Genetically modified non-humans with humanized gamma and delta TCR variable genes

Abstract

The invention provides a genetically modified non-human animal. The genetically modified non-human animal comprises exogenous gamma and delta T cell receptor variable gene coding fragments in a genome of the genetically modified non-human animal; as well as tissues, embryos and cells thereof. Also provided are constructs and methods for making the genetically modified non-human animals. Human T cell receptor (TCR) cloning and library analysis are also included. The use of the animals for infectious diseases is also provided.

Description

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to provisional application No. 63 / 397,633, filed on August 12, 2022, the entire disclosure of which is hereby incorporated by reference. Background Art Technical Field

[0002] The present invention relates to a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or a rat), wherein the genetically modified non-human animal comprises a human or humanized T cell receptor (TCR) variable region of a DNA encoding and / or a gene locus (e.g., TCRδ. and TCRγ. variable gene locus) in its genome. The present invention also relates to a non-human animal engineered to express human or humanized TCRδ or TCRγ on the surface of γ-δT cells. The present invention also relates to a non-human animal engineered to co-express both human or humanized TCRδ or TCRγ on the surface of γδT cells. Methods for cloning and analyzing human or humanized TCR libraries and human or humanized TCR amplification after infection are provided. Methods for expressing human or humanized γδT cell receptors or co-receptors for further development of therapies for human diseases are also provided. The present invention also relates to unrearranged human TCR variable region gene segments (e.g., human V, D and / or J segments) at endogenous non-human TCRγδ gene loci.

[0003] Discussions in related fields

[0004] Like conventional αβT cells and B cells, γδT cells use V(D)J gene rearrangement to generate a highly diverse set of receptors to recognize antigens. This diversity is mainly generated in the complementarity determining region 3 (CDR3) of the T cell antigen receptor (TCR). Unlike conventional αβT cells, γδT cells can be activated and expanded by non-peptide antigens. In contrast to conventional αβT cells, γδT cells do not rely on classical MHC molecules to present peptides. The efficacy of γδT cells does not rely on the recognition of classical HLA molecules.

[0005] These cells have unique properties in detecting antigenic disruption during various infections or cancers, a property that may be common between individuals and even between different animal species. Because γδT cells can quickly recognize antigenic interference, γδT cells are considered to be part of the first line of immune defense (innate immune system), but they can also generate immune memory (adaptive immune system) (Chien, Annu. Rev. Immunol. 32. 121-155 (2014)). Human γδT cells have been shown to kill infected cells and cancers.

[0006] However, non-human animals capable of producing diversified human γδCDR3 have not yet been developed. Therefore, there is a need in the art for non-human animals (e.g., rodents, e.g., rats or mice) comprising unrearranged human γδT cell variable region gene segments that can be rearranged to form genes encoding human γδT cell receptor variable domains (including domains that are homologous to each other, and including domains that specifically bind to an antigen of interest and subsequently initiate downstream chemical and / or biological activity). Summary of the invention

[0007] Embodiments of the present invention relate to non-human animals, e.g., rodents, comprising unrearranged human or humanized γδTCR encoding DNA segments and variable gene loci. The variable region further comprises human V, D and J segments. A method for producing the animal is also provided. The genome of the immune cells of the animal undergoes V(D)J recombination under the activity of V(D)J recombinase (e.g., RAG1 and / or RAG2). V(D)J recombination includes a process for producing a large amount of γ / δTCR diversity. This diversity increases the differences in the functional genes produced.

[0008] Provided herein is a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) comprising in its genome DNA encoding and / or gene loci (e.g., TCRδ and / or TCRγ variable gene loci) a human or humanized T cell receptor (TCR) variable region.

[0009] In various embodiments, the animal comprises a nucleotide sequence encoding a human TCR gamma and / or delta variable region. In one embodiment, the human variable region is fused to a non-human gamma and / or delta TCR coding region to form a chimeric human / non-human TCR. In one embodiment, the nucleotide sequence of the non-human animal is replaced by a human sequence at the endogenous locus of the gamma and / or delta T cell receptor.

[0010] In one embodiment, human V (D) J encoding as a part of the TCR CDR3 domain of a polypeptide, the polypeptide is connected to a non-human portion comprising a transmembrane and cytoplasmic domain. In one embodiment, the portion of the polypeptide containing the CDR3 domain on the surface of a γδ T cell is used as an extracellular domain. In another aspect, the chimeric TCR comprises a human variable region and a non-human constant region on the surface of a γδ T cell.

[0011] In one aspect, a genetically modified non-human animal (e.g., a rodent, e.g., a mouse or a rat) is provided, the genetically modified non-human animal comprising an unrearranged TCRγ and / or an unrearranged TCRδ nucleotide sequence in its genome. The unrearranged TCRγ comprises at least one human Vγ variable segment and at least one human Jγ segment. The unrearranged TCRδ comprises at least one human Vδ segment, at least one human Dδ, and at least one human Jδ segment.

