100] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 232).

[0101] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It contains DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 233).

[0102] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 234), and includes.

[0103] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 235), and includes.

[0104] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 236), and includes.

[0105] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 237), and includes.

[0106] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 238), and includes.

[0107] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 239), and includes.

[0108] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 240), and includes.

[0109] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 241), and includes.

[0110] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 242).

[0111] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 243).

[0112] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 244).

[0113] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 245), and includes.

[0114] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 246), and includes.

[0115] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 247), and includes.

[0116] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 248), and includes.

[0117] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 249), and includes.

[0118] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 250).

[0119] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 251).

[0120] In some embodiments, the anti-CD99 scFv has the amino acid sequence: It includes: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 252).

[0121] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS (SEQ ID NO: 253), and includes.

[0122] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 254), and includes.

[0123] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 255), and includes.

[0124] In some embodiments, the anti-CD99 scFv has the amino acid sequence: DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 256).

[0125] CLEC12A For example, an scFv that selectively binds to CLEC12A is described in US20200345779, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences. For example, in some embodiments, the anti-CLEC12A region of the disclosed antibody or CAR is derived from hybridoma 1F3, 1F8, 1G3, 2A10, 3F12, 4E3, 4E10, 5B2, 5F10, 6C7, 9A2, 11C7, 11H1, 12D6, or a combination thereof. In some embodiments, the anti-CLEC12A region (e.g., scFv) can have 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.

[0126] In some embodiments, the CDR1 sequence of the V H domain includes the amino acid sequence GFTFSSFA (SEQ ID NO: 257), SFAVS (SEQ ID NO: 258), or SHDMS (SEQ ID NO: 259), and the V HThe CDR2 sequence of the domain includes the amino acid sequence ISSGGAYT (SEQ ID NO: 260), or TISSGGAYTFYKDSVKGRFT (SEQ ID NO: 261), or YISGGGTNIYYSDTVKGRFT (SEQ ID NO: 262), and V H The CDR3 sequence of the domain includes the amino acid sequence ARHSGYDGYYLYAMDY (SEQ ID NO: 263), HSGYDGYYLYAMDY (SEQ ID NO: 264), or PNYNYGGSWFAY (SEQ ID NO: 265), and V L The CDR1 sequence of includes the amino acid sequence SSVHY (SEQ ID NO: 266), ASSSVHYMH (SEQ ID NO: 267), or SASSSVHYMH (SEQ ID NO: 268), and V L The CDR2 sequence of the domain includes the amino acid sequence DTS or DTSKLAS (SEQ ID NO: 270), and V L The CDR3 sequence of the domain includes the amino acid sequence QQWTSNPPT (SEQ ID NO: 271).

[0127] In some embodiments, the anti-CLEC12A V H domain includes the amino acid sequence ELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 272, 1F3H8).

[0128] Thus, in some embodiments, the anti-CLEC12A V H domain has the nucleic acid sequence It is encoded by GAACTAATACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTTCCTTTGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGAGCTTACACCTTCTATAAAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTGCAAGACATAGCGGCTATGATGGTTACTACCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 273, 1F3H8).

[0129] In some embodiments, the anti-CLEC12A V H domain comprises the amino acid sequence GVQCELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 274, 1F3A10).

[0130] Thus, in some embodiments, the anti-CLEC12A V H domain has the nucleic acid sequence It is encoded by GGTGTCCAGTGTGAACTAATACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTTCCTTTGCCGTGTCCTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGAGCTTACACCTTCTATAAAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTGCAAGACATAGCGGCTATGATGGTTACTACCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 275, 1F3A10).

[0131] In some embodiments, the anti-CLEC12A V H domain comprises the amino acid sequence EVQLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSA (SEQ ID NO: 276, 1F3F3).

[0132] Thus, in some embodiments, the anti-CLEC12A V H domain has the nucleic acid sequence It is encoded by GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTAGTGCAGCCGGGAGGGTCCCTGAAAGTCTCCTGTGCAGTTTCCGGACTCGCTTTCAGCAGCCATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGCGGCTGGAGTGGGTCGCATACATTAGTGGAGGTGGTACTAATATCTATTATTCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAAGACACAGCCATTTATTACTGTGCAAGACCCAATTATAATTACGGCGGTTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 277, 1F3F3).

[0133] In some embodiments, the anti-CLEC12A V L domain comprises the amino acid sequence QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 278, 1F3H8, 1F3F3, 1F3A10).

[0134] Thus, in some embodiments, the anti-CLEC12A V L domain has the nucleic acid sequence It is encoded by CAAATTGTTCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTACATTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGTCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATTAAACG (SEQ ID NO: 279, 1F3H8, 1F3F3, 1F3A10).

[0135] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: ELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 280, 1F3H8).

[0136] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: It contains GVQCELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 281, 1F3A10).

[0137] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: EVQLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 282, 1F3F3).

[0138] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 283).

[0139] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGVQCELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 284), and includes.

[0140] In some embodiments, the anti-CLEC12A scFv has the amino acid sequence: QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSA (SEQ ID NO: 285), and includes.

[0141] CD83 For example, an scFv that selectively binds to CD83 is described in US2020 / 0108098, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences.

[0142] For example, in some embodiments, the CDR1 sequence of the V H domain includes the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 286), SDGIS (SEQ ID NO: 287), or SNAMI (SEQ ID NO: 288), and the V HThe CDR2 sequence of the domain includes the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 289), IISSGGNTYYASWAKG (SEQ ID NO: 290), or AMDSNSRTYYATWAKG (SEQ ID NO: 291), and V H The CDR3 sequence of the domain includes the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 292), VVGGTYSI (SEQ ID NO: 293), or GDGGSSDYTEM (SEQ ID NO: 294), and V L The CDR1 sequence of V includes the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 295), QSSQSVYNNDFLS (SEQ ID NO: 296), or QSSQSVYGNNELS (SEQ ID NO: 297), and V L The CDR2 sequence of the domain includes the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 298), YASTLAS (SEQ ID NO: 299), or QASSLAS (SEQ ID NO: 300), and V L The CDR3 sequence of the domain includes the amino acid sequence GSSDSSGYV (SEQ ID NO: 301), TGTYGNSAWYEDA (SEQ ID NO: 302), or LGEYSISADNH (SEQ ID NO: 303).

[0143] For example, in some embodiments, V H The CDR1 sequence of the domain includes the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 304), and V H The CDR2 sequence of the domain includes the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 305), and V H The CDR3 sequence of the domain includes the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 306), and V L The CDR1 sequence of V includes the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 307), and V L The CDR2 sequence of the domain includes the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 308), and V L The CDR3 sequence of the domain includes the amino acid sequence GSSDSSGYV (SEQ ID NO: 309).

[0144] For example, in some embodiments, V HThe CDR1 sequence of the domain contains the amino acid sequence SDGIS (SEQ ID NO: 310), and V H The CDR2 sequence of the domain contains the amino acid sequence IISSGGNTYYASWAKG (SEQ ID NO: 311), and V H The CDR3 sequence of the domain contains the amino acid sequence VVGGTYSI (SEQ ID NO: 312), and V L The CDR1 sequence of contains the amino acid sequence QSSQSVYNNDFLS (SEQ ID NO: 313), and V L The CDR2 sequence of the domain contains the amino acid sequence YASTLAS (SEQ ID NO: 314), and V L The CDR3 sequence of the domain contains the amino acid sequence TGTYGNSAWYEDA (SEQ ID NO: 315).

[0145] For example, in some embodiments, V H The CDR1 sequence of the domain contains the amino acid sequence SNAMI (SEQ ID NO: 316), and V H The CDR2 sequence of the domain contains the amino acid sequence AMDSNSRTYYATWAKG (SEQ ID NO: 317), and V H The CDR3 sequence of the domain contains the amino acid sequence GDGGSSDYTEM (SEQ ID NO: 318), and V L The CDR1 sequence of contains the amino acid sequence QSSQSVYGNNELS (SEQ ID NO: 319), and V L The CDR2 sequence of the domain contains the amino acid sequence QASSLAS (SEQ ID NO: 320), and V L The CDR3 sequence of the domain contains the amino acid sequence LGEYSISADNH (SEQ ID NO: 321).

