Chimeric antigen receptor (CAR) constructs with NK receptor signaling domains

Abstract

Disclosed herein are chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to target and kill cancer. Like other CARs, the disclosed CAR polypeptides contain: an extracellular domain comprising a binding domain, a hinge domain, a transmembrane (TM) domain, and an intracellular domain comprising a signaling region. However, unlike other CARs, the intracellular domain of the disclosed CAR polypeptides contains the intracellular domain of the NK cell receptor and does not require a signaling domain from CD3 [zeta] (CD3 [zeta]). Immune effector cells, such as T cells or natural killer (NK) cells, engineered to express these CARs are also disclosed. Accordingly, also disclosed are methods of providing anti-tumor immunity in a subject suffering from a cancer expressing a tumor-associated antigen involving adoptive metastasis of the disclosed immune effector cells engineered to express the disclosed CARs.

Description

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

[0002] Sequence Listing

[0003] This application contains a sequence listing filed in ST.26 format, entitled "320803-2660 Sequence Listing", created on May 2, 2023. The contents of the sequence listing are incorporated herein in their entirety. Background Art

[0004] Surgery, radiation therapy and chemotherapy have become the standard recognized methods for treating cancer (including leukemia, solid tumors and metastases). Immunotherapy (sometimes called biological therapy, biotherapy or biological response modifier therapy) directly or indirectly uses the body's immune system to shrink or eradicate cancer and has been studied for many years as an adjunct to conventional cancer therapy. It is believed that the human immune system is an untapped resource for cancer therapy, and once the components of the immune system are properly utilized, effective treatment can be carried out.

[0005] A major advance in anticancer T cell therapy is the chimeric antigen receptor (CAR), which is a single-chain variable fragment (scFv) derived from an antibody fused to the signaling domain of the T cell receptor (TCR) (Davila, ML et al., Oncoimmunology, 2012.1 (9): 1577-1583). The intracellular domain of the first generation of CAR includes only CD3ζ, while the second generation of CAR also includes a co-stimulatory domain (such as CD28 or 41BB). These second generation CAR domains support efficient tumor killing in mice and have led to clinical evaluation of CAR T cell therapy in patients. The potential of CD19-targeted CAR T cells has been confirmed by reports of a 90% complete remission rate in patients with B cell acute lymphoblastic leukemia (B-ALL) (Davila, ML et al., Sci Transl Med, 2014.6 (224): 224ra25; Maude, SL et al., N Engl J Med, 2014.371 (16): 1507-17). However, poor CAR T cell persistence and excessive T cell activation lead to relapse and severe toxicity, respectively, and indicate an urgent need to understand CAR T cell biology (Gangadhar, TC and RH Vonderheide, Nat Rev Clin Oncol, 2014.11(2):91-9). In addition, all second-generation CARs have experienced relapse and toxicity, indicating that adding the costimulatory domain to CAR improves efficacy, but at the expense of biological complications. Summary of the invention

[0006] Disclosed herein are chimeric antigen receptor (CAR) polypeptides that can be used with adoptive cell transfer to target and kill cancer. Like other CARs, the disclosed CAR polypeptides contain: an extracellular domain containing a binding domain, a hinge domain, a transmembrane (TM) domain, and an intracellular domain containing a signaling region. However, unlike other CARs, the intracellular domain of the disclosed CAR polypeptide contains the intracellular domain of the NK cell receptor and does not require a signaling domain from CD3ζ (CD3ζ).

[0007] Thus, the disclosed CAR polypeptides utilize a completely different signaling pathway (NKG2D / DAP10) than current CAR-T cells (CD3, OX40, ICOS, etc.). Since the signaling pathway is not dependent on CD3ζ or ZAP70 signaling, it may be insensitive to PD-1-mediated inhibition. In addition, studies have shown that in T cells, NKG2D / DAP10 leads to target killing, 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 TGF-β immunosuppression, and enhance migration properties.

[0008] Therefore, disclosed herein is a CAR polypeptide having a ligand binding domain, a hinge domain, a transmembrane domain and an intracellular domain, the intracellular domain containing an NK cell receptor intracellular domain or a fragment thereof capable of activating NK cell killing. In some embodiments, the CAR polypeptide further contains an NK cell receptor extracellular domain or a fragment thereof.

[0009] In some embodiments, the disclosed polypeptides may further comprise additional motifs in the intracellular domain, such as one or more DAP10 or other signaling motifs, e.g., OX40, ICOS, CD3, etc. However, in some embodiments, the CAR polypeptide lacks any other intracellular signaling or co-stimulatory domains, such as CD3ζ, 41BB and / or CD28 domains.

[0010] NK receptors

[0011] NKG2D

[0012] In some embodiments, the NK cell receptor is hNKG2D. In some embodiments, the NK cell receptor intracellular (IC) domain comprises the amino acid sequence

[0013] 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 an hNKG2D TM / IC domain having the amino acid sequence

[0014] PFFFCCFIAVAMGIRFIIMVTIWMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS (SEQ ID NO: 3) or a variant / fragment thereof having a length of 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 3, which variant / fragment is capable of integrating into the plasma membrane and binding to DAP10 upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0015] 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 an amino acid sequence

[0016] 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.

[0017] CD16

[0018] In some embodiments, the NK cell receptor is hCD16 (FCGR3A). Thus, in some embodiments, the NK cell receptor intracellular domain 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 a hCD16 TM / IC domain having an amino acid sequence of VSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 6) or a variant / fragment thereof having a length of 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids and having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 6, which variant / fragment is capable of integrating into the plasma membrane and binding to FcεR1γ upon cross-linking. In some embodiments, the extracellular domain has an amino acid sequence of

[0019] 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

[0020] 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.

[0021] NKp30

[0022] In some embodiments, the NK cell receptor is hNKp30 (NCR3). Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0023] 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 a hNKp30 TM / IC domain having the amino acid sequence

[0024] AGTVLLLRAGFYAVSFLSVAVGSTVYYQGKCLTWKGPRRQLPAVVPAPLPPPCGSSAHLLPPVPG (SEQ ID NO: 9) or a variant / fragment thereof having a length of 55, 56, 57, 58, 59, 60, 61, 61, 62, 64 or 65 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 9, which variant / fragment is capable of integrating into the plasma membrane and binding to FcεR1γ upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0025] 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.

[0026] NKG2C

[0027] In some embodiments, the NK cell receptor is hNKG2C. Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0028] 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 an hNKG2C TM / IC domain having the amino acid sequence

[0029] LTAEVLGIICIVLMATVLKTIVLIPFLEQNNSSPNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLASILPSSWIGVFRNSSHHPWVTINGLAFKHKIKDSDNAELNCAVLQVNRLKSAQCGSSMIYHCKHKL (SEQ ID NO: 12), or a variant / fragment thereof having a length of 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 12, which variant / fragment is capable of integrating into the plasma membrane and binding DAP12 upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0030] 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 an amino acid sequence

[0031] 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.

[0032] 2B4

[0033] In some embodiments, the NK cell receptor is h2B4. Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0034] 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 an h2B4 TM / IC domain having the amino acid sequence

[0035] FLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 15) or a variant / fragment thereof having a length of 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 15, which variant / fragment is capable of signaling upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0036] 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

[0037] MLGQVVTLILLLLLKVYQGKGCQGSADHVVSISGVPLQLQPNSIQTKVDSIAWKKLLPSQNGFHHILKWENGSLPSNTSNDRFSFIVKNLSLL IKAAQQQDSGLYCLEVTSISGKVQTATFQVFVFESLLPDKVEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWYRGSKLIQTAGNLTYLDE EVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQRFWPFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS(SEQ ID NO:46) or it and SEQ Variants / fragments of ID NO: 46 having at least 70%, 85%, 90%, 95%, 98% or 99% identity.

[0038] DNAM-1

[0039] In some embodiments, the NK cell receptor is hDNAM-1. Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0040] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKT RV (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 an hDNAM TM / IC domain having the amino acid sequence

[0041] GGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 18) or a variant / fragment thereof having a length of 30, 31, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 18, which variant / fragment is capable of signaling upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0042] MTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTV SDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVA (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 an amino acid sequence

[0043] 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.

[0044] CD137

[0045] In some embodiments, the NK cell receptor is hCD137 (TNFRSF9). Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0046] 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 a hCD137 TM / IC domain having the amino acid sequence

[0047] IISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 21) or a variant / fragment thereof having a length of 59, 60, 61, 61, 62, 64, 65, 66, 67, 68 or 69 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 21, which variant / fragment is capable of signaling upon cross-linking. In some embodiments, the extracellular domain has an amino acid sequence

[0048] 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

[0049] 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.

[0050] OX-40

[0051] In some embodiments, the NK cell receptor is hOX-40. Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0052] 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 a hOX-40 TM / IC domain having the amino acid sequence

[0053] LLLVASVIQGLGLLLCFTYICLHFSALVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL (SEQ ID NO: 24) or a variant / fragment thereof having a length of 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 24, which variant / fragment is capable of signaling upon cross-linking. In some embodiments, the extracellular domain has an amino acid sequence

[0054] 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 an amino acid sequence

[0055] 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.

[0056] CD27

[0057] In some embodiments, the NK cell receptor is hCD27. Thus, in some embodiments, the NK cell receptor intracellular domain comprises the amino acid sequence

[0058] 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 a hCD27 TM / IC domain having the amino acid sequence

[0059] ILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 27) or a variant / fragment thereof having a length of 59, 60, 61, 61, 62, 64, 65, 66, 67, 68 or 69 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 27, which variant / fragment is capable of integrating into the plasma membrane and binding TRAF upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0060] 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 an amino acid sequence

[0061] 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.

[0062] 2DS5

[0063] In some embodiments, the NK cell receptor is killer cell immunoglobulin-like receptor 2DS5 (KIR2DS5). In some embodiments, the NK cell receptor intracellular (IC) domain comprises the amino acid sequence

[0064] 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 a hNKG2D TM / IC domain having an amino acid sequence of VLIGTSVVKLPFTILLFFLLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVS YA (SEQ ID NO: 30) or a variant / fragment thereof having a length of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 30, which variant / fragment is capable of integrating into the plasma membrane and binding to DAP12 upon cross-linking. In some embodiments, the extracellular domain has an amino acid sequence of

[0065] 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

[0066] MSLMVISMACVAFFLLQGAWPHEGFRRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGTFNHTLRLIGEHIDGVSKGNFSIGRMTQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSS YDMYHLSREGEAHERRLPAGPKVNRTFQADFPLDPATHGGTYRCFGGSFRDSPYEWSKSSDPLLVSVTGNSSNSWPSPTEPSSETGNPRHLHVLIGTSVVKLPFTILLFFLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVSYA(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.

[0067] KIR3DS1

[0068] In some embodiments, the NK cell receptor is KIR3DS1. In some embodiments, the NK cell receptor intracellular (IC) domain 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 comprises an hNKG2DTM / IC domain having the amino acid sequence

[0069] 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 variant / fragment is capable of integrating into the plasma membrane and binding to DAP12 upon cross-linking. In some embodiments, the extracellular domain has the amino acid sequence

[0070] MLLMVVSMACVGLFLVQRAGPHMGGQDKPFLSAWPSAVVPRGGHVTLRCHYRHRFNNFMLYKEDRIHVPIFHGRIFQEGFNMSPVTTAHAGNYTCRGSHPHSPTGWSAPSNPMVIMVTGNHRKPSLLAHPGPLVKSGERVILQCWSDIMFEHFFLHREWISKDPSRLVGQIH DGVSKANFSIGSMMRALAGTYRCYGSVTHTPYQLSAPSDPLDIVVTGLYEKPSLSAQPGPKVQAGESVTLSCSSRSSYDMYHLSREGGAHERRLPAVRKVNRTFQADFPLGPATHGGTYRCFGSFRHSPYEWSDPSDPLLVSVTGNPSSSWPSPTEPSSKSGNLRHLH(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

[0071] MLLMVVSMACVGLFLVQRAGPHMGGQDKPFLSAWPSAVVPRGGHVTLRCHYRHRFNNFMLYKEDRIHVPIFHGRIFQEGFNMSPVTTAHAGNYTCRGSHPHSPTGWSAPSNPMVIMVTGNHRKPSLLAHPGPLVKSGERVILQCWSDIMFEHFFLHREWISKDPSRLVGQIHDGVSKANFSIGSMMRALAGTY RCYGSVTHTPYQLSAPSDPLDIVVTGLYEKPSLSAQPGPKVQAGESVTLSCSSRSSYDMYHLSREGGAHERRLPAVRKVNRTFQADFPLGPATHGGTYRCFGSFRHSPYEWSDPSDPLLVSVTGNPSSSWPSPTEPSSKSGNLRHLHILIGTSVVKIPFTILLFFLLHRWCSNKKKCCCNGPRACREQK(SEQ ID NO:58) or its combination with SEQ ID NO:58 variants / fragments having at least 70%, 85%, 90%, 95%, 98% or 99% identity.