[0012] In one embodiment, a non-human animal (e.g., a rodent, e.g., a mouse or a rat) comprises in its genome at least one human Vγ variable segment and at least one human Jγ segment operably linked to a non-human (e.g., a rodent, e.g., a mouse or a rat) TCRγ constant gene sequence. The genome of the non-human animal also comprises at least one human Vδ segment, at least one human Dδ, and at least one human Jδ segment operably linked to a non-human (e.g., a rodent, e.g., a mouse or a rat) TCRδ constant gene sequence.

[0013] In one embodiment, a human unrearranged TCR gamma variable gene locus replaces one or more non-human TCR gamma variable genes at the endogenous TCR gamma variable gene locus.

[0014] In one embodiment, a human unrearranged TCRδ variable gene locus replaces one or more non-human TCRδ variable genes at the endogenous TCRδ variable gene locus.

[0015] In one aspect, the human variable region comprising Vγ and Jγ segments can be rearranged in whole or in part to form a rearranged Vγ and Jγ sequence. In another aspect, the human variable region comprising Vδ, Dδ and Jδ segments can be rearranged in whole or in part to form a rearranged V(D)Jδ sequence.

[0016] In some embodiments, the result of rearranging the human Vγ / Jγ sequence upon activation of the recombinase is the generation of a functional CDR3 domain. In some embodiments, the result of rearranging the human Vδ / Dδ / Jδ sequence upon activation of the recombinase is the generation of a functional CDR3 domain.

[0017] In some aspects, the T cells of the non-human animal undergo T cell development into human γδ-positive T cells in the thymus.

[0018] In various embodiments, the non-human animal generates a γδ T cell population in the periphery, spleen, lung, liver, kidney, intestine, and skin.

[0019] In one aspect, the invention relates to the expression of a sequence segment of at least 10 amino acids in human TCRγ and / or human TCRγ of a non-human animal.

[0020] In some embodiments, the human TCR comprises an amino acid sequence of one of the following: (a) human variable Vγ9, 10, 11 having SEQ ID NO (amino acid): 62, 63, 64, respectively; (b) a conservative amino acid sequence having at least 90% identity with SEQ ID NO (amino acid): 62 or 63; and (c) a conservative amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 62, 63 or 64.

[0021] In one aspect, the invention relates to the expression of a sequence segment of at least 10 amino acids in human TCRδ and / or human TCRδ of a non-human animal. In some embodiments, the human TCR comprises an amino acid sequence of one of the following: (a) a human variable V(δ) 1, 2, 3, 4, 5, 6, 7 or 8 having SEQ ID No: 65, 66, 67, 68, 69, 70, 71 or 72, respectively; (b) a conservative amino acid sequence having at least 90% identity with SEQ ID (amino acid) NO: 62 or 63; and (c) a conservative amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with SEQ ID (amino acid) NO: 65, 66, 67, 68, 69, 70, 71 or 72.

[0022] In one aspect, the disclosure relates to a nucleic acid encoding all or part of the amino acid sequence of an endogenous non-human animal Vγ constant region (C) at an endogenous locus. In some embodiments, the VγC region is from other species and can be functional when the VγC region is connected to human VJγ. Human Vγ, Jγ and functional C regions can be introduced into endogenous loci or randomly integrated into the genome of non-human animals.

[0023] In some aspects, the constant region of TCRδ comprises one or more of the conserved amino acid sequences: PSVF, MKNG, GTNVACL, SAVKLGQ, SVTCSV, KVNMMSL, VLGLR, LFAK and / or NFLL.

[0024] In some aspects, the constant region of TCRγ comprises one or more conserved amino acid sequences: PKPT, LCLL, KTKD, MKFSWLT, TSAYY and / or LLLLLKS.

[0025] In one embodiment, the unrearranged TCRγ gene locus in the non-human animal described herein comprises 3 human Vγ segments and 3 human Jγ segments.

[0026] In one embodiment, the non-human animal described herein further comprises 8 human Vδ segments, 3 human Dδ segments, and 4 human Jδ segments.

[0027] In another embodiment, the unrearranged TCRγ gene loci in non-human animals can form a functional repertoire of human γCDR3 domains under the activity of Rag1 and / or Rag2 or a combination of both or other relevant enzymes.

[0028] In another embodiment, the unrearranged TCRδ gene locus in a non-human animal can form a functional repertoire of human γCDR3 domains under the activity of Rag1 and / or Rag2 or a combination of both or other relevant enzymes.

[0029] In an additional embodiment, the non-human animal (e.g., rodent) described herein further comprises a nucleotide sequence of a human TCR δ variable segment at a humanized TCR δ locus. In one embodiment, the non-human animal (e.g., rodent) further comprises at least one human V δ, D δ and / or J δ segment.

[0030] In one embodiment, human TCRγ and mouse TCRγ can form a receptor complex and be expressed on mouse T cells. In one embodiment, human delta T cell receptor can form a receptor complex with mouse gamma T cell receptor for expression on mouse gamma / delta T cells.