[0146] In some embodiments, the anti-CD83 scFv V H domain contains the amino acid sequence: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 322, VH-GBM00).

[0147] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence: QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 323, VL-GBM00).

[0148] In some embodiments, the anti-CD83 scFv V H domain comprises the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSNNAINWVRQAPGKGLEWIGYIWSGGLTYYANWAEGRFTISKTSTTVDLKMTSPTIEDTATYFCARGINNSALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 324, 20D04).

[0149] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDMRAPTQLLGLLLLWLPGARCADVVMTQTPASVSAAVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYRTSTLASGVSSRFKGSGSGTEYTLTISGVQCDDVATYYCQCTSGGKFISDGAAFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 325, 20D04).

[0150] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFTISDYDLSWVRQAPGEGLKYIGFIAIDGNPYYATWAKGRFTISKTSTTVDLKITAPTTEDTATYFCARGAGDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 326, 11G05).

[0151] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDTREPTQLLGLLLLWLPGARCADVVMTQTPASVSAAVGGTVTINCQSSKNVYNNNWLSWFQQKPGQPPKLLIYYASTLASGVPSRFRGSGSGTQFTLTISDVQCDDAATYYCAGDYSSSSDNGFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC SEQ ID NO: 327, 11G05).

[0152] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVHCQSVEESGGRLVTPGTPLTLTCTASGFSRSSYDMSWVRQAPGKGLEWVGVISTAYNSHYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGGSWLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 328, 14C12).

[0153] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDXRAPTQLLGLLLLWLPGARCALVMTQTPASVSAAVGGTVTINCQSSQSVYDNDELSWYQQKPGQPPKLLIYALASKLASGVPSRFKGSGSGTQFALTISGVQCDDAATYYCQATHYSSDWYLTFGGGTEVVVKGFPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGTENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 329, 14C12).

[0154] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYDMTWVRQAPGKGLEWIGIIYASGTTYYANWAKGRFTISKTSTTVDLKVTSPTIGDTATYFCAREGAGVSMTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 330, 020B08).

[0155] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDMRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQASQSISTYLDWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQQGYTHSNVDNVFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 331, 020B08).

[0156] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVSPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTSTTVDLEVTSLTTEDTATYFCSREHAGYSGDTGHLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVGIGPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 332, 006G05).

[0157] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDMRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVAIKCQASQSVSSYLAWYQQKPGQPPKPLIYEASMLAAGVSSRFKGSGSGTDFTLTISDLECDDAATYYCQQGYSISDIDNAFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC (SEQ ID NO: 333, 006G05).

[0158] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSDGISWVRQAPGKGLEWIGIISSGGNTYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARVVGGTYSIWGQGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 334, 96G08).

[0159] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAPVGGTVTINCQSSQSVYNNDFLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCTGTYGNSAWYEDAFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC (SEQ ID NO: 335, 96G08).

[0160] In some embodiments, anti-CD83 scFv V H The domain contains the amino acid sequence: METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSNAMIWVRQAPREGLEWIGAMDSNSRTYYATWAKGRFTISRTSSITVDLKITSPTTEDTATYFCARGDGGSSDYTEMWGPGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 336, 95F04).

[0161] In some embodiments, anti-CD83 scFv V LThe domain contains the amino acid sequence: MDTRAPTQLLGLLLLWLPGATFAQAVVTQTTSPVSAPVGGTVTINCQSSQSVYGNNELSWYQQKPGQPPKLLIYQASSLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCLGEYSISADNHFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC (SEQ ID NO: 337, 95F04).

[0162] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence: QVQLVQSGGAVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAAVSYDGSNKYYADFVKGRFTISRDNPKNTLYLQMNSLRADDTAVYYCARRGGLDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA (SEQ ID NO: 338).

[0163] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence: LTQPPPASGTPGQQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYYGNDQRPSGVPDRFSASKSGTSASLAISGLQSEDEAHYYCAAWDGSLNGGVIFGGGTKVTLG (SEQ ID NO: 339).

[0164] In some embodiments, the anti-CD83 scFv V LThe domain includes the amino acid sequence: VTQPPSASGTPGQRVTISCSGSSSNIGTNPVNWYQQLPGTAPKLLIYTTDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLSGLYVFGTGTKVTVLG (SEQ ID NO: 340).

[0165] In some embodiments, the anti-CD83 scFv V L The domain includes the amino acid sequence: MTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQRPGQSPQPLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVQAEDVGVYYCMQSLQLWTFGQGTKVEIKR (SEQ ID NO: 341).

[0166] In some embodiments, the anti-CD83 scFv V L The domain includes the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLIHSDGNTYLDWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLRISRVEAEDIGVYYCMQATHWPRTFGQGTKVEIKR (SEQ ID NO: 342).

[0167] In some embodiments, the anti-CD83 scFv V L The domain includes the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVDSAGNTFLHWFHQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 343).

[0168] In some embodiments, the anti-CD83 scFv V L The domain includes the amino acid sequence: LTQSPLSLPVTLGQPASISCKSSQSLVDSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 344).

[0169] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence: MTQSPLSLPVTLGQPASISCRSSQSLVHSDGNMYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQATQPTWTFGQGTKLEIKR (SEQ ID NO: 345).

[0170] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence: MTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSATYYCQQTYQGTKLEIKR (SEQ ID NO: 346).

[0171] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence: MTQSPSSLSASVGHPVTITCRASQSLISYLNWYHQKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLQPENFASYYCQHTDSFPRTFGHGTKVEIKR (SEQ ID NO: 347).

[0172] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence: LTQPPSASGTPGQGVTISCRGSTSNIGNNVVNWYQHVPGSAPKLLIWSNIQRPSGIPDRFSGSKSGTSASLAISGLQSEDQAVYYCAVWDDGLAGWVFGGGTTVTVLS (SEQ ID NO: 348).

[0173] In some embodiments, the anti-CD83 scFv V LThe domain contains the amino acid sequence: MTQAPVVSVALEQTVRITCQGDSLAIYYDFWYQHKPGQAPVLVIYGKNNRPSGIPHRFSGSSSNTDSLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTNLTVLG (SEQ ID NO: 349).

[0174] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence: LTQSPLSLPVTLGQPASISCKSNQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYCMQGTQWPRTFGGQGTKLDIKR (SEQ ID NO: 350).

[0175] In some embodiments, the anti-CD83 scFv V H The domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 351, VH-GBM01).

[0176] In some embodiments, the anti-CD83 scFv V H The domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 352, VH-GBM02).

[0177] In some embodiments, the anti-CD83 scFv V HThe domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 353, VH-GBM03).

[0178] In some embodiments, the anti-CD83 scFv V H The domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 354, VH-GBM04).

[0179] In some embodiments, the anti-CD83 scFv V H The domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 355, VH-GBM05).

[0180] In some embodiments, the anti-CD83 scFv V H The domain is humanized and contains the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 356, VH-GBM06).

[0181] In some embodiments, the anti-CD83 scFv V LThe domain is humanized and contains the amino acid sequence: QLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 357, VL-GBM01).

[0182] In some embodiments, the anti-CD83 scFv V L The domain is humanized and contains the amino acid sequence: LPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 358, VL-GBM02).

[0183] In some embodiments, the anti-CD83 scFv has the amino acid sequence: QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAASSGGGGSGGGGSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAA (SEQ ID NO: 359).

[0184] In some embodiments, the anti-CD83 scFv has the amino acid sequence: It includes: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTV (SEQ ID NO: 360).

[0185] In some embodiments, the anti-CD83 scFv includes the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 361).

[0186] In some embodiments, the anti-CD83 scFv includes the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 362).