[0072] NKp80 / KLRF1

[0073] In some embodiments, the NK cell receptor is NKp80 / KLRF1. In some embodiments, the NK cell receptor intracellular (IC) domain comprises the amino acid sequence

[0074] 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 an hNKG2D TM / IC domain having the amino acid sequence

[0075] MQDEERYMTLNVQSKKRSSAQTSQLTFKDYSVTLHWYKILLGISGTVNGILTLTLISLI (SEQ ID NO: 36) or a variant / fragment thereof having a length of 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 amino acids and having at least 70%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 36, which variant / fragment is capable of signaling upon cross-linking. In some embodiments, the extracellular domain has an amino acid sequence

[0076] 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

[0077] 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.

[0078] Binding domain

[0079] 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 a single chain variable fragment (scFv) of an antibody that specifically binds to a target molecule. In some embodiments, the binding domain is an aptamer that specifically binds to a 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 a target molecule. The binding domain can also be a natural ligand of a target molecule or a variant and / or fragment thereof that can bind to the target molecule.

[0080] CD33

[0081] For example, scFvs that selectively bind to CD33 are described in US2020 / 0223920, which is incorporated by reference in its entirety for describing these antibodies and their sequences. For example, in some embodiments, the anti-CD33 region of the disclosed antibody or CAR is derived from hybridomas 27A3, 33G3, 36C2, 6A11, 35D5, 38G5, or a combination thereof. In some embodiments, the anti-CD33 region (e.g., scFv) may comprise a variable heavy (V) region having CDR1, CDR2, and CDR3 sequences. H ) domain and a variable light (V L ) domain.

[0082] For example, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GFTFSNYG (SEQ ID NO: 61), GYTFTSYW (SEQ ID NO: 62) or GFSLSRYS (SEQ ID NO: 63), wherein the V H The CDR2 sequence of the domain comprises the amino acid sequence ISSGGGDT (SEQ ID NO: 64), IHPSDSET (SEQ ID NO: 65) or IWGGGYT (SEQ ID NO: 66), wherein the V H The CDR3 sequence of the domain comprises the amino acid sequence ARDYGGTWDYFDY (SEQ ID NO: 67), AREEGQLGHGGAMDY (SEQ ID NO: 68) or ARYIDSSGYDY (SEQ ID NO: 69), wherein V L The CDR1 sequence comprises 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), wherein the V LThe CDR2 sequence of the domain comprises the amino acid sequence YTS, YVS, DTS or NAK, wherein the V L The CDR3 sequence of the domain comprises 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.

[0083] Thus, in some embodiments, the anti-CD33 scFv V H The domain contains the amino acid sequence

[0084] EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQTSDKRLEWVASISSGGGDTYYPDNVKGRFTISRENAKNTLYLQMSSLNSEDTALYYCARDYGGTWDYFDYWGQGTTLTVSS(SEQ ID NO:82),

[0085] or

[0086] QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSS (SEQ ID NO: 84).

[0087] In some embodiments, the anti-CD33 scFv V L The domain contains the amino acid sequence

[0088] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK(SEQ ID NO:85), SIVMTQTPKFLLVSAGDRVTITCKASQTVNDDVAWYQQKPGQSPKLLIYYVSNRHTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK(SEQ ID NO:86),

[0089] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(SEQ ID NO:87) or

[0090] DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIK (SEQ ID NO: 88).

[0091] The heavy chain and light chain are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 89).

[0092] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence:

[0093] EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQTSDKRLEWVASISSGGGDTYYPDNVKGRFTISRENAKNTLYLQMSSLNSEDTALYYCARDYGGTWDYFDYWGQGTTLTVSSGGG GSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK(SEQ ID NO:90,6A11HC1_LC).

[0094] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence:

[0095] QVQLQQPGAELVRPGVSVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKAILTVDKSSSTAYMQLSSPTSEDSAVYYCAREEGQLGHGAMDYWGQGTSVTVSSGG GGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPWTFGGGTKLEIK(SEQ ID NO:91,6A11HC2_LC).

[0096] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence:

[0097] QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGG SGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQTVNDDVAWYQQKPGQSPKLLIYYVSNRHTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK(SEQ ID NO:92,27A3HC_LC1).

[0098] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence:

[0099] QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGG SGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(SEQ ID NO:93,27A3HC_LC2).

[0100] In some embodiments, the anti-CD33 scFv comprises the amino acid sequence:

[0101] QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGYTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARYIDSSGYDYWGQGTTLTVSSGGGG SGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIK(SEQ ID NO:94,27A3HC_LC3).

[0102] CD123

[0103] For example, scFvs that selectively bind to CD123 are described in 2020 / 0165348, which is incorporated by reference in its entirety for describing these antibodies and their sequences. For example, in some embodiments, the anti-CD123 scFv is derived from hybridomas 3F5, 4E10, 12H5, 15A12, 17E7, 12H11, or a combination thereof. In some embodiments, the anti-CD123 scFv may include a variable heavy (V) having CDR1, CDR2, and CDR3 sequences. H ) domain and a variable light (V L ) domain. For example, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GYTFTDYN (SEQ ID NO: 95), the V HThe CDR2 sequence of the domain comprises the amino acid sequence INPNNGGT (SEQ ID NO: 96), the V H The CDR3 sequence of the domain comprises the amino acid sequence ARKGYGGNYDYFDY (SEQ ID NO: 97), V L The CDR1 sequence of the present invention comprises the amino acid sequence QSIGTS (SEQ ID NO: 98), the V L The CDR2 sequence of the domain comprises the amino acid sequence YASx (SEQ ID NO: 99), and the V L The CDR3 sequence of the domain comprises the amino acid sequence QQSNSWPYT (SEQ ID NO: 100).

[0104] In some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GFNIKDTY (SEQ ID NO: 101) or GFSLSTYGMG (SEQ ID NO: 102), the V H The CDR2 sequence of the domain comprises the amino acid sequence IDPANGNT (SEQ ID NO: 103) or IYWDDDK (SEQ ID NO: 104), the V H The CDR3 sequence of the domain comprises the amino acid sequence ALYYYGGSLDY (SEQ ID NO: 105) or AQSLIYDGYYGFAY ​​(SEQ ID NO: 106), V L The CDR1 sequence of the domain comprises the amino acid sequence QSLLYSGNQKNY (SEQ ID NO: 107), the V L The CDR2 sequence of the domain comprises the amino acid sequence WASx (SEQ ID NO: 108), the V L The CDR3 sequence of the domain comprises the amino acid sequence QQYYSYPRT (SEQ ID NO: 109).

[0105] In some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GYTFTYYG (SEQ ID NO: 110), the V H The CDR2 sequence of the domain comprises the amino acid sequence INTYSGVP (SEQ ID NO: 111), the V H The CDR3 sequence of the domain comprises the amino acid sequence ARWIYYSDLYGMDY (SEQ ID NO: 112), V L The CDR1 sequence of the present invention comprises the amino acid sequence QSIVHSNGDTY (SEQ ID NO: 113), the V LThe CDR2 sequence of the domain comprises the amino acid sequence KVSx (SEQ ID NO: 114), and the V L The CDR3 sequence of the domain comprises the amino acid sequence FQGSHVPWT (SEQ ID NO: 115).

[0106] In some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GYTFSSYW (SEQ ID NO: 116) or GYTLTTYL (SEQ ID NO: 117), the V H The CDR2 sequence of the domain comprises the amino acid sequence INPSSGYT (SEQ ID NO: 118) or INPNSGSS (SEQ ID NO: 119), the V H The CDR3 sequence comprises the amino acid sequence ARDGNYDHWYFDV (SEQ ID NO: 120) or AIRHYGGSLFDY (SEQ ID NO: 121), V L The CDR1 sequence of the domain comprises the amino acid sequence QDINSY (SEQ ID NO: 122) or QSLLNSRTRKNY (SEQ ID NO: 123), the V L The CDR2 sequence of the V L The CDR3 sequence of the domain comprises the amino acid sequence LQYDELLT (SEQ ID NO: 126) or EQSYNLFT (SEQ ID NO: 127).

[0107] 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); 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); HThe 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); V L The CDR1 sequence 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); V L The CDR2 sequence of the domain comprises the amino acid sequence YASx, WASx, KVSx or RAN; 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.

[0108] Thus, in some embodiments, the anti-CD123 scFv V H The domain contains the amino acid sequence:

[0109] EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGTINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARKGYGGNYDYFDYWGQGTTLTVSS(SEQ ID NO:142,3F5HC1),

[0110] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTIYASKFQGKATITADTSSNTAYMQLSSLTSGDTAVYYCALYYYGGSLDYWGQGTTLTVSS(SEQ ID NO:143,12H1HC1),

[0111] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSNNQVFLKITSVDTADTATYYCAQSLIYDGYYGFAYWGQGTLVTVSA(SEQ ID NO:144,12H1HC2),

[0112] QIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLEWMGWINTYSGVPTYADDFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARWIYYSDLYGMDYWGQGTSVTVSS(SEQ ID NO:145,12H2HC1),

[0113] QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSS(SEQ ID NO:146,15A12HC1) or

[0114] QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSS(SEQ ID NO:147,15A12HC2).

[0115] In some embodiments, the anti-CD123 scFv V L domain comprises the amino acid sequence:

[0116] DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK(SEQ ID NO:148,3F5LC1),

[0117] DIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK(SEQ ID NO:149,12H1LC1),

[0118] DVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGDTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYHCFQGSHVPWTFGGGTKLEIK(SEQ ID NO:150,12H2LC),

[0119] DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK(SEQ ID NO:151,15A12LC1) or

[0120] DIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK(SEQ ID NO:152,15A12LC2).

[0121] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0122] EVQLQQSGPELVKPGSSVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGTINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARKGYGGNYDYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK(SEQ ID NO:153,3F5HC1_LC).

[0123] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0124] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTIYASKFQGKATITADTSSNTAYMQLSSLTSGDTAVYYCALYYYGGSLDYWGQGTTLTVSSGGGGSGG GGSGGGGSDIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK(SEQ ID NO:154,12H1HC1_LC1).

[0125] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0126] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTYGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSNNQVFLKITSVDTADTATYYCAQSLIYDGYYGFAYWGQGTLVTVSAGGGGS GGGGSGGGGSDIVMSQSPSSLAVSVGERVTMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPRTFGGGTKLEIK(SEQ ID NO:155,12H1HC2_LC1).

[0127] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0128] QIQLVQSGPELKKPGETVKISCKASGYTFTYYGMNWVKQAPGKGLEWMGWINTYSGVPTYADDFKGRFAFSLETSVSTAYLQINNLKNEDTATYFCARWIYYSDLYGMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGDTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYHCFQGSHVPWTFGGGTKLEIK(SEQ ID NO:156,12H2HC1_LC1).