[0187] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 363).

[0188] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 364).

[0189] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 365).

[0190] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 366).

[0191] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 367).

[0192] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 368).

[0193] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 369).

[0194] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 370).

[0195] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 371).

[0196] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 372).

[0197] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence: QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 373).

[0198] PSCA For example, the scFv that selectively binds to PSCA is described in US2021 / 0379108, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences. For example, in some embodiments, the anti-PSCA scFv has the amino acid sequence: MVLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWIWIRQHPGKGLEWIGYIYYNGNTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGITMIRGYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSVSASVGDRVTITCRASRGISSWLAWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPRTFGQGTKVEIKAAAFV (SEQ ID NO: 374).

[0199] OR2H1 For example, the scFv that selectively binds to OR2H1 is described in WO2021257905, which is hereby incorporated by reference in its entirety for the description of these antibodies and their sequences.

[0200] In some embodiments of the anti-OR2H1 scFv, the CDR1 sequence of the V H domain contains the amino acid sequence GYIFTGYY (SEQ ID NO: 375), and the CDR2 sequence of the V H domain contains the amino acid sequence NAKSGE (SEQ ID NO: 376), and the CDR3 sequence of the V H domain contains the amino acid sequence GPLL (SEQ ID NO: 377). The CDR1 sequence of the L contains the amino acid sequence SSGSSNIGSNFVS (SEQ ID NO: 378), and the CDR2 sequence of the V L domain contains the amino acid sequence RNNQRPS (SEQ ID NO: 379), and the CDR3 sequence of the V L domain contains the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 380). In some embodiments, the anti-OR2H1 scFv V HThe domain is humanized and has the amino acid sequence: QVQLQQSGAEVKKPGESLKISCKGSGYIFTGYYMHWVRQAPGQRPEWLGRMNAKSGEADSAQRFQGRVTMTRDTSINTAYMELRDLRSDDTAVYYCTRGPLLWGQGTLVTVS (SEQ ID NO: 381). In some embodiments, anti-OR2H1 V L The domain is humanized and has the amino acid sequence: QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKTGTSASLAISGLRSEDEADYYCAAWDDSVRGPVFGGGTELTVLAAA (SEQ ID NO: 382).

[0201] In some embodiments of the anti-OR2H1 scFv, V H The CDR1 sequence of the domain includes the amino acid sequence GYIFTGYY (SEQ ID NO: 383), and V H The CDR2 sequence of the domain includes the amino acid sequence MNAKSGEA (SEQ ID NO: 384), and V H The CDR3 sequence of the domain includes the amino acid sequence TRGPLL (SEQ ID NO: 385), and V L The CDR1 sequence of includes the amino acid sequence SSNIGSNF (SEQ ID NO: 386), and V L The CDR2 sequence of the domain includes the amino acid sequence RNN, and V L The CDR3 sequence of the domain includes the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 74). In some embodiments, the anti-OR2H1 scFv V H The domain is humanized and has the amino acid sequence: MAQVQLQQSGAEVKKPGESLKISCKGSGYIFTGYYMHWVRQAPGQRPEWLGRMNAKSGEADSAQRFQGRVTMTRDTSINTAYMELRDLRSDDTAVYYCTRGPLLWGQGTLVTVSS (SEQ ID NO: 75). In some embodiments, anti-OR2H1 V LThe domain is humanized and contains the amino acid sequence: QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKTGTSASLAISGLRSEDEADYYCAAWDDSVRGPVFGGGTELTVL (SEQ ID NO: 75).

[0202] In some embodiments of the anti-OR2H1 scFv, the V H domain CDR1 sequence contains the amino acid sequence SSNIGSNF (SEQ ID NO: 77), and the V H domain CDR2 sequence contains the amino acid sequence RNN, and the V H domain CDR3 sequence contains the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 125), and the V L CDR1 sequence of contains the amino acid sequence GYTFTSNY (SEQ ID NO: 139), and the V L domain CDR2 sequence contains the amino acid sequence INPSGGRT (SEQ ID NO: 176), and the V L domain CDR3 sequence contains the amino acid sequence ARSHCSGGSCYSIDY (SEQ ID NO: 269). In some embodiments, the anti-OR2H1 scFv V H domain is humanized and contains the amino acid sequence: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGIINPSGGRTSYAQKFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARSHCSGGSCYSIDYWGQGTLVTVSS (SEQ ID NO: 387). In some embodiments, the anti-OR2H1 V L domain is humanized and contains the amino acid sequence: QPVLTQSSSASASLGSSVKLTCTLSSGHSGYIIAWHQQQPGKAPRYLMKVEGSGSYNKGSGIPERFSGSSSGADRYLTISNLQSEDEADYYCETWDSNTHVFGTGTKVTVL (SEQ ID NO: 124).

[0203] Additional scFv sequences are disclosed in 7,575,742, 8,298,525, 8,636,997, 9,394,368, 9,447,194, 9,624,306, 9,765,342, 10,767,184, 10,815,487, 10,738,312, 10,738,313, 10,844,387, 10,829,767, 10,900,042, 10,876,123, 10,815,488, 10,829,768, 10,837,019, and 10,829,769, US2013 / 0071414, US2014 / 0023647, US2014 / 0271635, US2014 / 0286987, US2015 / 0283178, US2015 / 0344844, US2016 / 0046724, US2016 / 0046700, US2016 / 0152723, US2016,0297884, US2016,0303230, US2016,0376375, US2017,0183418, US2017,0218337, US2017,0226216, US2017 / 0283504, US2017 / 0368101, WO2016 / 130598, and WO2019 / 195017, which are hereby incorporated by reference in their entirety for their teachings of these scFv sequences.

[0204] In some embodiments, the scFv is the anti-BCMA02 scFv (ABECMA; idecabtagene vicleucel) disclosed in 9,765,342. In some embodiments, the scFv is the anti-CD19 scFv (BREYANZI; lisocabtagene maraleucel) disclosed in 9,701,758. In some embodiments, the scFv is the anti-CD19 scFv (KYMRIAH; tisagenlecleucel) disclosed in US2014 / 0271635. In some embodiments, the scFv is the anti-CD19 scFv (TECARTUS; brexucabtagene autoleucel) disclosed in US2015 / 0344844. In some embodiments, the scFv is the anti-CD19 scFv (YESCARTA; axicabtagene ciloleucel) disclosed in US2015 / 0344844.

[0205] Also disclosed are isolated nucleic acid sequences encoding the disclosed CAR polypeptides, vectors containing these isolated nucleic acids, and cells containing these vectors. For example, the cells can be immune effector cells selected from the group consisting of alpha-beta T cells, gamma-delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, and regulatory T cells.

[0206] In some embodiments, when the antigen-binding domain of the CAR binds to a TAA on the tumor, the cells exhibit anti-tumor immunity.

[0207] Also disclosed is a method of providing anti-tumor immunity in a subject having a cancer that expresses a TAA, comprising administering to the subject an effective amount of immune effector cells genetically modified with the disclosed TAA-specific CAR.

[0208] Details of one or more embodiments of the present invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of the Drawings

[0209]

Figure 1A

Figure 1B

Figure 2

Modes for Carrying Out the Invention

[0210] Before explaining the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus can of course vary. It should also be understood that the terminology used herein is for the purpose of describing only the particular embodiments and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0211] When a range of values is provided, each intervening value, to one tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated value or intervening value within the stated range, is to be understood as being included within the present disclosure, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the present disclosure, subject to any specifically excluded limitations within the stated range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the present disclosure.

[0212] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

[0213] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference, and are incorporated herein by reference to disclose and describe methods and / or materials in connection with the cited publications. Any citation of a publication is for its disclosure prior to the filing date, and the present disclosure should not be construed as admitting that the present disclosure has a right to antedate such a publication for its prior disclosure. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently verified.

[0214] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the present disclosure. Any of the recited methods can be performed in the order of the recited events, or in any other order that is logically possible.

[0215] Embodiments of the present disclosure use techniques such as chemistry, biology, etc., within the scope of the relevant art, unless otherwise indicated.

[0216] The following examples are provided to fully disclose and describe to those skilled in the art the methods and the use of the probes disclosed and claimed herein. Although efforts have been made to ensure accuracy with respect to numerical values (e.g., amounts, temperatures, etc.), some errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, temperature is in °C, and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0217] Before the embodiments of the present disclosure are described in detail, it will be understood that the present disclosure is not limited to specific materials, reagents, reactants, manufacturing processes, etc., and can vary, unless otherwise indicated. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In the present disclosure, the steps can also be performed in a different order if logically possible.

[0218] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0219] Definitions 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 codes for amino acids and are represented as follows. A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0220] The term "antibody" refers to an immunoglobulin, its derivatives that maintain specific binding ability, and proteins having a binding domain that is homologous or nearly homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources or can be produced partially or completely synthetically. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, included within the term "antibody" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind to a target antigen.

[0221] The term "aptamer" refers to an oligonucleic acid or peptide molecule that binds to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamer can recognize the target molecule with high affinity and specificity by adapting to its unique three-dimensional structure. A "nucleic acid aptamer" is a DNA or RNA oligonucleic acid that binds to a target molecule through its three-dimensional structure, thereby inhibiting or suppressing the function of such a molecule. Nucleic acid aptamers can be composed of DNA, RNA, or combinations thereof. A "peptide aptamer" is a combinatorial protein molecule having a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast two-hybrid conditions, which enhance the likelihood that the selected peptide aptamer will be stably expressed and correctly folded in an intracellular context.

[0222] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and any harmful side effects in the subject.

[0223] The term "chimeric molecule" refers to a single molecule created by joining two or more molecules that exist separately in their natural state. A single chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecule is a fusion protein.

[0224] The term "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. A fusion protein can be formed by chemical coupling of the constituent polypeptides or 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 conventional techniques in molecular biology by ligating two genes in-frame into a single nucleic acid and then expressing the nucleic acid in a suitable host cell under conditions in which the fusion protein is produced.

[0225] The term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that can be aligned for purposes of comparison. If a position in the comparison sequences is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of 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, e.g., FASTA, which is available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings, or BLAST. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to a particular polypeptide described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, percent similarity is based on the BLASTP positive score and percent sequence identity is based on the BLASTP match score. A BLASTP "match" indicates the number and portions of the total residues in a high scoring sequence pair that are identical, and a BLASTP "positive" indicates the number and portions of residues that have an alignment score having a positive value and are similar to each other. 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 are contemplated and encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides can be deduced using the genetic code and obtained by conventional means, particularly by back-translating their amino acid sequences using the genetic code.

[0226] The term "nucleic acid" refers to a natural or synthetic molecule that contains a single nucleotide or two or more nucleotides linked by a phosphate group at the 3'-position of one nucleotide to the 5'-end of another nucleotide. Nucleic acids are not limited by length and thus can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0227] The term "operably linked" 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, an operative binding of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds, and transcribes the DNA.

[0228] The terms "peptide", "protein", and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule that contains two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another amino acid.

[0229] The term "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0230] The term "protein domain" refers to a part of a protein, a part of multiple proteins, or the entire protein that exhibits structural integrity, and this determination can be based on the amino acid composition of a part of a protein, a part of multiple proteins, or the entire protein.

[0231] As used herein, "spacer" refers to a peptide that binds a protein, including a fusion protein. Generally, a spacer has no specific biological activity other than binding a protein or maintaining some minimum distance or other spatial relationship between proteins. However, the constituent amino acids of a spacer can be selected to affect some properties of the molecule, such as the folding of the molecule, the net charge, or the hydrophobicity.

[0232] As used herein, the term "specifically binds" when referring to a polypeptide (including an antibody) or a receptor refers to a binding reaction that determines that a protein or polypeptide or receptor is present in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions (e.g., immunoassay conditions in the case of an antibody), if a particular ligand or antibody does not bind appreciably to other proteins present in a sample, or to other proteins that the ligand or antibody might encounter in vivo, then it "specifically binds" to that particular "target" (e.g., an antibody specifically binds to an endothelial antigen). Generally, a first molecule that "specifically binds" to a second molecule has an affinity constant (Ka) greater than about 10 5 M -1 (e.g., 10 6 M -1 、10 7 M -1 、10 8 M -1 、10 9 M -1 、10 10 M -1 、10 11 M -1 、and 10 12 M -1 or greater).

[0233] As used herein, the term "specific delivery" refers to the preferential association of a molecule with a cell or tissue having a particular target molecule or marker, and is not applicable to cells or tissues lacking that target molecule. Of course, it is recognized that some non-specific interaction can occur between the molecule and non-target cells or tissues. Nevertheless, specific delivery can be distinguished as being mediated through specific recognition of the target molecule. Typically, specific delivery results in a much stronger association between the delivered molecule and cells having the target molecule than between the delivered molecule and cells lacking the target molecule.

[0234] As used herein, the term "subject" refers to any individual that is the target of administration or treatment. A subject can be a vertebrate, for example, a mammal. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, e.g., a physician.

[0235] As used herein, the term "therapeutically effective" means that the amount of the composition used is sufficient to reduce one or more causes or symptoms of a disease or disorder. Such reduction only needs to be a reduction or modification and does not need to be an elimination.

[0236] As used herein, the terms "transformation" and "transfection" mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell, including the introduction of the nucleic acid into the chromosomal DNA of the cell.

[0237] The term "treatment" refers to the medical management of a patient with the intention of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at improving a disease, condition, or disorder, and also causal treatment, i.e., treatment directed at removing the cause of the related disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed for the alleviation of symptoms rather than the cure of a disease, condition, or disorder; prophylactic treatment, i.e., treatment directed at minimizing the onset of, or partially or completely inhibiting, the related disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed at improving the related disease, condition, or disorder.

[0238] The term "variant" refers to a conservative amino acid substitution, a non-conservative amino acid substitution (i.e., a degenerate variant), a substitution within the wobble position of each codon (i.e., DNA and RNA) encoding an amino acid, an amino acid added to the C-terminus of a peptide, or an amino acid or peptide sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0239] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid linked to the vector sequence into a cell. The term "expression vector" includes any vector (e.g., plasmid, cosmid, or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to transcriptional control elements).

[0240] Chimeric antigen receptor (CAR) having an NKG2D intracellular domain CARs generally incorporate an antigen recognition domain derived from the 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). Chimeric antigen receptors (CARs) that can be expressed in immune effector cells are disclosed herein to enhance anti-tumor activity against cancer.

[0241] The disclosed CARs generally consist of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain contains a binding region and is involved in antigen recognition. Optionally, the CAR contains a signal peptide (SP) so that it can be glycosylated and anchored to the cell membrane of immune effector cells. The transmembrane (TM) domain, as its name implies, binds the ectodomain to the endodomain and is present within the cell membrane when expressed by the cell. The endodomain is the tip of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. The disclosed CARs contain the intracellular domain of NKG2D in the endodomain.

[0242] In some embodiments, the disclosed CARs have the formula: SP-BD-HG-TM-NKRIC as defined by wherein "SP" represents an optional signal peptide, "BD" represents a target binding domain, "HG" represents an optional hinge domain, "TM" represents a transmembrane domain, "NKRIC" represents the intracellular domain of an NK cell receptor, and "-" represents a peptide bond or a linker.

[0243] 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, which is hereby incorporated by reference in its entirety for the teachings of these CAR models.

[0244] For example, the CAR can be a TRUCK, universal CAR, self-driving CAR, armored CAR, self-destructive CAR, conditional CAR, labeled CAR, TenCAR, dual CAR, or sCAR.