[0129] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0130] QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK(SEQ ID NO:157,15A12HC1_LC1).

[0131] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0132] QVQLQQSGAELAKPGASVKMSCKASGYTFSSYWMHWLKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDGNYDHWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK(SEQ ID NO:158,15A12HC1_LC2).

[0133] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0134] QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSSGGG GSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDELLTFGAGTKLELK(SEQ ID NO:159,15A12HC2_LC1).

[0135] In some embodiments, the anti-CD123 scFv comprises the amino acid sequence:

[0136] QVQLQQPGAELVRPGASVKMSCKASGYTLTTYLMDWVKQRLGQGFEWIGNINPNSGSSNYNEKFKGKAKLTVDKSSSTAYMQLSSSLTSEDSAVYYCAIRHYGGSLFDYWGQGTTLTVSSGGGGSG GGGSGGGGSDIVMSQSPSSLAVSAGERVTMSCRSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCEQSYNLFTFGSGTKLEIK(SEQ ID NO:160,15A12HC2_LC2).

[0137] CD99

[0138] For example, scFvs that selectively bind to CD99 are described in US20200397882, which is incorporated by reference in its entirety for a description of these antibodies and their sequences.

[0139] In some embodiments, the anti-CD99 region of the disclosed antibodies or CARs is derived from hybridomas 1H3, 4C5, 9G12, 3C7, 2F11, 4D5, 4F4, 6A10, or a combination thereof. In some embodiments, the anti-CD99 region (e.g., scFv) may comprise a variable heavy (V) region having CDR1, CDR2, and CDR3 sequences. H ) domain and a variable light (VL ) domain.

[0140] In some embodiments, V H The CDR1 sequence of the 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); H The CDR2 sequence of the domain comprises the amino acid sequence IDPANGDT (SEQ ID NO: 165), RIRSKVNNYATYYADSVKDRFT (SEQ ID NO: 166) or TIYPGDDDTRYTQKFKGRAT (SEQ ID NO: 167); V H The CDR3 sequence of the 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); V L The CDR1 sequence 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); V L The CDR2 sequence of the 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).

[0141] Thus, in some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0142] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS (SEQ ID NO: 182, 1H3H7).

[0143] Thus, in some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0144] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSS (SEQ ID NO: 183, 4C5E2).

[0145] Thus, in some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0146] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSS (SEQ ID NO: 184, 4C5H10).

[0147] Thus, in some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0148] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 185, 9G12C9).

[0149] Thus, in some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0150] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS (SEQ ID NO: 186, 9G12G6HB1).

[0151] In some embodiments, anti-CD99 V H The domain contains the amino acid sequence

[0152] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGT TLTVSS (SEQ ID NO: 187,9G12G6HB3).

[0153] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0154] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 188,1H3H9).

[0155] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0156] GNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 189, 1H3H7 LC1).

[0157] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0158] GNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 190, 1H3H7 LC2).

[0159] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0160] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 191, 4C5E2).

[0161] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0162] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO: 192, 4C5H10).

[0163] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0164] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 193, 9G12C9).

[0165] In some embodiments, anti-CD99 V L The domain contains the amino acid sequence

[0166] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO: 194, 9G12G6).

[0167] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0168] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSG GGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:195).

[0169] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0170] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGG GSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:196).

[0171] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0172] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGG GSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:197).

[0173] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0174] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGG GSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK(SEQ ID NO:198).

[0175] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0176] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGG SGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:199).

[0177] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0178] EVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSSGGGGSGGGG SGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:200).

[0179] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0180] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGS GGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:201).

[0181] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0182] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGG GGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:202).

[0183] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0184] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGG GGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:203).

[0185] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0186] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO:204).

[0187] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0188] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO:205).

[0189] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0190] EVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO:206).

[0191] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0192] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGS GGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:207).

[0193] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0194] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGG GGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:208).

[0195] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0196] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGG GGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:209).

[0197] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0198] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGG GGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK(SEQ ID NO:210).

[0199] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0200] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGGG GSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:211).

[0201] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0202] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSSGGGGSGGG GSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:212).

[0203] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0204] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGG GGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:213).

[0205] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0206] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:214).

[0207] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0208] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:215).

[0209] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0210] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK(SEQ ID NO:216).

[0211] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0212] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:217).

[0213] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0214] QVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:218).

[0215] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0216] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:219).

[0217] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0218] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:220).

[0219] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0220] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:221).

[0221] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0222] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK(SEQ ID NO:222).

[0223] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0224] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK(SEQ ID NO:223).

[0225] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0226] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:224).

[0227] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0228] DVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:225).

[0229] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0230] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGG GGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK(SEQ ID NO:226).

[0231] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0232] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSGNSWSHSLRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:227).

[0233] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0234] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSGNSWRHSPRSLSVTIGQPASISCKSSQSLLDGNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:228).

[0235] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0236] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO:229).

[0237] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0238] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIK (SEQ ID NO:230).

[0239] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0240] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGSGGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK (SEQ ID NO:231).

[0241] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0242] QVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSSGGGGS GGGGSGGGGSDTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK(SEQ ID NO:232).

[0243] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0244] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGG GSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS(SEQ ID NO:233).

[0245] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0246] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGG SEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSS(SEQ ID NO:234).

[0247] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0248] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGG SEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPPMDYWGQGISVTVSS(SEQ ID NO:235).

[0249] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0250] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSQ VQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:236).

[0251] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0252] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSD VKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:237).

[0253] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0254] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSQ VQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:238).

[0255] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0256] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGS GGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS(SEQ ID NO:239).

[0257] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0258] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSG GGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:240).

[0259] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0260] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSG GGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:241).

[0261] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0262] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGG SQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:242).

[0263] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0264] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:243).

[0265] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0266] DIVMTQAAPSVPVTPGESVSISCGSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:244).

[0267] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0268] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS(SEQ ID NO:245).

[0269] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0270] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:246).

[0271] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0272] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:247).

[0273] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0274] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:248).

[0275] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0276] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGG GSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:249).

[0277] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0278] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLCRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGG GSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:250).

[0279] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0280] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPEFQGKASLTADTSSNTAYLQFSNLTSEDTAVYYCARRGGLSWGQGTTLTVSS(SEQ ID NO:251).

[0281] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0282] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLEESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:252).

[0283] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0284] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMYWVCQAPGKGLKWVARIRSKVNNYATYYADSVKDRFTISRDDSQNMLFLHMNNLKTEDTAIYFCVRDPMDYWGQGISVTVSS(SEQ ID NO:253).

[0285] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0286] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKLSCKASGYTFTTFWMQWVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:254).

[0287] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0288] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSDVKLQESGAELARPGASVKLSCKASGYTFTTFWMQRVKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:255).

[0289] In some embodiments, the anti-CD99 scFv comprises the amino acid sequence:

[0290] DTVMSQSPSSLAVSVGEKITMSCKSSQSLLYRSNQKNYLAWYQQKPGQSPKQLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLKESGAELARPGASVKLSCKASGYTFTTFWMQWAKQRPGQGLEWIGTIYPGDDDTRYTQKFKGRATLTADKSSTTAYMQLSNLSSEDSAVYYCARSGYERGPYYFDSWGQGTTLTVSS(SEQ ID NO:256).

[0291] CLEC12A

[0292] For example, scFvs that selectively bind to CLEC12A are described in US20200345779, which is incorporated by reference in its entirety for describing these antibodies and their sequences. For example, in some embodiments, the anti-CLEC12A region of the disclosed antibody or CAR is derived from hybridomas 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) may comprise a variable heavy (V) region having CDR1, CDR2, and CDR3 sequences. H ) domain and a variable light (V L ) domain.

[0293] In some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GFTFSSFA (SEQ ID NO: 257) SFAVS (SEQ ID NO: 258) or SHDMS (SEQ ID NO: 259); H The CDR2 sequence of the domain comprises the amino acid sequence ISSGGAYT (SEQ ID NO: 260) or TISSGGAYTFYKDSVKGRFT (SEQ ID NO: 261) or YISGGGTNIYYSDTVKGRFT (SEQ ID NO: 262); V H The CDR3 sequence of the domain comprises the amino acid sequence ARHSGYDGYYLYAMDY (SEQ ID NO: 263), HSGYDGYYLYAMDY (SEQ ID NO: 264) or PNYNYGGSWFAY (SEQ ID NO: 265); V L The CDR1 sequence comprises the amino acid sequence SSVHY (SEQ ID NO: 266), ASSSVHYMH (SEQ ID NO: 267) or SASSSVHYMH (SEQ ID NO: 268); V L The CDR2 sequence of the V domain comprises the amino acid sequence DTS or DTSKLAS (SEQ ID NO: 270); and L The CDR3 sequence of the domain comprises the amino acid sequence QQWTSNPPT (SEQ ID NO: 271).

[0294] In some embodiments, anti-CLEC12A V H The domain contains the amino acid sequence

[0295] ELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 272, 1F3H8).

[0296] Therefore, in some embodiments, anti-CLEC12A V H The domain is encoded by the following nucleic acid sequence

[0297] GAACTAATACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTTCCTTTGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGAGCTTACACCTTCTATAAAGACA GTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTGCAAGACATAGCGGCTATGATGGTTACTACCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQID NO:273,1F3H8).

[0298] In some embodiments, anti-CLEC12A V H The domain contains the amino acid sequence

[0299] GVQCELILVESGGGLVKPGGSLKLSCAVSGFFTSSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS (SEQ ID NO: 274, 1F3A10).

[0300] Therefore, in some embodiments, anti-CLEC12A V H The domain is encoded by the following nucleic acid sequence

[0301] GGTGTCCAGTGTGAACTAATACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTTCCTTTGCCGTGTCCTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGAGCTTACACCTTCTAT AAAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACTCGGCCATGTATTACTGTGCAAGACATAGCGGCTATGATGGTTACTACCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:275,1F3A10).

[0302] In some embodiments, anti-CLEC12A V H The domain contains the amino acid sequence

[0303] EVQLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISSRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSA (SEQ ID NO: 276, 1F3F3).

[0304] Therefore, in some embodiments, anti-CLEC12A V H The domain is encoded by the following nucleic acid sequence

[0305] GAGGTGCAGCTGGAGGAGTCTGGGGGAGGCTTAGTGCAGCCGGGAGGGTCCCTGAAAGTCTCCTGTGCAGTTTCCGGACTCGCTTTCAGCAGCCATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGCGGCTGGAGTGGGTCGCATACATTAGTGGAGGTGGTACTAATATCTATTATTCA GACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAAGACACAGCCATTTATTACTGTGCAAGACCCAATTATAATTACGGCGGTTCCTGGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ IDNO:277,1F3F3).

[0306] In some embodiments, anti-CLEC12A V L The domain contains the amino acid sequence

[0307] QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 278,1F3H8,1F3F3,1F3A10).

[0308] Therefore, in some embodiments, anti-CLEC12A V L The domain is encoded by the following nucleic acid sequence

[0309] CAAATTGTTCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTACATTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCT TCTGGAGTCCCTGGTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGTCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATTAAACG(SEQ ID NO:279,1F3H8,1F3F3,1F3A10).

[0310] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0311] ELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK(SEQ ID NO:280,1F3H8).

[0312] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0313] GVQCELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK(SEQ ID NO:281,1F3A10).

[0314] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0315] EVQLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSQIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIK(SEQ ID NO:282,1F3F3).

[0316] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0317] QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSELILVESGGGLVKPGGSLKLSCAVSGFTFSSFAMSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS(SEQ ID NO:283).

[0318] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0319] QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGVQCE LILVESGGGLVKPGGSLKLSCAVSGFFTSSSFAVSWVRQTPEKRLEWVATISSGGAYTFYKDSVKGRFTISRDNAKNTLYLQMSSLRSEDSAMYYCARHSGYDGYYLYAMDYWGQGTSVTVSS(SEQ ID NO:284).