[0245] TRUCK (T cells redirected for universal cytokine killing) co-expresses a chimeric antigen receptor (CAR) and an anti-tumor cytokine. Expression of the cytokine can be constitutive or induced by T cell activation. When targeted by the specificity of the CAR, pro-inflammatory cytokines are locally produced to recruit endogenous immune cells to the tumor site, enhancing the anti-tumor response.

[0246] Universal allogeneic CAR T cells are modified to not express the endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0247] Self-driving CAR co-expresses a CAR that binds to tumor ligands and a chemokine receptor, thereby enhancing tumor homing.

[0248] CAR T cells engineered to be resistant to immunosuppression (armored CARs) can be genetically engineered to no longer express various immune checkpoint molecules (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)), along with immune checkpoint switch receptors, or can be administered with monoclonal antibodies that block immune checkpoint signaling.

[0249] Self-destructive CARs can be designed to encode the CAR using RNA delivered by electroporation. Alternatively, inducible apoptosis of T cells can be achieved based on ganciclovir binding to thymidine kinase in genetically modified lymphocytes, or the recently described activation system of human caspase 9 by a small molecule dimerizer.

[0250] Conditional CAR T cells do not respond by default or are switched "off" until the addition of a small molecule to complete the circuit, allowing for complete transduction of both signal 1 and signal 2, thereby activating the CAR T cells. Alternatively, T cells can be engineered to express an adapter-specific receptor that has affinity for a secondary antibody administered subsequently that targets the target antigen.

[0251] Labeled CAR T cells express a CAR and a tumor epitope to which existing monoclonal antibody agents bind. In settings of intolerable side effects, administration of the monoclonal antibody removes the CAR T cells and alleviates symptoms without further off-tumor effects.

[0252] Tandem CAR (TanCAR) T cells express a single CAR consisting of two linked single-chain variable fragments (scFvs) with different affinities fused to an intracellular co-stimulatory domain(s) and a CD3ζ domain. TanCAR T cell activation is achieved only when the target cell co-expresses both targets.

[0253] The safety chimeric antigen receptor (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells that co-express a standard CAR are only activated when they encounter target cells that have the standard CAR target but lack the sCAR target.

[0254] The binding 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 having more exogenous recognition components such as simple ectodomains (e.g., the CD4 ectodomain for recognizing HIV-infected cells) and linked cytokines (leading to the recognition of cells having cytokine receptors). In fact, almost anything that binds to a given target with high affinity can be used as the antigen recognition region.

[0255] The end domain is the tip of the CAR that transmits a signal to immune effector cells after antigen recognition and activates at least one of the normal effector functions of immune effector cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity including cytokine secretion. The disclosed CAR includes, in the end domain, the intracellular domain of NKG2D. However, in some embodiments, the end domain may further include the “intracellular signaling domain” of the T cell receptor (TCR) and an optional co-receptor. For example, the end domain of the CAR can further include the CD3ζ signaling domain by itself or can be designed to be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the cytoplasmic domain of the CAR can further include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and ligands that specifically bind to NKG2D. Thus, while the CAR is primarily exemplified with CD28 as the co-stimulatory signaling element, other co-stimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0256] In some embodiments, the CAR comprises a hinge sequence. The hinge sequence is a short sequence of amino acids that promotes flexibility of the antibody (see, e.g., Woof et al. Nat. Rev. Immunol., 4(2):89-99 (2004)). The hinge sequence can be positioned between the antigen recognition portion 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 the CD8a molecule or the CD28 molecule. In some embodiments, the hinge sequence is derived from an NK receptor, preferably the same NK receptor from which the intracellular domain is derived.

[0257] The transmembrane domain can be derived from either a natural or a synthetic source. When 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 the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, and PAG / Cbp (i.e., including at least these transmembrane regions). Alternatively, the transmembrane domain can be synthetic, in which case it contains mainly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. A short oligolinker or polypeptide linker, such as 2-10 amino acids in length, can form a bond between the transmembrane domain and the endoplasmic domain of the CAR.

[0258] In some embodiments, the CAR can have two or more transmembrane domains that can be repeats of the same transmembrane domain or can be different transmembrane domains.

[0259] In some embodiments, the CAR is a multi-chain CAR as described in WO2015 / 039523, which is incorporated by reference for the purposes of this teaching. The multi-chain CAR can include distinct extracellular ligand-binding and signaling domains within different transmembrane polypeptides. The signaling domain can be designed to be constructed in a juxtamembrane position, which forms a flexible structure similar to that of a native receptor and confers optimal signaling.

[0260] In some embodiments, the binding domain is a single-chain variable fragment (scFv) antibody. The affinity / specificity of the scFv is largely determined by specific sequences within the complementarity-determining regions (CDRs) in the heavy chain (V H ) and the light chain (V L ). Each V H sequence and V L sequence has three CDRs (CDR1, CDR2, CDR3).

[0261] In some cases, the binding domain is an affinity-matured scFv. In some cases, the binding domain has a dissociation constant (KD) for the TAA of less than 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, or 10 nM.

[0262] In some embodiments, the binding domain is derived from a native antibody such as a monoclonal antibody. In some cases, the antibody is human. In some cases, the antibody has been altered to reduce immunogenicity when administered to a human. For example, the alteration can include one or more techniques selected from the group consisting of chimerization, humanization, CDR grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequences.

[0263] Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T cell-mediated immune response. The binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the antigen-binding domain will depend on the specific type of cancer being treated. Tumor antigens are known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA IX, CS-1, MUC1, BCMA, bcr-abl, HER2, beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, Cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid, PLAC1, RU1, RU2(AS), intestinal carboxylesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYP1B1, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 variants, Ras variants, gp100, prostain, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, Melan-A / MART1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUCl, HER2, and any combination thereof.,

[0264] Non-limiting examples of tumor antigens include the following: differentiation antigens such as tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed fetal antigens such as CEA; overexpressed cancer genes such as p53, Ras, HER-2 / neu, and mutant tumor suppressor genes; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other major, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASl, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0265] Nucleic acids and vectors Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed CARs that enable the expression of the CARs in the disclosed immune effector cells.

[0266] The nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art using standard techniques such as screening a library from cells expressing the gene, deriving from vectors known to contain the gene, or directly isolating them from cells and tissues containing them. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0267] Expression of the nucleic acid encoding the CAR 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 include transcriptional and translational termination factors, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0268] The disclosed nucleic acids can be cloned into several 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. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0269] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vector is a lentiviral or retroviral vector.

[0270] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into the vector using techniques known in the art and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to the cells of interest either in vivo or ex vivo.

[0271] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the MND (myeloproliferative sarcoma virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters including but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include but are not limited to the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0272] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30 to 110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. Since the spacing between promoter elements is often flexible, promoter function is retained when the elements are inverted or moved relative to each other.

[0273] To evaluate the expression of a CAR polypeptide or a part thereof, the expression vector introduced into cells can also contain either, or both, a selectable marker gene and / or a reporter gene, which can facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker can be carried on a separate DNA fragment and can be used in a co-transfection procedure. Appropriate regulatory sequences can be adjacent to both the selectable marker and the reporter gene to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0274] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and whose expression encodes a polypeptide that manifests by some readily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes include those encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct having a minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as a promoter. Such promoter regions can be ligated to a reporter gene and used to evaluate agents for their ability to regulate promoter-driven transcription.

[0275] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0276] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0277] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.

[0278] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0279] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid can associate with a lipid. The nucleic acid associated with a lipid can be encapsulated within the aqueous interior of a liposome, can be interspersed within the lipid bilayer of a liposome, can be attached to a liposome via a linking molecule that associates with both the liposome and the oligonucleotide, can be entrapped within a liposome, can be complexed with a liposome, can be dispersed in a lipid-containing solution, can be mixed with a lipid, can be combined with a lipid, can be contained as a suspension in a lipid, can contain micelles, or can be complexed with micelles, or in other cases, can associate with a lipid. Lipid, lipid / DNA or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "disrupted" structure. They can also simply be interspersed in solution and, in some cases, form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be of natural or synthetic origin. For example, lipids include lipid droplets that naturally occur within the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, N.Y.), cholesterol ("Choi") can be obtained from Calbiochem-Behring, dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0280] immune effector cells Also disclosed are immune effector cells that are modified to express a disclosed CAR (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, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from several sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spinal cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed against surface markers specific to the cells to be positively selected, for example, by incubating with antibody-conjugated beads for a time sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of an immune effector cell population can be achieved by negative selection using a combination of antibodies directed against surface markers specific to the cells to be negatively selected.

[0281] In some embodiments, immune effector cells include any white blood cells involved in the body's defense against infection and foreign substances. For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells can include T lymphocytes.

[0282] T cells, or T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer (NK) cells, by the presence of a T cell receptor (TCR) on their 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 different functions.

[0283] Helper T cells (T H cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). When activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response. These cells can differentiate into one of several subtypes, including T H 1, T H 2, T H 3, T H 17, T H 9, or T FH and secrete different cytokines to promote different types of immune responses.

[0284] Cytotoxic T cells (T C cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8 + T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8+ cells can be inactivated into an anergic state via IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases.

[0285] Memory T cells are a subset of antigen-specific T cells that persist for long periods after an infection has resolved. When re-exposed to cognate antigen, they rapidly expand into large numbers of effector T cells and thus provide the immune system with "memory" of past infections. Memory cells can be either CD4 + or CD8 + . Memory T cells typically express the cell surface protein CD45RO.

[0286] Regulatory T cells (T reg cells) were previously known as suppressor T cells and are important for maintaining immunological tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that escaped the negative selection process in the thymus. Two major classes of CD4 + T reg cell-intrinsic T reg cells and adaptive T reg cells have been described.

[0287] Natural killer T (NKT) cells (so as not to be confused with natural killer (NK) cells) bridge the adaptive and innate immune systems. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0288] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T is a cytotoxic CD8 + T lymphocyte. In some embodiments, the T cells include γδ T cells and have a different T cell receptor (TCR) with one γ chain and one δ chain instead of an α chain and a β chain.

[0289] Natural killer (NK) cells can kill virus-infected cells and transformed cells and constitute an important cell subset of the innate immune system, CD56 + CD3 - are large granular lymphocytes (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+ T lymphocytes, NK cells do not require prior sensitization, can exert cytotoxicity against tumor cells, 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 cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and potentially lethal complications of off-target, off-tumor effects. NK cells have a known role as killers of cancer cells, and impairment of NK cells has been widely documented to be important for the progression of MM (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676, Fauriat C, et al. Leukemia 2006 20:732-733), but the means by which NK cell-mediated anti-MM activity can be enhanced were largely uninvestigated prior to the disclosure of CARs.

[0290] Treatment methods Immune effector cells expressing the disclosed CARs can induce an anti-tumor immune response against TAA-expressing cancer cells. The anti-tumor immune response induced by the disclosed CAR-modified immune effector cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific for the TAA.

[0291] The adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anti-cancer therapeutic. After collection of the patient's immune effector cells, the cells can be genetically engineered to express the disclosed CAR and then infused back into the patient.

[0292] The disclosed CAR-modified immune effector cells can be administered as a pharmaceutical composition, alone or in combination with a diluent and / or in combination with other components such as IL-2, IL-15, or other cytokines or cell populations. Briefly, the pharmaceutical composition can include the target cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. In some embodiments, the compositions used in the disclosed methods are formulated for intravenous administration. The pharmaceutical composition can be administered by any method appropriate for treating MM. The amount and frequency of administration are determined by factors such as the patient's condition and the severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0293] When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount", or "therapeutic amount" is indicated, the exact amount of the composition of the invention administered can be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). The pharmaceutical composition containing T cells described herein is 10 4 ~10 9 cells / kg body weight, e.g., 10 5 ~10 6It can generally be stated that the dosage can be administered at a dosage of cells / kg body weight, including all integer values within those ranges. The T cell composition can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0294] In certain embodiments, it may be desirable to administer activated T cells to a subject, then re-collect blood (or perform apheresis), activate T cells therefrom according to the disclosed method, 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, the T cells can be activated from a blood collection of 10 cc to 400 cc. In certain embodiments, the T cells are activated from a blood collection of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood collection / multiple re-infusion protocol can help select out a particular population of T cells.

[0295] Administration of the disclosed composition can be performed by any convenient method, including injection, transfusion, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed composition is administered to the patient by intradermal or subcutaneous injection. In some embodiments, the disclosed composition is administered by intravenous injection. The composition can also be injected directly into a tumor, lymph node, or site of infection.

[0296] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) any 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 used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immune-depleting agents such as CAM PATH, anti-CD3 antibodies, or other antibody therapies, cytokines, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation methods. In some embodiments, the CAR-modified immune effector cells are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using any of antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after a B cell depletion therapy with an agent that reacts with CD20, e.g., rituxan. For example, in some embodiments, a subject can receive high-dose chemotherapy followed by standard treatment with peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In further embodiments, the expanded cells are administered before or after surgery.

[0297] The cancer of the disclosed method can be any TAA-expressing cell in a subject that undergoes uncontrolled growth, invasion, or metastasis. In some embodiments, the cancer can be any neoplasm or tumor for which radiation therapy is currently being used. Alternatively, the cancer can be a neoplasm or tumor that is not sufficiently sensitive to radiation therapy using standard methods. Thus, the cancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. A representative but non-limiting list of cancers for which the disclosed compositions can be used to treat includes lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, larynx, pharynx, and lung, endometrial cancer, cervical cancer, uterine cancer, breast cancer, epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colorectal cancer, prostate cancer, and pancreatic cancer.

[0298] The disclosed CAR can be combined with any compound, moiety, or group having a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents that can function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators, particularly those used in cancer therapy.

[0299] The disclosed CARs can be used in combination with checkpoint inhibitors. Two known inhibitory checkpoint pathways involve signaling through the cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. While PD-L1 is the dominant ligand, PD-L2 has a much more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is transmitted to the T cell, reducing cytokine production and suppressing T cell proliferation. Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0300] Methods for treating cancer using human monoclonal antibodies against programmed death 1 (PD-1) and anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are described in U.S. Patent No. 8,008,449, which is incorporated herein by reference for these antibodies. Anti-PD-L1 antibodies and their use are described in U.S. Patent No. 8,552,154, which is incorporated herein by reference for these antibodies. Anti-cancer agents containing anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which is incorporated herein by reference for these antibodies.

[0301] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds to PD1, such as pembrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Methods for treating cancer using human monoclonal antibodies against PD-1, and anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are described in U.S. Patent No. 8,008,449, which is incorporated by reference with respect to these antibodies. Anti-PD-L1 antibodies and their use are described in U.S. Patent No. 8,552,154, which is incorporated by reference with respect to these antibodies. Anti-cancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which is incorporated by reference with respect to these antibodies.

[0302] The CARs disclosed herein can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapies: passive immunotherapy uses components of the immune system to direct target cell cytotoxic activity against cancer cells without necessarily initiating an immune response in the patient, while active immunotherapy actively induces an endogenous immune response. Passive strategies include the use of monoclonal antibodies (mAbs) produced by B cells in response to specific antigens. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens enabled the pharmaceutical development of mAbs that can specifically target tumor cells for destruction by the immune system. To date, mAbs have been the greatest success story in immunotherapy, and the top 3 best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein highly expressed on the surface of B cell malignancies such as non-Hodgkin lymphoma (NHL). Rituximab has been approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0303] To generate an optimal “killer” CD8 T cell response, T cell receptor activation + co-stimulation is also required, which can be provided by ligation of tumor necrosis factor receptor family members including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with an activating (agonist) anti-OX40 mAb enhances T cell differentiation and cytolytic function, resulting in enhanced antitumor immunity against various tumors.