[0320] In some embodiments, the anti-CLEC12A scFv comprises the amino acid sequence:

[0321] QIVLTQSPEIMSASPGEKVTMTCSASSSVHYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGTSYSLTISSMESEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEV QLEESGGGLVQPGGSLKVSCAVSGLAFSSHDMSWVRQTPEKRLEWVAYISGGGTNIYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARPNYNYGGSWFAYWGQGTLVTVSA(SEQ ID NO:285).

[0322] CD83

[0323] For example, scFvs that selectively bind to CD83 are described in US2020 / 0108098, which is incorporated by reference in its entirety for a description of these antibodies and their sequences.

[0324] For example, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 286), SDGIS (SEQ ID NO: 287) or SNAMI (SEQ ID NO: 288); V HThe CDR2 sequence of the domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 289), IISSGGNTYYASWAKG (SEQ ID NO: 290) or AMDSNSRTYYATWAKG (SEQ ID NO: 291); V H The CDR3 sequence of the domain comprises the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 292), VVGGTYSI (SEQ ID NO: 293) or GDGGSSDYTEM (SEQ ID NO: 294); V L The CDR1 sequence comprises the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 295), QSSQSVYNNDFLS (SEQ ID NO: 296) or QSSQSVYGNNELS (SEQ ID NO: 297); V L The CDR2 sequence of the domain comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 298), YASTLAS (SEQ ID NO: 299) or QASSLAS (SEQ ID NO: 300), and the V L The CDR3 sequence of the domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO: 301), TGTYGNSAWYEDA (SEQ ID NO: 302), or LGEYSISADNH (SEQ ID NO: 303).

[0325] For example, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO: 304), the V H The CDR2 sequence of the domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO: 305), the V H The CDR3 sequence of the domain comprises the amino acid sequence CARAYGKLGFDY (SEQ ID NO: 306), V L The CDR1 sequence of the amino acid sequence TLSSQHSTYTIG (SEQ ID NO: 307), the V L The CDR2 sequence of the domain comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO: 308), and the V L The CDR3 sequence of the domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO: 309).

[0326] For example, in some embodiments, V HThe CDR1 sequence of the domain comprises the amino acid sequence SDGIS (SEQ ID NO: 310), the V H The CDR2 sequence of the domain comprises the amino acid sequence IISSGGNTYYASWAKG (SEQ ID NO: 311), the V H The CDR3 sequence of the domain comprises the amino acid sequence VVGGTYSI (SEQ ID NO: 312), V L The CDR1 sequence of the invention comprises the amino acid sequence QSSQSVYNNDFLS (SEQ ID NO: 313), wherein V L The CDR2 sequence of the V L The CDR3 sequence of the domain comprises the amino acid sequence TGTYGNSAWYEDA (SEQ ID NO: 315).

[0327] For example, in some embodiments, V H The CDR1 sequence of the domain comprises the amino acid sequence SNAMI (SEQ ID NO: 316), the V H The CDR2 sequence of the domain comprises the amino acid sequence AMDSNSRTYYATWAKG (SEQ ID NO: 317), the V H The CDR3 sequence of the domain comprises the amino acid sequence GDGGSSDYTEM (SEQ ID NO: 318), V L The CDR1 sequence of the domain comprises the amino acid sequence QSSQSVYGNNELS (SEQ ID NO: 319), the V L The CDR2 sequence of the domain comprises the amino acid sequence QASSLAS (SEQ ID NO: 320), and the V L The CDR3 sequence of the domain comprises the amino acid sequence LGEYSISADNH (SEQ ID NO: 321).

[0328] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0329] QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 322, VH-GBM00).

[0330] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0331] QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL (SEQ ID NO: 323, VL-GBM00).

[0332] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0333] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSNNAINWVRQAPGKGLEWIGYIWSGGLTYYANWAEGRFTISKTSTTVDLKMTSPTIEDTATYFCARG INNSALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPS TCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTI SKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:324,20D04).

[0334] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0335] MDMRAPTQLLGLLLLWLPGARCADVVMTQTPASVSAAVGGTVTINCQASESISNYLSWYQQKPGQPPKLLIYRTSTLASGVSSRFKGSGSGTEYTLTISGVQCDDVATYYCQCTSGGKFISDGAAFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC(SEQ ID NO:325,20D04).

[0336] In some embodiments, the anti-CD83 scFv V H domain comprises the amino acid sequence:

[0337] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFTISDYDLSWVRQAPGEGLKYIGFIAIDGNPYYATWAKGRFTISKTSTTVDLKITAPTTEDTATYFCARGAGDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:326,11G05).

[0338] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence:

[0339] MDTREPTQLLGLLLLWLPGARCADVVMTQTPASVSAAVGGTVTINCQSSKNVYNNNWLSWFQQKPGQPPKLLIYYASTLASGVPSRFRGSGSGTQFTLTISDVQCDDAATYYCAGDYSSSSDNGFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC SEQ ID NO:327,11G05)

[0340] In some embodiments, the anti-CD83 scFv V H domain comprises the amino acid sequence:

[0341] METGLRWLLLVAVLKGVHCQSVEESGGRLVTPGTPLTLTCTASGFSRSSYDMSWVRQAPGKGLEWVGVISTAYNSHYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGGSWLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:328,14C12).

[0342] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence:

[0343] MDXRAPTQLLGLLLLWLPGARCALVMTQTPASVSAAVGGTVTINCQSSQSVYDNDELSWYQQKPGQPPKLLIYALASKLASGVPSRFKGSGSGTQFALTISGVQCDDAATYYCQATHYSSDWYLTFGGGTEVVVKGFPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGTENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC(SEQ ID NO:329,14C12).

[0344] In some embodiments, the anti-CD83 scFv V H domain comprises the amino acid sequence:

[0345] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYDMTWVRQAPGKGLEWIGIIYASGTTYYANWAKGRFTISKTSTTVDLKVTSPTIGDTATYFCAREGAGVSMTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:330,020B08).

[0346] In some embodiments, the anti-CD83 scFv V L domain comprises the amino acid sequence:

[0347] MDMRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQASQSISTYLDWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQQGYTHSNVD NVFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC(SEQ ID NO:331,020B08)

[0348] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0349] METGLRWLLLVAVLKGVQCQSVEESGGRLVSPGTPLTLTCTASGFSLSSYDMSWVRQAPGKGLEYIGIISSSGSTYYASWAKGRFTISKTSTTVDLEVTSLTTEDTATYFCSREH AGYSGDTGHLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTV APSTCSKPTCPPPELLGGPSVGIGPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKT ISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:332,006G05).

[0350] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0351] MDMRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVAIKCQASQSVSSYLAWYQQKPGQPPKPLIYEASMLAAGVSSRFKGSGSGTDFTLTISDLECDDAATYYCQQGYSISDID NAFGGGTEVVVKGDPVAPTVLLFPPSSDEVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC(SEQ ID NO:333,006G05)

[0352] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0353] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSDGISWVRQAPGKGLEWIGIISSGGNTYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARV VGGTYSIWGQGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKI VPRDCGCKPCICTVPEVSSVFIFPPKPDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:334,96G08).

[0354] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0355] MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAPVGGTVTINCQSSQSVYNNDFLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCTGTYGNSAWY EDAFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC(SEQ ID NO:335,96G08)

[0356] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0357] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSNAMIWVRQAPREGLEWIGAMDSNSRTYYATWAKGRFTISRTSSITVDLKITSPTTEDTATYFCARGD GGSSDYTEMWGPGTLVTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKK IVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:336,95F04).

[0358] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0359] MDTRAPTQLLGLLLLWLPGATFAQAVVTQTTSPVSAPVGGTVTINCQSSQSVYGNNELSWYQQKPGQPPKLLIYQASSLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCLGEYSISA DNHFGGGTEVVVKRTPVAPTVLLFPPSSAELATGTATIVCVANKYFPDGTVTWKVDGITQSSGINNSRTPQNSADCTYNLSSTLTLSSDEYNSHDEYTCQVAQDSGSPVVQSFSRKSC(SEQ ID NO:337,95F04)

[0360] In some embodiments, the anti-CD83 scFv V H The domain contains the amino acid sequence:

[0361] QVQLVQSGGAVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAAVSYDGSNKYYADFVKGRFTISRDNPKNTLYLQMNSLRADDTAVYYCARRGGLDIWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA(SEQ ID NO:338).

[0362] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0363] LTQPPPASGTPGQQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYYGNDQRPSGVPDRFSASKSGTSASLAISGLQSEDEAHYYCAAWDGSLNGGVIFGGGTKVTLG (SEQ ID NO: 339).

[0364] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0365] VTQPPSASGTPGQRVTISSCSGSSSNIGTNPVNWYQQLPGTAPKLLIYTTDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLSGLYVFGTGTKVTVLG (SEQ ID NO: 340).

[0366] In some embodiments, the anti-CD83 scFv VL domain comprises the amino acid sequence:

[0367] MTHTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQRPGQSPQPLIYEVSNRFSGV PDRFSGSGSGTDFTLKISRVQAEDVGVYYCMQSLQLWTFGQGTKVEIKR (SEQ ID NO: 341).

[0368] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0369] MTQSPLSLPVTLGQPASISCRSSQSLIHSDGNTYLDWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLRISRVEAEDIGVYYCMQATHWPRTFGQGTKVEIKR (SEQ ID NO: 342).

[0370] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0371] MTQSPLSLPVTLGQPASISCRSSQSLVDSAGNTFLHWFHQRPGQSPRRLIYKVSNRDSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 343).

[0372] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0373] LTQSPLSLPVTLGQPASISCKSSQSLVDSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR (SEQ ID NO: 344).

[0374] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0375] MTQSPLSLPVTLGQPASISCRSSQSLVHSDGNMYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQATQPTWTFGQGTKLEIKR (SEQ ID NO: 345).

[0376] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0377] MTQSPSSLSSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSATYYCQQTYQGTKLEIKR (SEQ ID NO: 346).

[0378] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0379] MTQSPSSLSSASVGHPVTITCRASQSLISYLNWYHQKPGKAPKLLIYAASILQSGVPSRFSGSGSGTDFTLTISSLQPENFASYYCQHTDSFPRTFGHGTKVEIKR (SEQ ID NO: 347).

[0380] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0381] LTQPPSASGTPGQGVTISCRGSTSNIGNNVVNWYQHVPGSAPKLLIWSNIQRPSGIPDRFSGSKSGTSASLAISGLQSEDQAVYYCAVWDDGLAGWVFGGGTTVTVLS (SEQ ID NO: 348).

[0382] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0383] MTQAPVVSVALEQTVRITCQGDSLAIYYDFWYQHKPGQAPVLVIYGKNNRPSGIPHRFSGSSSNTDSLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTNLTVLG (SEQ ID NO: 349).

[0384] In some embodiments, the anti-CD83 scFv V L The domain contains the amino acid sequence:

[0385] LTQSPLSLPVTLGQPASISCKSNQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYCMQGTQWPRTFGGQGTKLDIKR (S SEQ ID NO: 350).

[0386] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0387] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 351, VH-GBM01).

[0388] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0389] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 352, VH-GBM02).

[0390] In some embodiments, the anti-CD83 scFv V HThe domain has been humanized and comprises the amino acid sequence:

[0391] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 353, VH-GBM03).

[0392] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0393] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 354, VH-GBM04).

[0394] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0395] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISSVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 355, VH-GBM05).

[0396] In some embodiments, the anti-CD83 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0397] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS (SEQ ID NO: 356, VH-GBM06).

[0398] In some embodiments, the anti-CD83 scFv V LThe domain has been humanized and comprises the amino acid sequence:

[0399] QLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 357, VL-GBM01).

[0400] In some embodiments, the anti-CD83 scFv V L The domain has been humanized and comprises the amino acid sequence:

[0401] LPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL (SEQ ID NO: 358, VL-GBM02).