[0304] In some embodiments, such additional therapeutic agents can be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0305] In some embodiments, such additional therapeutic agents may be selected from alkylating agents such as mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, and other platinum derivatives such as cisplatin and carboplatin.

[0306] In some embodiments, such additional therapeutic agents may be selected from mitotic inhibitors such as taxanes, for example, docetaxel, and paclitaxel, and vinca alkaloids, for example, vindesine, vincristine, vinblastine, and vinorelbine.

[0307] In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors such as topotecan or irinotecan, or cell division inhibitors such as etoposide and teniposide.

[0308] In some embodiments, such additional therapeutic agents may be selected from growth factor inhibitors, for example, inhibitors of ErbBl (EGFR) (EGFR antibodies, for example, zalutumumab, cetuximab, panitumumab or nimotuzumab, or other EGFR inhibitors, for example, gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (for example, HER2 antibodies, for example, trastuzumab, trastuzumab-DM1 or pertuzumab), or inhibitors of both EGFR and HER2, for example, lapatinib).

[0309] In some embodiments, such additional therapeutic agents may be selected from tyrosine kinase inhibitors such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0310] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanilimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0311] In some embodiments, the therapeutic agent for use in combination with a CAR for treating the disorders described above can be an anti-cancer cytokine, chemokine, or a combination thereof. Examples of suitable cytokines and growth factors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (e.g., IFNα2b), IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, anastim, and TNFα. Suitable chemokines can include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and C-C chemokine families. Suitable cytokines can include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0312] In some embodiments, the therapeutic agent for use in combination with the CAR for treating the above disorders can be a cell cycle control / apoptosis regulator (or "regulator"). The cell cycle / apoptosis regulator can include molecules that modulate cell cycle / apoptosis regulators targeting (i) cdc-25 (such as NSC663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modifiers (such as BIBR1532, SOT-095, GRN163, and compositions described, for example, in US6,440,735 and US6,713,055). Non-limiting examples of molecules that interfere with the apoptosis pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), TRAIL receptors, IFN, and antibodies that activate antisense Bcl-2.

[0313] In some embodiments, the therapeutic agent for use in combination with the CAR for treating the above disorders can be a hormone regulator such as an agent useful in anti-androgen and anti-estrogen therapies. Examples of such hormone regulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / ethinyl, anti-androgens (e.g., flutamide / euflex), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone / provera, megestrol acetate / megace), adrenal corticosteroids (e.g., hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and its analogs, and other LHRH agonists such as buserelin and goserelin), aromatase inhibitors (e.g., anastrozole / arimidex, aminoglutethimide / cytadren, exemestane), or hormone inhibitors (e.g., octreotide / sandostatin).

[0314] In some embodiments, the therapeutic agent for use in combination with the CAR for treating the diseases described above can be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0315] As described above, the combined administration can be simultaneous, separate, or sequential. For simultaneous administration, the agents can be administered as one composition or as separate compositions, as needed.

[0316] In some embodiments, the disclosed CAR is administered in combination with radiation therapy. Radiation therapy can include the administration of radiation or related radiopharmaceuticals to a patient. The radiation source can be either external or internal to the patient being treated (radiation therapy can be in the form of, for example, external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in practicing such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

[0317] In some embodiments, the disclosed CAR is administered in combination with surgery.

[0318] CAR-T cells can be engineered in several ways to enhance tumor cell cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and extend their therapeutic half-lives. TRUCK (T cells redirected for universal cytokine killing) T cells, for example, carry a CAR but are also engineered to secrete cytokines such as IL-12 that promote tumor killing. Since these cells are designed to release a molecular payload upon activation of the CAR once localized in the tumor environment, these CAR-T cells are sometimes also referred to as "armed CARs". Several cytokines as cancer therapies have been studied preclinically and clinically and can also be shown to be useful when incorporated into the TRUCK form of CAR-T therapy. These include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-α, IFN-γ, TNF-α, TRAIL, FLT3 ligand, lymphotactin, and TGF-β (Dranoff 2004). "Self-driving" or "homing" CAR-T cells are engineered to express chemokine receptors in addition to their CARs. Since certain chemokines can be increased in tumors, the incorporation of chemokine receptors helps with the transport of tumors to adoptive T cells and infiltration by adoptive T cells, thereby enhancing both the specificity and functionality of CAR-T (Moon 2011). Universal CAR-T cells also carry a CAR but are engineered not to express the endogenous TCR (T cell receptor) or MHC (major histocompatibility complex) proteins. Removing these two proteins from the signaling repertoire of adoptive T cell therapy prevents graft-versus-host disease and rejection, respectively. Furthermore, armed CAR-T cells are so named in reference to their ability to avoid tumor immunosuppression and tumor-induced CAR-T hypofunction. These particular CAR-Ts can carry a CAR and be engineered not to express checkpoint inhibitors. Alternatively, these CAR-Ts can be co-administered with monoclonal antibodies (mAbs) that block checkpoint signaling.Administration of anti-PDL1 antibody significantly restored the killing ability of CAR TIL (tumor infiltrating lymphocytes). Although the PD1-PDL1 and CTLA-4-CD80 / CD86 signaling pathways have been studied, it is possible to target other immune checkpoint signaling molecules in the design of armored CAR-Ts, including LAG-3, Tim-3, IDO-1, 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatase (SHP1), ubiquitin ligase (i.e., cbl-b), and kinase (i.e., diacylglycerol kinase). Armored CAR-Ts can also be engineered to express proteins or receptors that protect them from or make them resistant to the effects of tumor-secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with the double-negative form of the TGF-β receptor are resistant to immunosuppression by TGF-β secreted by lymphoma. These transduced cells showed a significant increase in antitumor activity in vivo when compared to their control counterparts.

[0319] A tandem CAR contains two consecutive antigen-binding domains facing the extracellular environment, coupled to an intracellular co-stimulatory domain and a stimulatory domain.

[0320] One of the main concerns regarding CAR-T cells as a form of "living therapeutics" is their in vivo manipulability and their potential immune-stimulatory side effects. To better control CAR-T therapies and prevent unwanted side effects, various features including off-switches, safety mechanisms, and conditional control mechanisms have been engineered. For example, both self-destructive and marked / tagged CAR-T cells have been engineered to have an "off-switch" that promotes clearance of CAR-expressing T cells. Self-destructive CAR-Ts contain the CAR but are also engineered to express an apoptosis-promoting suicide gene or "kill switch" that is inducible upon administration of an exogenous molecule. For this purpose, various suicide genes can be used, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC tag, and truncated EGFR (epidermal growth factor receptor). For example, HSK converts the prodrug ganciclovir (GCV) to GCV-triphosphate, which incorporates itself into replicating DNA and ultimately leads to cell death. iCasp9 is a chimeric protein containing a component of the FK506-binding protein that binds to the small molecule AP1903, resulting in caspase 9 dimerization and apoptosis. However, marked / tagged CAR-T cells have the CAR but are also engineered to express a selectable marker. Administration of an mAb against this selectable marker promotes clearance of the CAR-T cells. Truncated EGFR is one such targetable antigen by an anti-EGFR mAb, and administration of cetuximab functions to promote elimination of CAR-T cells. CARs engineered to have these features are also referred to as sCARs for "switchable CARs" and RCARs for "regulatable CARs". "Safety CARs", also known as "inhibitory CARs" (iCARs), are engineered to express two antigen-binding domains. One of these extracellular domains is directed against a tumor-associated antigen and is linked to an intracellular co-stimulatory and stimulatory domain. However, the second extracellular antigen-binding domain is specific for normal tissue and is linked to an intracellular checkpoint domain such as CTLA4, PD1, or CD45.It is also possible to incorporate multiple intracellular inhibitory domains into iCAR. Some inhibitory molecules that can provide these inhibitory domains include B7-H1, B7-1, CD160, PIH, 2B4, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1, and TGFβ-R. In the presence of normal tissue, stimulation of this second antigen-binding domain functions to inhibit the CAR. It should be noted that due to this dual antigen specificity, iCAR is also in the form of a bispecific CAR-T cell. Safety CAR-T engineering enhances the specificity of CAR-T cells for tumor tissue and is advantageous in situations where certain normal tissues that cause off-target effects with standard CARs may express very low levels of tumor-associated antigens (Morgan 2010). Conditional CAR-T cells express an extracellular antigen-binding domain coupled to an intracellular co-stimulatory domain and a separate intracellular co-stimulatory factor. The sequences of the co-stimulatory domain and the stimulatory domain are engineered such that upon administration of an exogenous molecule, the resulting protein aggregates intracellularly to complete the CAR circuit. In this way, CAR-T activation can be regulated and, in some cases, "fine-tuned" or individualized for a particular patient. Similar to the dual CAR design, the stimulatory domain and the co-stimulatory domain are physically separated when inactive in the conditional CAR, and for this reason, they are also referred to as "split-type CARs".