[0402] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0403] QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAASSGGGGSGGG GSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRAAA(SEQ ID NO:359).

[0404] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0405] QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGG GSGGGGSQVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTV(SEQ ID NO:360).

[0406] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0407] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:361).

[0408] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0409] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:362).

[0410] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0411] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:363).

[0412] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0413] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:364).

[0414] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0415] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:365).

[0416] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0417] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSQHSTYTIGWHQQQPEKGPRYLMKVNSDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:366).

[0418] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0419] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:367).

[0420] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0421] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQHPGKGLEWIGYIFSSGNTNYNPSIKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:368).

[0422] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0423] QVQLQESGPGLVKPSQTLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:369).

[0424] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0425] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARAYGKLGFDYWGQGTLVTVSSGGGGS GGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:370).

[0426] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0427] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRVTISVDTSKNQFSLKLSSVTAADTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:371).

[0428] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0429] QVQLQESGPGLVKPSETLSLTCTVSGFSITTGGYWWTWIRQPPGKGLEWIGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSLPVLTQPPSASALLGASIKLTCTLSSQHSTYTIGWYQQRPGRSPQYIMKVNSDGSHSKGDGIPDRFMGSSSGADRYLTFSNLQSDDEAEYHCGSSDSSGYVFGSGTKVTVL(SEQ ID NO:372).

[0430] In some embodiments, the anti-CD83 scFv comprises the amino acid sequence:

[0431] QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRISITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGSSSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL(SEQ ID NO:373).

[0432] PSCA

[0433] For example, scFvs that selectively bind to PSCA are described in US2021 / 0379108, which is incorporated by reference in its entirety for describing these antibodies and their sequences. For example, in some embodiments, the anti-PSCA scFv has the amino acid sequence:

[0434] MVLLVTSLLLCELPHPAFLLIPQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWIWIRQHPGKGLEWIGYIYYNGNTYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGITMIRGYYYGMDVWGQ GTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSVSASVGDRVTITCRASRGISSWLAWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPRTFGQGTKVEIKAAAFV(SEQ ID NO:374).

[0435] OR2H1

[0436] For example, scFvs that selectively bind to OR2H1 are described in WO2021257905, which is incorporated by reference in its entirety for describing these antibodies and their sequences.

[0437] In some embodiments of anti-OR2H1 scFv, V H The CDR1 sequence of the domain comprises the amino acid sequence GYIFTGYY (SEQ ID NO: 375); V H The CDR2 sequence of the domain comprises the amino acid sequence NAKSGE (SEQ ID NO: 376); V H The CDR3 sequence of the domain comprises the amino acid sequence GPLL (SEQ ID NO: 377); V L The CDR1 sequence comprises the amino acid sequence SSGSSNIGSNFVS (SEQ ID NO: 378); V L The CDR2 sequence of the domain comprises the amino acid sequence RNNQRPS (SEQ ID NO: 379); and V L The CDR3 sequence of the domain comprises the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 380). In some embodiments, the anti-OR2H1 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0438] QVQLQQSGAEVKKPGESLKISCKGSGYIFTGYYMHWVRQAPGQRPEWLGRMNAKSGEADSAQRFQGRVTMTRDTSINTAYMELRDLRSDDTAVYYCTRGPLLWGQGTLVTVS (SEQ ID NO: 381). In some embodiments, the anti-OR2H1 V L The domain has been humanized and comprises the amino acid sequence:

[0439] QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKTGTSASLAISGLRSEDEADYYCAAWDDSVRGPVFGGGTELTVLAAA (SEQ ID NO: 382).

[0440] In some embodiments of anti-OR2H1 scFv, V H The CDR1 sequence of the domain comprises the amino acid sequence GYIFTGYY (SEQ ID NO: 383); V H The CDR2 sequence of the domain comprises the amino acid sequence MNAKSGEA (SEQ ID NO: 384); V H The CDR3 sequence of the domain comprises the amino acid sequence TRGPLL (SEQ ID NO: 385); L The CDR1 sequence comprises the amino acid sequence SSNIGSNF (SEQ ID NO: 386); V L The CDR2 sequence of the domain comprises the amino acid sequence RNN; and V L The CDR3 sequence of the domain comprises the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 74). In some embodiments, the anti-OR2H1 scFv V H The domain has been humanized and comprises the amino acid sequence:

[0441] MAQVQLQQSGAEVKKPGESLKISCKGSGYIFTGYYMHWVRQAPGQRPEWLGRMNAKSGEADSAQRFQGRVTMTRDTSINTAYMELRDLRSDDTAVYYCTRGPLLWGQGTLVTVSS (SEQ ID NO: 75). In some embodiments, anti-OR2H1 V LThe domain has been humanized and comprises the amino acid sequence: QPVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVSWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKTGTSASLAISGLRSEDEADYYCAAWDDSVRGPVFGGGTELTVL (SEQ ID NO: 75).

[0442] In some embodiments of anti-OR2H1 scFv, V H The CDR1 sequence of the domain comprises the amino acid sequence SSNIGSNF (SEQ ID NO: 77); V H The CDR2 sequence of the domain comprises the amino acid sequence RNN; V H The CDR3 sequence of the domain comprises the amino acid sequence AAWDDSVRGPV (SEQ ID NO: 125); V L The CDR1 sequence comprises the amino acid sequence GYTFTSNY (SEQ ID NO: 139); V L The CDR2 sequence of the domain comprises the amino acid sequence INPSGGRT (SEQ ID NO: 176); V L The CDR3 sequence of the domain comprises the amino acid sequence ARSHCSGGSCYSIDY (SEQ ID NO: 269). In some embodiments, the anti-OR2H1 scFvV H The domain is humanized and contains the amino acid sequence:

[0443] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGIINPSGGRTSYAQKFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARSHCSGGSCYSIDYWGQGTLVTVSS (SEQ ID NO: 387). In some embodiments, the anti-OR2H1 V L The domain has been humanized and comprises the amino acid sequence:

[0444] QPVLTQSSSASASLGSSVKLTCTLSSGHSGYIIAWHQQQPGKAPRYLMKVEGSGSYNKGSGIPERFSGSSSGADRYLTISNLQSEDEADYYCETWDSNTHVFGTGTKVTVL (SEQ ID NO: 124).

[0445] 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, US20 14 / 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 incorporated by reference in their entirety for teachings of these scFv sequences.

[0446] In some embodiments, the scFv is an anti-BCMA02 scFv (ABECMA; idecabtagene vicleucel), disclosed in din 9,765,342. In some embodiments, the scFv is an anti-CD19 scFv (BREYANZI; lisocabtagene maraleucel), disclosed in 9,701,758. In some embodiments, the scFv is an anti-CD19 scFv (KYMRIAH; tisagenlecleucel), disclosed in US2014 / 0271635. In some embodiments, the scFv is an anti-CD19 scFvscFv (TECARTUS; brexucabtagene autoleucel), disclosed in US2015 / 0344844. In some embodiments, the scFv is an anti-CD19 scFv (YESCARTA; axicabtagene ciloleucel), disclosed in US2015 / 0344844.

[0447] Also disclosed are the nucleic acid sequences encoding the separation of the disclosed CAR polypeptides, vectors comprising these separated nucleic acids, and cells containing these vectors. For example, cells can be immune effector cells selected from the group consisting of: α-βT cells, γ-δT 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.

[0448] In some embodiments, when the antigen binding domain of the CAR binds to a TAA on a tumor, the cell exhibits anti-tumor immunity.

[0449] Also disclosed is a method of providing anti-tumor immunity in a subject having a cancer expressing a TAA, the method involving administering to the subject an effective amount of immune effector cells genetically modified with a disclosed TAA-specific CAR.

[0450] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0451] Figure 1A and 1B Hela ( Figure 1A ) and BT-549( Figure 1B ) Cell killing (%).

[0452] Figure 2 Shown are HeLa tumor volumes after treatment with OR5V1 or OR5V1-NKG2D CAR. DETAILED DESCRIPTION

[0453] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, as these embodiments may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0454] Where a range of values ​​is provided, it is understood that each intervening value (to one-tenth of the unit of the lower limit, unless expressly stated otherwise) between the upper and lower limits of the range, as well as any other stated or intervening value in the range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specific exclusions within the ranges. Where the range includes one or both of the limits, ranges excluding either or both of the included limits are also encompassed within the disclosure.

[0455] 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 invention 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, preferred methods and materials are now described.

[0456] All publications and patents cited in this specification are incorporated herein by reference, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials related to the cited publications. Reference to any publication should not be construed as an admission that the present disclosure is not entitled to antedate such publications due to existing disclosure. Further, the publication date provided may be different from the actual publication date, which may need to be independently confirmed.

[0457] Those skilled in the art will appreciate after reading this disclosure that each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recorded method can be performed in the order of events recorded or in any other order that is logically possible.

[0458] Unless otherwise stated, the embodiments of the present disclosure will employ techniques of chemistry, biology, etc., which are within the technical scope of the art.

[0459] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to implement the methods disclosed and claimed herein and to use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, temperatures are ° C, and pressures are at or near atmospheric pressure. Standard temperature and pressure are defined as 20 ° C and 1 atmosphere.

[0460] Before describing the embodiments of the present disclosure in detail, it should be understood that, unless otherwise stated, the present disclosure is not limited to specific materials, reagents, reaction materials, manufacturing processes, etc., as they may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. In the present disclosure, it is also possible that steps can be performed in different orders where logically possible.

[0461] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0462] definition

[0463] 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 single-letter codes for amino acids and are represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0464] The term "antibody" refers to an immunoglobulin, a derivative thereof that maintains specific binding ability, and a protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or may be partially or completely synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class from any species, including any one of the human classes: IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibody used with the methods and compositions described herein is a derivative of the IgG class. In addition to complete immunoglobulin molecules, the term "antibody" also includes fragments or polymers of these immunoglobulin molecules and immunoglobulin molecules of a human or humanized form that selectively binds to a target antigen.

[0465] The term "aptamer" refers to an oligonucleic acid or peptide molecule that binds to a specific target molecule. These molecules are usually selected from a random sequence pool. The selected aptamer can adapt to a unique tertiary structure and recognize the target molecule with high affinity and specificity. "Nucleic acid aptamers" are DNA or RNA oligonucleic acids that bind to a target molecule via its conformation and thus inhibit or suppress the function of such molecules. Nucleic acid aptamers can be composed of DNA, RNA or a combination thereof. "Peptide aptamers" are combined protein molecules with variable peptide sequences inserted into a constant scaffold protein. The identification of peptide aptamers is usually carried out under strict yeast two-hybrid conditions, which increases the possibility of stable expression and correct folding of the selected peptide aptamers in the intracellular environment.

[0466] The term "carrier" refers to a compound, composition, substance or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity or any other characteristic of a compound or composition to achieve its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects to a subject.

[0467] The term "chimeric molecule" refers to a single molecule produced 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.

[0468] The term "fusion protein" refers to a polypeptide formed by connecting 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 it can be expressed as a single polypeptide by a nucleic acid sequence encoding a single continuous fusion protein. A single-chain fusion protein is a fusion protein with a single continuous polypeptide backbone. Conventional techniques in molecular biology can be used to connect two genes in frame into a single nucleic acid and then express the nucleic acid in an appropriate host cell under conditions that produce a fusion protein to prepare a fusion protein.

[0469] The term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the position in each sequence that can be compared for comparison purposes. When the position in the comparison sequence is occupied by the same base, the molecule is identical at that position. The similarity or degree of identity between nucleic acids or amino acid sequences is a function of the number of identical or matching nucleotides at the position shared by the nucleic acid sequence. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which can be obtained as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity with specific polypeptides described herein and preferably showing substantially the same function and polynucleotides encoding such polypeptides are contemplated. Unless otherwise stated, similarity scores will be based on the use of BLOSUM62. When using BLASTP, the similarity percentage is based on the BLASTP similarity score, and the sequence identity percentage is based on the BLASTP identity score. BLASTP "identity" shows the number and fraction of total residues that are identical in a high-scoring sequence pair; and BLASTP "similarity" shows the number and fraction of residues whose alignment scores have similarity values ​​and are similar to each other. Amino acid sequences having these degrees of identity or similarity, or any intermediate degrees of identity or similarity, to the amino acid sequences disclosed herein are contemplated and encompassed by the present disclosure. The polynucleotide sequences of similar polypeptides are inferred using the genetic code and can be obtained by conventional means, particularly by reverse translating their amino acid sequences using the genetic code.

[0470] The term "nucleic acid" refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked to the 5' end of another nucleotide via a phosphate group at the 3' position of one nucleotide. Nucleic acids are not limited in length, and thus nucleic acids may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0471] The term "operably linked to" refers to the functional relationship of a nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, the operably linked DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and the promoter, such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0472] The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprised of two or more amino acids linked through the carboxyl group of one amino acid to the alpha amino group of another amino acid.

[0473] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0474] The term "protein domain" refers to a portion of a protein, multiple portions of a protein, or an entire protein that exhibits structural integrity; the determination can be based on the amino acid composition of the portion of a protein, multiple portions of a protein, or an entire protein.

[0475] As used herein, "spacer" refers to a peptide that connects the proteins that comprise the fusion protein. Typically, the spacer has no specific biological activity other than connecting the proteins or maintaining a certain minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer can be selected to affect some properties of the molecule, such as the folding, net charge or hydrophobicity of the molecule.

[0476] As used herein, the term "specifically binds" in relation to polypeptides (including antibodies) or receptors refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a specified ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen) when it does not bind in significant amounts to other proteins present in the sample or other proteins that the ligand or antibody may contact in the organism. Typically, a first molecule that "specifically binds" to a second molecule has a specific binding affinity greater than about 10 5 M -1 (For example, 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 more) of affinity constant (Ka).

[0477] As used herein, the term "specific delivery" refers to the preferential association of a molecule with a cell or tissue carrying a specific target molecule or marker, rather than with a cell or tissue lacking the target molecule. Of course, it is recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. However, specific delivery can be distinguished as mediated by specific recognition of a target molecule. Typically, specific delivery results in a much stronger association between the delivered molecule and a cell carrying the target molecule than between the delivered molecule and a cell lacking the target molecule.

[0478] The term "subject" refers to any individual that is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. Thus, the subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician (e.g., a physician).

[0479] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only requires reduction or modification, not elimination.

[0480] The terms "transformation" and "transfection" mean the introduction of a nucleic acid (eg, an expression vector) into a recipient cell, including the introduction of a nucleic acid into the chromosomal DNA of the cell.

[0481] The term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. This term encompasses active treatment, i.e. treatment specifically directed to the improvement of a disease, pathological condition or disorder, and also encompasses causal treatment, i.e. treatment directed to the removal of the cause of the relevant disease, pathological condition or disorder. In addition, the term also includes: palliative treatment, i.e. treatment aimed at relieving symptoms rather than curing the disease, pathological condition or disorder; preventive treatment, i.e. treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e. treatment used to supplement another specific therapy aimed at improving the relevant disease, pathological condition or disorder.

[0482] The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e., degenerate variants), substitutions within the wobble position of each codon (i.e., DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of the peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0483] The term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid to which the vector sequence has been linked into a cell. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0484] Chimeric antigen receptor (CAR) with NKG2D intracellular domain

[0485] CAR generally comprises an antigen recognition domain of a single chain variable fragment (scFv) from a monoclonal antibody (mAb) having a transmembrane signaling motif involved in lymphocyte activation (Sadelain M et al. Nat Rev Cancer 20033: 35–45). Disclosed herein is a chimeric antigen receptor (CAR) that can be expressed in immune effector cells to enhance anti-tumor activity against cancer.

[0486] The disclosed CAR is generally composed of three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains a binding region and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that CAR can be glycosylated and anchored in the cell membrane of immune effector cells. The transmembrane (TM) domain, as the name implies, connects the extracellular domain to the intracellular domain and resides in the cell membrane when expressed by the cell. The intracellular domain is the working end of CAR, which transmits activation signals to immune effector cells after antigen recognition. The disclosed CAR contains the intracellular domain of NKG2D in the intracellular domain.

[0487] In some embodiments, the disclosed CAR is defined by the formula:

[0488] SP-BD-HG-TM-NKRIC;

[0489] wherein "SP" represents an optional signal peptide,

[0490] Where "BD" stands for target binding domain,

[0491] wherein "HG" represents an optional hinge domain,

[0492] "TM" stands for transmembrane domain.

[0493] where "NKRIC" stands for NK cell receptor intracellular domain, and

[0494] Wherein “-” represents a peptide bond or a linker.

[0495] For example, additional CAR constructs are described in, e.g., Fresnak AD et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81, which is incorporated by reference in its entirety for teachings of these CAR models.

[0496] For example, the CAR may be a TRUCK, a universal CAR, a self-driving CAR, an armored CAR, a self-destructing CAR, a conditional CAR, a marking CAR, a TenCAR, a dual CAR, or an sCAR.

[0497] TRUCK (T cells redirected for universal cytokine killing) co-express chimeric antigen receptors (CARs) and anti-tumor cytokines. Cytokine expression can be constitutive or induced by T cell activation. Local production of pro-inflammatory cytokines specifically targeted by CARs recruits endogenous immune cells to the tumor site and may enhance the anti-tumor response.

[0498] Universal allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0499] Self-actuated CARs co-express CAR and chemokine receptors, which bind to tumor ligands, thereby enhancing tumor homing.

[0500] CAR T cells engineered to resist immunosuppression (armored CARs) can be genetically modified to no longer express various immune checkpoint molecules with immune checkpoint switch receptors (e.g., cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)) or can be administered with monoclonal antibodies that block immune checkpoint signaling.

[0501] Self-destructive CARs can be designed using RNA encoding CARs 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 system of activating human caspase 9 by small molecule dimers.

[0502] Conditioned CAR T cells are unresponsive or "off" by default until a small molecule is added to complete the circuit, achieving full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells can be engineered to express an adaptor-specific receptor with affinity for a subsequently administered secondary antibody against the target antigen.

[0503] CAR T cells are tagged to express the CAR and a tumor epitope that is bound by an existing monoclonal antibody reagent. In the event of intolerable adverse events, administration of the monoclonal antibody eliminates the CAR T cells and alleviates symptoms without producing additional off-tumor effects.

[0504] Tandem CAR (TanCAR) T cells express a single CAR composed of two linked single-chain variable fragments (scFv) with different affinities fused to an intracellular co-stimulatory domain and a CD3ζ domain. TanCAR T cell activation is achieved only when the target cell co-expresses both targets.

[0505] The safe CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR will only be activated when they encounter a target cell that possesses the standard CAR target but lacks the sCAR target.

[0506] The binding domain of the disclosed CAR is usually scFv. However, there are many alternatives. Antigen recognition domains from natural T cell receptor (TCR) α and β single chains have been described, as well as simple extracellular domains (e.g., CD4 extracellular domains for identifying HIV infected cells) and more exotic recognition components, such as connected cytokines (which lead to recognition of cells carrying cytokine receptors). In fact, almost anything that binds to a given target with high affinity can be used as an antigen recognition region.

[0507] The intracellular domain is the working end of CAR, which transmits signals to immune effector cells after antigen recognition, thereby activating at least one of the normal effector functions of immune effector cells. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. The disclosed CAR contains the intracellular domain of NKG2D in the intracellular domain. However, in some embodiments, the intracellular domain may further include an "intracellular signaling domain" of a T cell receptor (TCR) and an optional co-receptor. For example, the intracellular domain of CAR can be designed to further include the CD3ζ signaling domain itself or in combination with any other desired cytoplasmic domain useful in the context of CAR. For example, the cytoplasmic domain of CAR may further include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the intracellular domain of CAR that contains a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are required for the effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Therefore, although CAR is mainly exemplified with CD28 as a costimulatory signaling element, other costimulatory elements can be used alone or in combination with other costimulatory signaling elements.

[0508] In some embodiments, CAR comprises a hinge sequence. Hinge sequence is a short amino acid sequence that promotes antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4 (2): 89-99 (2004)). Hinge sequence can be located between antigen recognition portion and transmembrane domain. Hinge sequence can be any suitable sequence, which is derived from or obtained from any suitable molecule. In some embodiments, for example, hinge sequence is derived from CD8a molecule or CD28 molecule. In some embodiments, hinge sequence is derived from NK receptor, preferably, the same as NK receptor derived from intracellular domain.

[0509] Transmembrane domains may be derived from natural or synthetic sources. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be derived from (i.e., at least comprises the transmembrane region of) the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), 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, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, IT GA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD 11c, 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. Alternatively, the transmembrane domain can be synthetic, in which case it will contain mainly hydrophobic residues, such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. A short oligopeptide or polypeptide linker, such as 2 to 10 amino acids in length, can form a connection between the transmembrane domain of the CAR and the endoplasmic domain domain.

[0510] In some embodiments, the CAR has more than one transmembrane domain, which may be repeats of the same transmembrane domain, or may be different transmembrane domains.

[0511] In some embodiments, CAR is a multi-chain CAR, as described in WO2015 / 039523, which is incorporated herein by reference for this teaching. Multi-chain CAR can include separate extracellular ligand binding domains and signaling domains in different transmembrane polypeptides. The signaling domain can be designed to be assembled in a near-membrane position, which forms a flexible structure closer to the natural receptor, thereby achieving optimal signal transduction.

[0512] In some embodiments, the binding domain is a single chain variable fragment (scFv) antibody. The affinity / specificity of the scFv is largely determined by the heavy chain (V H ) and light chain (V L ) in the complementarity determining region (CDR). Each V H and V L The sequence will have three CDRs (CDR1, CDR2, CDR3).

[0513] In some cases, the binding domain is an affinity matured scFv. In some cases, the binding domain has a dissociation constant (K) for TAA of less than 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, or 10 nM. D ).

[0514] In some embodiments, the binding domain is derived from a natural antibody, such as a monoclonal antibody. In some cases, the antibody is human. In some cases, the antibody has been altered to reduce its immunogenicity when administered to humans. For example, the alteration includes one or more techniques selected from the group consisting of: chimerization, humanization, CDR grafting, deimmunization, and framework amino acid mutations corresponding to the closest human germline sequence.

[0515] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. The binding domain can be an antibody or a natural ligand for the tumor antigen. The choice of antigen binding domain will depend on the specific type of cancer to be treated.Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-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, CYPIBI, BORIS, prostate enzymes, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostate-specific protein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, asparagine endopeptidase, 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, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, 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, Tie 2, 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.

[0516] 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, pi 5); overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens generated by 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 large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA15-3\CA 27.29\BCAA, CA 195, CA 242, 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, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6 and MET.

[0517] Nucleic acids and vectors

[0518] Also disclosed are polynucleotides encoding the disclosed CARs and polynucleotide vectors that allow expression of the CARs in the disclosed immune effector cells.

[0519] The nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene using standard techniques, by being derived from vectors known to include the gene, or by being isolated directly from cells and tissues containing the gene. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0520] The expression of nucleic acid encoding CAR is usually achieved by operably linking the nucleic acid encoding CAR polypeptide to a promoter and integrating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of desired nucleic acid sequences.

[0521] The disclosed nucleic acids can be cloned into various types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0522] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, a suitable vector is included in at least one organism, a promoter sequence, a convenient restriction endonuclease site and one or more selective markers that work as a replication origin. In certain embodiments, the polynucleotide vector is a slow virus or a retroviral vector.

[0523] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.

[0524] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongated growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, MND (bone marrow and extracellular myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. Alternatively, the promoter may be an inducible promoter. Examples of inducible promoters include but are not limited to metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

[0525] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in the 30 to 110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible so that promoter function is maintained when the elements are inverted or moved relative to each other.