[0321] In some embodiments, two or more of these engineered features can be combined to create enhanced multifunctional CAR-Ts. For example, it is possible to generate CAR-T cells having either a dual CAR design that also releases cytokines, such as a TRUCK, or a conditional CAR design. In some embodiments, dual-conditional CAR-T cells can be generated to express two CARs having two distinct antigen-binding domains for two distinct cancer antigens, each binding to their respective co-stimulatory domains. The co-stimulatory domains become functional with the stimulatory domains only after an activating molecule has been administered. For this CAR-T cell to be effective, the cancer must express both cancer antigens and the activating molecule must be administered to the patient. This design thereby incorporates the features of both dual CAR-T cells and conditional CAR-T cells.

[0322] Typically, CAR-T cells are generated using α-β T cells, but γ-δ T cells can also be used. In some embodiments, the CAR constructs, domains, and engineered features used to generate the CAR-T cells described can similarly be used to generate other types of CAR-expressing immune cells, including NK (natural killer) cells, B cells, mast cells, myeloid-derived phagocytes, and NKT cells. Alternatively, CAR-expressing cells can be generated to have the properties of both T cells and NK cells. In additional embodiments, those transduced with the CAR can be autologous or allogeneic.

[0323] Several different methods for CAR expression can be used, including retroviral transduction (including γ-retrovirus), lentiviral transduction, transposon / transposase (Sleeping Beauty system and PiggyBac system), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) is also becoming increasingly important with respect to the possibility of engineering CAR-T cells. The CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator-like effector nuclease) systems are three potential methods by which CAR-T cells can be generated.

[0324] Some embodiments of the invention have been described. Nevertheless, it is to be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Examples

[0325] Example 1: NKG2D is a transmembrane stimulatory receptor expressed on the surface of natural killer (NK) cells, NKT cells, human CD8 T cells, and mouse CD8 T cells (Raulet DH. Nat Rev Immunol 2003 3:781-90). In CD8 T cells, NKG2D is expressed in association with its adapter molecule DAP10 (Wu J, et al. Science 1999 285:730-2). The ligands for the NKG2D receptor are ULBP1-4, MICA, MICB in humans, and Rae1, MULT1, and H60 in mice (Garrity D, et al. Proc Natl Acad Sci 2005 102:7641-6). In CD8 T cells, ligation of the NKG2D receptor to its ligand enhances the activation, proliferation, and production of inflammatory cytokines and effector molecules 1. NKG2D can mediate the survival of memory CD8 T cells and is sufficient to maintain memory formation in CD8 T cells in the absence of CD4 T cell "help" (Zloza A, et al. Nat Med 2012 18:422-8).

[0326] In CD8 T cells, DAP10 expresses a YxNM domain that recruits p85 to induce PI3K signaling, and the YxNX domain is involved in the recruitment and signaling of Grb2 (Okkenhaug K, et al. Nat Rev Immunol 2003 3:317-30, Upshaw JL, et al. Nat Immunol 2006 7:524-32).

[0327] The aim of this study was to engineer scFv CAR T cells with the characteristics (death, survival, and migration characteristics) of pro-T cells by utilizing the NKG2D / DAP10 signaling pathway. To achieve this aim, an scFv against OR5V1 was linked to the transmembrane domain (Tm) and intracellular domain (IC) of NKG2D (Figure 1A). In this design, the scFv recognizes an antigen that leads to the activation of NKG2D / DAP10 signaling, which in turn will initiate the death and memory phases in the carrier T cells.

[0328] The designed construct was expressed in human T cells and tested alongside second-generation CAR-T cells for its ability to recognize tumor cells expressing the scFv target. The ability of ORV1-NKG2D-CAR to kill target cells (HeLa, cervical cancer) and BT-549 cells (papillary, invasive ductal tumor) was indistinguishable from that of normal CAR-T cells (Figures 2A and 2B). These data indicate that NKG2D (TM-IC) can replace normal CAR-T cell signaling that utilizes the CD3 domain.

[0329] Next, it was determined whether ORV1-NKG2D-CAR could kill tumors in vivo compared to normal CAR-T cells. Figure 3 shows that ORV1-NKG2D-CAR controls tumor growth similarly to normal CAR-T. In this experiment, immunodeficient mice (NSG mice) were tumor challenged with flank HeLa cells and treated with mock T cells, normal CAR-T, and CAR-T NKG2D. Tumor growth was measured manually and plotted. These data indicate that scFv-NKG2D (TM-IC) can replace normal CAR-T cell signaling that utilizes the CD3 domain and mediates an in vivo anti-tumor response against human tumors.

[0330] 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. Publications cited herein and the substances they cite are specifically incorporated herein by reference.

[0331] One of ordinary skill 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 chimeric antigen receptor (CAR) polypeptide comprising a ligand-binding domain, a hinge domain, a transmembrane domain, and an endodomain containing an intracellular domain of an NK cell receptor or a fragment thereof capable of activating a target cell death pathway.

2. The polypeptide according to claim 1, wherein the NK cell receptor is hNKG2D, hCD16, hNKp30, hNKG2C, h2B4, hDNAM-1, hCD137, hOX-40, hCD27, KIR2DS5, KIR3DS1, or NKp80 / KLRF1.

3. The polypeptide according to claim 2, wherein the intracellular domain includes the amino acid sequence numbers SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO:

34.

4. The polypeptide according to claim 3, wherein the transmembrane domain comprises the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO:

35.

5. The polypeptide according to claim 2, comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, or SEQ ID NO:

36.

6. The polypeptide according to claim 1, wherein the end domain does not contain a CD3 zeta (CD3ζ) signaling domain.

7. The aforementioned CAR polypeptide is of the formula: SP-BD-HG-TM-NKRIC Defined by, In the formula, "SP" represents an optional signal peptide. "BD" represents the target binding domain. "HG" represents an optional hinge domain. "TM" represents the transmembrane domain. "NKRIC" represents the intracellular domain of the NK cell receptor. The polypeptide according to claim 1, wherein "-" represents a peptide bond or a linker.

8. An isolated nucleic acid sequence encoding a recombinant polypeptide according to any one of claims 1 to 7.

9. A vector comprising the isolated nucleic acid sequence described in claim 8.

10. A cell comprising the vector according to claim 9.

11. The aforementioned cells include αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, and regulatory T (T) cells. ｒｅｇ The cells according to claim 10, selected from the group consisting of ) cells or any combination thereof.

12. The cell according to claim 11, wherein when the antigen-binding domain of the CAR binds to TAA, the cell exhibits antitumor immunity.

13. A method for providing antitumor immunity in a subject having TAA-expressing cancer, the method comprising administering to the subject an effective amount of immunoeffector cells genetically modified to express a CAR polypeptide according to any one of claims 1 to 7, thereby providing antitumor immunity in a mammal.

14. The aforementioned immune effector cells include αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, and regulatory T (T) cells. ｒｅｇ The method according to claim 13, comprising a selection from the group consisting of cells or any combination thereof.

15. The method according to claim 13, further comprising administering a checkpoint inhibitor to the subject.

16. The method according to claim 15, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

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