[0526] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, the selective marker can be carried on a separate DNA fragment and used for a co-transfection procedure. Both the selective marker and the reporter gene can be flanked by suitable regulatory sequences to enable them to be expressed in host cells. Useful selective markers include, for example, antibiotic resistance genes.

[0527] Reporter gene is used to identify cells that may be transfected, and is used to assess the function of regulatory sequences. Generally, reporter gene is a gene that is not present in or is not expressed by a receptor organism or tissue, and the expression of its encoded polypeptide is demonstrated by some easily detectable characteristics (e.g., enzymatic activity). The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein gene. Suitable expression system is well-known and can be prepared or commercially available using known technology. Generally, the construct with the minimum 5' flanking region showing the highest level of reporter gene expression is identified as a promoter. Such promoter region can be connected with reporter gene and used to assess the ability of transcription driven by promoter regulating agent.

[0528] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical or biological methods.

[0529] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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).

[0530] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (eg, human) cells.

[0531] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0532] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes via connecting molecules associated with both liposomes and oligonucleotides, captured in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, merged with lipids, contained as a suspension in lipids, containing micelles or compounded with micelles, or otherwise associated with lipids. Compositions associated with lipids, lipid / DNA or lipid / expression vectors are not limited to any specific structure in solution. For example, they can exist as follows: bilayer structure, micelle, or "collapsed" structure. They can also be simply dispersed in solution, possibly forming aggregates that are not uniform in size or properties. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids are included in naturally occurring fat droplets in the cytoplasm, and the categories 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, dimyristoylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0533] Immune effector cells

[0534] Also disclosed are immune effector cells (also referred to herein as "CAR-T cells") engineered to express the disclosed CAR. These cells are preferably obtained from the subject to be treated (ie, autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Any number of techniques known to those skilled in the art, such as FicollTM separation, can be used to obtain immune effector cells from blood collected from the subject. For example, cells from the circulating blood of an individual can be obtained by apheresis. In some embodiments, immune effector cells are separated from peripheral blood lymphocytes by lysing red blood cells and removing mononuclear cells, for example, by PERCOLLTM gradient centrifugation or by countercurrent centrifugal elutriation. Specific subsets of immune effector cells can be further separated by positive selection techniques or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed against surface markers specific to positively selected cells, such as by incubating with antibody-conjugated beads for a time sufficient to positively select desired immune effector cells. Alternatively, the enrichment of immune effector cell populations can be accomplished by negative selection using a combination of antibodies directed against surface markers specific to negatively selected cells.

[0535] In some embodiments, immune effector cells include any white blood cells involved in defending the body against infectious diseases and foreign substances. For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells can include T lymphocytes.

[0536] T cells, or T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a different function.

[0537] T helper cells (T HHelper T cells assist other white blood cells in immune 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 called CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines, which regulate or help active immune responses. These cells can differentiate into one of several subtypes, including T H 1. T H 2. T H 3. T H 17. T H 9 or T FH , which secrete different cytokines to promote different types of immune responses.

[0538] Cytotoxic T cells (T C CTLs (CD8 T cells) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. + T cells, because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated to an anergic state by IL-10, adenosine, and other molecules secreted by regulatory T cells, which prevents autoimmune diseases.

[0539] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has resolved. When they are re-exposed to their cognate antigen, they rapidly expand into large numbers of effector T cells, thus providing the immune system with a "memory" of past infections. Memory cells can be CD4 + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0540] Regulatory T cells (T reg CD4 T cells, formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus. Two major classes of CD4 T cells have been described. + T reg Cells - Naturally occurring T reg Cells and adaptive T reg cell.

[0541] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) link the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0542] In some embodiments, the T cells include a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subpopulations based on cell surface expression. For example, in some cases, the T cells include cytotoxic CD8 + T lymphocytes. In some embodiments, the T cells include γδ T cells, which possess a unique T-cell receptor (TCR) having a γ chain and a δ chain instead of an α chain and a β chain.

[0543] Natural killer (NK) cells are CD56 + CD3 - Large granular lymphocytes, which can kill virus-infected and transformed cells and constitute a key cell subset of the innate immune system (Godfrey J et al. Leuk Lymphoma 201253:1666–1676). + Unlike T lymphocytes, NK cells can be cytotoxic to tumor cells without prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E et al. Int Immunol 2011 23:427–431). NK cells are safer effector cells because they can avoid the potentially fatal complications of cytokine storm (Morgan RA et al. Mol Ther 2010 18:843–851), tumor lysis syndrome (Porter DL et al. N Engl J Med 2011 365:725–733) and on-target, off-tumor effects. Despite the well-known role of NK cells as cancer cell killers, NK cell damage has been extensively documented to be critical for MM progression (Godfrey J et al. LeukLymphoma 2012 53:1666–1676; Fauriat C et al. Leukemia 2006 20:732–733), and prior to the disclosed CARs, the means by which one might enhance NK cell-mediated anti-MM activity had been largely unexplored.

[0544] Treatment

[0545] Immune effector cells expressing the disclosed CARs can elicit an anti-tumor immune response against cancer cells expressing TAAs. The anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells can be an active or passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach, in which the CAR-modified immune effector cells induce an immune response specific for TAAs.

[0546] Adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anticancer therapeutic approach. After a patient's immune effector cells are collected, the cells can be genetically engineered to express the disclosed CARs and then infused back into the patient.

[0547] The disclosed CAR modified immune effector cells can be administered alone or as a pharmaceutical composition combined with a diluent and / or other components (such as IL-2, IL-15 or other cytokines or cell populations). In short, the pharmaceutical composition may include a target cell population as described herein and one or more pharmaceutical or physiological carriers, diluents or excipients. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; 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 composition used in the disclosed method is formulated for intravenous administration. The pharmaceutical composition can be administered in any manner suitable for treating MM. The amount and frequency of administration will be determined by factors such as the patient's condition and the severity of the patient's disease, but the appropriate dose can be determined by clinical trials.

[0548] When "immunologically effective amount", "anti-tumor effective amount", "tumor suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally speaking, the pharmaceutical composition comprising the T cells described herein can be 10 4 Up to 10 9 The dosage of cells / kg body weight is, for example, 10 5 Up to 10 6Cells / kg body weight, including all integer values ​​within these ranges. T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Those skilled in the art of medicine can easily determine the optimal dose and treatment regimen for a particular patient by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0549] In certain embodiments, it may be necessary to administer activated T cells to a subject, and then redraw blood (or perform apheresis), activate T cells therefrom according to the disclosed method, and re-infuse these activated and amplified T cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10cc to 400cc. In certain embodiments, T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Using this multiple blood draw / multiple re-infusion scheme can be used to select out certain T cell populations.

[0550] Administration of the disclosed compositions can be carried out in any convenient manner, including by injection, transfusion or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions can also be injected directly into a tumor, lymph node or infection site.

[0551] In certain embodiments, with any number of related treatment modalities (including but not limited to thalidomide, dexamethasone, bortezomib and lenalidomide) together (for example, before, simultaneously or afterwards) the disclosed CAR modified immune effector cells are applied to the patient.In further embodiments, the CAR modified immune effector cells can be used in combination with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506), antibodies or other immune scavengers (such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation).In certain embodiments, with bone marrow transplantation, T cell ablation therapy using chemotherapeutics (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide or antibodies (such as OKT3 or CAMPATH) (for example, before, simultaneously or afterwards) the CAR modified immune effector cells are applied to the patient. In another embodiment, the cell composition of the invention is administered after B cell ablative therapy, such as an agent reactive with CD20 (e.g., Rituxan). For example, in some embodiments, the subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after the transplant, the subject receives an infusion of the immune cells expanded by the invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0552] The cancer of the disclosed methods can be any TAA expressing cell in a patient that undergoes unregulated growth, invasion or metastasis. In some aspects, the cancer can be any neoplasm or tumor currently treated with radiation therapy. 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 that can be treated using the disclosed compositions includes: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer 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, throat, larynx and lung, endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, kidney cancer, genitourinary cancer, pulmonary cancer, esophageal cancer, head and neck carcinoma, colorectal cancer, hematopoietic cancer; testicular cancer; colon and rectal cancer, prostate cancer, and pancreatic cancer.

[0553] The disclosed CARs can be used in combination with any compound, moiety or group having a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents that can act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors or DNA intercalators, and particularly those used for cancer treatment.

[0554] The disclosed CAR can be used in combination with checkpoint inhibitors. Two known inhibitory checkpoint pathways involve signal transduction through 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, which play an important role in 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. PD-L1 is the dominant ligand, while the expression pattern of PD-L2 is more limited. When the ligand binds to PD-1, the inhibitory signal is transmitted to the T cell, which reduces cytokine production and inhibits 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).

[0555] Human monoclonal antibodies against programmed death 1 (PD-1) and methods of treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are described in U.S. Pat. No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and their uses are described in U.S. Pat. No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agents including anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Pat. No. 8,617,546, which is incorporated by reference for these antibodies.

[0556] In some embodiments, PDL1 inhibitors include antibodies that specifically bind to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, PD1 inhibitors include antibodies that specifically bind to PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb) or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods of treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and their uses are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agents including anti-PD-1 antibodies or anti-PD-L1 antibodies are described in U.S. Pat. No. 8,617,546, which is incorporated by reference for these antibodies.

[0557] The disclosed CAR can be used in combination with other cancer immunotherapies. There are two different types of immunotherapy: passive immunotherapy uses components of the immune system to guide targeted cytotoxic activity against cancer cells, and does not necessarily induce an immune response in the patient, while active immunotherapy actively induces endogenous immune responses. 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 recognition of tumor-specific antigens allowed the development of mAbs (which can specifically target tumor cells for destruction by the immune system). So far, monoclonal antibodies are the most successful cases of immunotherapy; the three best-selling anticancer drugs in 2012 are monoclonal antibodies. Among them are rituximab (Rituxan, Genentech), which binds to CD20 proteins highly expressed on the surface of B cell malignancies such as non-Hodgkin's lymphoma (NHL). Rituximab is approved by the FDA for combined treatment of NHL and chronic lymphocytic leukemia (CLL) with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which has revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting HER2 expression.

[0558] Generating an optimal “killer” CD8 T cell response also requires T cell receptor activation and co-stimulation, which can be provided by ligating 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, thereby enhancing antitumor immunity against a variety of tumors.

[0559] In some embodiments, such additional therapeutic agents may be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0560] In some embodiments, such additional therapeutic agents can be selected from alkylating agents such as nitrogen mustard, thiophene, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin.

[0561] In some embodiments, such additional therapeutic agents may be selected from anti-mitotic agents such as taxanes, eg, docetaxel and paclitaxel, and vinca alkaloids, eg, vindesine, vincristine, vinblastine, and vinorelbine.

[0562] In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors, such as topotecan or irinotecan; or cytostatic drugs, such as etoposide and teniposide.

[0563] In some embodiments, such additional therapeutic agents may be selected from a growth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such as an EGFR antibody, e.g., zalutumumab, cetuximab, panitumumab, or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as a HER2 antibody, e.g., trastuzumab, trastuzumab-DM 1, or pertuzumab), or an inhibitor of both EGFR and HER2 (such as lapatinib).

[0564] In some embodiments, such additional therapeutic agents may be selected from tyrosine kinase inhibitors, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0565] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0566] In some embodiments, the therapeutic agent used in combination with CAR to treat the disease as described above can be an anticancer factor, a chemokine or a combination thereof. Suitable examples of cytokines and growth factors include IFN γ, 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, IFNa (eg, INFa2b), IFN, GM-CSF, CD40L, Flt3 ligands, stem cell factors, Ancistim and TNF α. Suitable chemokines can include Glu-Leu-Arg (ELR)-negative chemokines, such as IP-10, MCP-3, MIG and SDF-1a from human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments and cytokine fusion proteins.

[0567] In some embodiments, the therapeutic agent used in combination with CAR to treat a disorder as described above can be a cell cycle control / apoptosis regulator (or "regulatory agent"). Cell cycle control / apoptosis regulators can include molecules that target and regulate cell cycle control / apoptosis regulators, such as: (i) cdc-25 (such as NSC 663284); (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 regulators (such as BIBR1532, SOT-095, GRN163 and compositions described in, for example, US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0568] In certain embodiments, the therapeutic agent used for treating the disease as described above in combination with CAR can be a hormone regulator, such as a medicament that can be used for anti-androgen and anti-estrogen therapy. Examples of such hormone regulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinylestradiol / estradiol, anti-androgen (such as flutamine / flutamide), progesterone (such as hydroxyprogesterone caproate, medroxyprogesterone / medroxyprogesterone acetate / Meg), adrenal cortical steroids (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and its analogs and other LHRH agonists, such as buserelin and goserelin), aromatase inhibitors (such as anastrozole / Arimidex, aminoglutethimide / cytraden, exemestane) or hormone inhibitors (such as octreotide / sandostatin).

[0569] In some embodiments, the therapeutic agent for use in combination with CAR to treat a condition as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0570] As mentioned above, the combined administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0571] In some embodiments, the disclosed CAR is administered in combination with radiotherapy. Radiotherapy may include providing radiation or related radiopharmaceutical administration to the patient. The radiation source may be external or internal to the patient being treated (radiation therapy may be, for example, in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used to practice 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.

[0572] In some embodiments, the disclosed CARs are administered in combination with surgery.

[0573] CAR-T cells can be designed in a variety of ways to enhance tumor cell toxicity and specificity, escape tumor immunosuppression, avoid host rejection, and extend their therapeutic half-life. TRUCK (T cells redirected for universal cytokine killing) for example has CAR, but is also engineered to release cytokines, such as IL-12, which promote tumor killing. Because these cells are designed to release molecular payloads when CAR is activated once localized to the tumor environment, these CAR-T cells are sometimes also referred to as "armored CARs." Several cytokines are being studied preclinically and clinically as cancer therapies, and may also prove useful when similarly 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, lymphocyte chemokine, and TGF-β (Dranoff 2004). "Self-driving" or "homing" CAR-T cells are engineered to express chemokine receptors in addition to CAR. Since some chemokines may be upregulated in tumors, the incorporation of chemokine receptors facilitates tumor transport to and infiltration by adoptive T cells, thereby enhancing both the specificity and functionality of CAR-T (Moon 2011). Universal CAR-T cells also have CARs, but are engineered so that they do not express 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. In addition, armored CAR-T cells are named for their ability to evade tumor immunosuppression and tumor-induced CAR-T dysfunction. These specific CAR-Ts possess CARs and can 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 antibodies significantly restored the killing ability of CAR TILs (tumor-infiltrating lymphocytes). Although the PD1-PDL1 and CTLA-4-CD80 / CD86 signaling pathways have been studied, other immune checkpoint signaling molecules, including LAG-3, Tim-3, IDO-1, 2B4, and KIR, can be targeted in the design of armored CAR-Ts. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinases). Armored CAR-Ts can also be engineered to express proteins or receptors that protect them from or render them resistant to the effects of cytokines secreted by tumors.For example, CTLs (cytotoxic T lymphocytes) transduced with a double negative form of the TGF-β receptor are resistant to immunosuppression by TGF-β secreted by lymphomas. When compared to their control cells, these transduced cells exhibit significantly increased antitumor activity in vivo.

[0574] Tandem CARs contain two consecutive antigen-binding domains facing the extracellular environment, which are connected to intracellular co-stimulatory and stimulatory domains.

[0575] The main problem of CAR-T cells as a form of "living treatment" is their operability in vivo and its potential immunostimulatory side effects. In order to better control CAR-T therapy and prevent unnecessary side effects, a variety of features have been engineered, including off switches, safety mechanisms, and conditional control mechanisms. For example, both self-destructive cells and labeled / tagged CAR-T cells are engineered to have a "off switch" that promotes the removal of T cells expressing CAR. Self-destructive CAR-T contains CAR, but is also engineered to express pro-apoptotic suicide genes or "elimination genes" that can be induced after the administration of exogenous molecules. A variety of suicide genes can be used for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC tags, and truncated EGFR (endothelial growth factor receptor). For example, HSK converts the prodrug ganciclovir (GCV) into GCV-triphosphate that integrates itself into the replicating DNA, ultimately leading to cell death. iCasp9 is a chimeric protein containing a component of a FK506 binding protein that binds to the small molecule AP1903, leading to caspase 9 dimerization and apoptosis. However, the tagged / tagged CAR-T cells not only have CAR, but are also engineered to express a selection marker. Administration of mAbs for the selection marker will promote the elimination of CAR-T cells. Truncated EGFR is such a targetable antigen for anti-EGFR mAb, and the administration of cetuximab promotes the elimination of CAR-T cells. CARs created for these features are also referred to as sCARs for "switchable CARs" and RCARs for "regulatable CARs." "Safety CAR," also known as "inhibitory CAR" (iCAR), is engineered to express two antigen binding domains. One of these extracellular domains is directed against tumor-associated antigens and binds to intracellular co-stimulatory and stimulatory domains. However, the second extracellular antigen binding domain is specific to normal tissues and binds to intracellular checkpoint domains (such as CTLA4, PD1, or CD45). It is also possible to incorporate multiple intracellular inhibitory domains into iCAR. Some inhibitory molecules that may 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 will inhibit CAR. It is worth noting that due to this dual antigen specificity, iCAR is also a form of bispecific CAR-T cells.Safe CAR-T engineering enhances the specificity of CAR-T cells for tumor tissues and is advantageous in situations where certain normal tissues may express very low levels of tumor-associated antigens, which can result in off-target effects of standard CARs (Morgan 2010). Conditional CAR-T cells express an extracellular antigen-binding domain linked to an intracellular co-stimulatory domain and a separate intracellular co-stimulator. The co-stimulatory and stimulatory domain sequences are engineered in such a way that the resulting proteins will aggregate together within the cell to complete the CAR circuit after administration of the exogenous molecule. In this way, CAR-T activation can be regulated and may even be “fine-tuned” or personalized for specific patients. Similar to the dual CAR design, when the conditional CAR is inactive, the stimulatory and co-stimulatory domains are physically separated; therefore, these are also called “split CARs.”

[0576] In some embodiments, two or more of these engineered features can be combined to create an enhanced multifunctional CAR-T. For example, a CAR-T cell with a dual or conditional CAR design can be created that also releases cytokines like TRUCK. In some embodiments, a dual conditional CAR-T cell can be prepared so that it expresses two CARs with two separate antigen binding domains for two different cancer antigens, each of which binds to their respective co-stimulatory domains. The co-stimulatory domain functions with the stimulatory domain only after the activation molecule is administered. In order for this CAR-T cell to be effective, the cancer must express two cancer antigens and the activation molecule must be administered to the patient; the design therefore incorporates the features of dual and conditional CAR-T cells.

[0577] Typically, α-βT cells are used to create CAR-T cells, however, γ-δT cells can also be used. In some embodiments, the CAR constructs, domains, and engineered features described for generating CAR-T cells can be similarly used to generate other types of CAR-expressing immune cells, including NK (natural killer) cells, B cells, mast cells, bone marrow-derived phagocytes, and NKT cells. Alternatively, cells expressing CAR can be created to have the characteristics of both T cells and NK cells. In other embodiments, transduction with CAR can be autologous or allogeneic.

[0578] Several different methods of CAR expression can be used, including retroviral transduction (including gamma-retrovirus), lentiviral transduction, transposon / transposase (Sleeping Beauty and PiggyBac systems), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) is also becoming increasingly important for the possibility of engineering CAR-T cells. CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator-like effector nuclease) systems are three possible methods for generating CAR-T cells.

[0579] Several embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are within the scope of the following claims.

[0580] Examples

[0581] Example 1:

[0582] 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 adaptor 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 Rael, MULT1 and H60 in mice (Garrity D et al. Proc Natl Acad Sci 2005 102:7641–6). In CD8 T cells, the ligation of the NKG2D receptor with its ligand enhances the activation, proliferation and production of inflammatory cytokines and effector molecules 1. NKG2D mediates the survival of memory CD8 T cells and is sufficient to maintain CD8 T cell memory in the absence of CD4 T cell “help” (Zloza A et al. Nat Med 2012 18:422–8).

[0583] In CD8 T cells, DAP10 expresses a YxNM domain that recruits p85 to induce PI3K signaling, and the YxNX domain is associated with Grb2 recruitment and signaling (Okkenhaug K et al. Nat Rev Immunol 2003 3:317–30; Upshaw JL et al. Nat Immunol 2006 7:524–32).

[0584] The goal of this study was to engineer scFv CAR T cells with Pro-T cell properties (killing, survival, and migration properties) by exploiting the NKG2D / DAP10 signaling pathway. To achieve this goal, an scFv targeting OR5V1 was linked to the transmembrane structure (Tm) and intracellular domain (IC) of NKG2D ( Figure 1A ). In this design, the scFv will recognize the antigen, leading to the activation of NKG2D / DAP10 signaling, which in turn will initiate the killing and memory phases in the carrier T cells.

[0585] The designed constructs were expressed in human T cells and tested side by side with second generation CAR-T cells for their 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 tumors) was no different from that of conventional CAR-T cells ( Figure 2 A and 2B). These data suggest that NKG2D(TM-IC) can replace conventional CAR-T cell signaling utilizing the CD3 domain.

[0586] Next, it was determined whether ORV1-NKG2D-CAR could kill tumors in vivo compared to conventional CAR-T cells. Figure 3 shows that ORV1-NKG2D-CAR controls tumor growth similarly to conventional CAR-T. In this experiment, immunodeficient mice (NSG mice) were attacked by tumors of HeLa cells in the flank and treated with simulated engineered T cells, conventional CAR-T and CAR-TNKG2D. Tumor growth was measured and plotted manually. These data indicate that scFv-NKG2D (TM-IC) can replace conventional CAR-T cell signaling using the CD3 domain and mediate in vivo anti-tumor responses against human tumors.

[0587] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference.

[0588] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising a ligand binding domain, a hinge domain, a transmembrane domain and an intracellular domain, wherein the intracellular domain comprises an NK cell receptor intracellular domain or a fragment thereof capable of activating a target killing 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 of claim 2, wherein the intracellular domain comprises the amino acid sequence of 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 of claim 3, wherein the transmembrane domain comprises the amino acid sequence of 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 of 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 of any one of claims 1 to 5, wherein the intracellular domain does not contain a CD3ζ (CD3ζ) signaling domain.

7. The polypeptide according to any one of claims 1 to 6, wherein the CAR polypeptide is defined by the following formula: SP-BD-HG-TM-NKRIC; wherein "SP" represents an optional signal peptide, Where "BD" stands for target binding domain, wherein "HG" represents an optional hinge domain, "TM" stands for transmembrane domain. wherein "NKRIC" stands for NK cell receptor intracellular domain, and Wherein "-" represents a peptide bond or a linker.

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

9. A vector comprising the isolated nucleic acid sequence according to claim 8.

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

11. The cell according to claim 10, wherein the cell is selected from the group consisting of: 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, regulatory T (T reg ) cells or any combination thereof.

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

13. A method of providing anti-tumor immunity in a subject having a cancer expressing a TAA, the method comprising: An effective amount of immune effector cells genetically modified to express the CAR polypeptide according to any one of claims 1 to 7 is administered to the subject, thereby providing anti-tumor immunity in the mammal.

14. The method of claim 13, wherein the immune effector cells are selected from the group consisting of 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, regulatory T (T reg ) cells or any combination thereof.

15. The method according to claim 13 or 14, further comprising: A checkpoint inhibitor is administered to the subject.

16. The method of 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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