[0031] In some embodiments, the present invention relates to a method comprising performing the replacement of endogenous non-human TCRγ with human TCRγ variable gene loci as described herein in a single ES (embryonic stem) cell, and introducing the single ES cell into a non-human (e.g., rodent, e.g., mouse or rat) embryo to prepare a genetically modified non-human animal (i.e., a first non-human animal, e.g., a human Vγ rodent); and performing the replacement of endogenous non-human TCRδ with human Vδ variable gene loci as described herein in a single ES cell, and introducing the single ES cell into a non-human (e.g., rodent, e.g., mouse or rat) embryo to prepare a genetically modified non-human animal (i.e., Vδ rodent). In one embodiment, Vγ rodents and Vδ rodents are hybridized to form offspring, wherein the offspring comprises a humanized TCRγ variable locus and a humanized TCRδ variable locus in its germline.

[0032] In the methods, the non-human animal is a rodent, eg, a mouse, a genetically modified mouse.

[0033] The present invention also provides herein cells derived from non-human animals described herein (e.g., rodents, e.g., mice or rats), e.g., isolated T cells (e.g., γ / δ T cells, helper T cells, memory T cells, etc.). Tissues and embryos derived from non-human animals described herein are also provided.

[0034] In one aspect, a method for preparing a human TCR variable domain, the method comprising: genetically modifying a rodent as described herein so that it comprises a humanized TCRγ locus and / or a humanized TCRδ locus: maintaining the rodent under conditions sufficient to form T cells, wherein the T cells express the human TCRγ and / or human TCRδ variable domains.

[0035] In some aspects, different non-human promoters for human Vδ are also provided, including promoters from animals (e.g., cattle, rabbits) rich in γ / δT cells. In other aspects, non-human promoters for human Vγ are also provided, including promoters from rodents (e.g., rats or mice). In one aspect, non-human animals include promoters directly from human sources and sequences that are not rearranged. In some other aspects, other human Vδ is inserted into the vicinity of unrearranged Vδ sequences.

[0036] In some aspects, methods are provided for amplifying DNA fragments encoding human TCRγ and TCRδ CDR3 after reverse transcription from mRNA. Methods for sequencing PCR products containing CDR3 by next generation sequencing are also provided. Methods for human γ / δ TCR library analysis are also provided.

[0037] In some aspects, sequences of TCR (γ / δ) CDR3 from human Vδ1, Vδ2, Vδ3, Vδ4, Vδ6 are provided. In some aspects, sequences of TCRγ CDR3 from human Vγ9 and Vγ10 are provided. In other aspects, the present invention also discloses human γ and δ TCRs, and their CDR3s are obtained from various tissues of non-human animals (including blood, intestine, thymus, liver, spleen, heart, skin and lung).

[0038] In some aspects, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR1 from human Vδ1 comprises a conserved amino acid sequence comprising TSWWSYY, or at least one S, one W, one Y or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ1 comprises QGS, or at least one Q, one G or one S. In another aspect, the peptide sequence of the CDR3 from human Vδ1 comprises a conserved start and end sequence. The preferred amino acids for the first three amino acids of the CDR3 of Vδ1 are ALG. The further preferred amino acids for the first four amino acids are ALGE, wherein position E can be any one of G, A, D, V, E or R. For the termination sequence, two of the last five amino acids of the CDR3 of Vδ1 are DK, LI, KL, TD, DT, PI, TA, TR, QL or WD. In some aspects, the preferred amino acids for the last three amino acids of the CDR3 of Vδ1 are KLI.

[0039] In one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR1 from human Vδ2 comprises a conserved amino acid sequence comprising GEAIGNYY, or at least one G, one E, one A, one I, one N, one Y within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ2 comprises EKD, or at least one E, one K, or one D. In some aspects, for the starting sequence, the amino acids at the first three positions of the CDR3 of Vδ2 are ACD, ACG, ACE, ARD, or ASD. In some aspects, the first two amino acids of the CDR3 of Vδ2 are AW, PV, or DC. Further preferred amino acids for the first four amino acids are ACDS, wherein the fourth position can be any one of T, K, I, P, Y, C, L, M, or R. For the termination sequence, two of the last five amino acids of the CDR3 of Vδ2 are DK, LI, KL, TD, DT, PI, TA, TR, QL, SS, HI, TH, or PD. In some aspects, the preferred amino acids for the last three amino acids of CDR3 of Vδ2 are KLI.

[0040] CDRs of human Vδ3

[0041] In one aspect, in a non-human animal having human or humanized γ / δT cells, the peptide sequence of the CDR1 from human Vδ3 comprises a conserved amino acid sequence comprising TVYSNPD, or at least one T, one V, one Y, one S, one N, one P or one D within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ3 comprises GDNSR, or at least one G, one D, one N, one S or one R. The peptide sequence of the CDR3 from human Vδ3 comprises a conserved start sequence. The first two starting amino acid sequences of the CDR3 of Vδ3 are AF. In some aspects, the preferred amino acids of the first three amino acids of the CDR3 of Vδ3 are AL, AS, AC, AY, AI or AV. The further preferred amino acids of the first two amino acids are PF, TF, LF, GF or GC.

[0042] CDRs of human Vδ4

[0043] In one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of CDR1 from human Vδ4 comprises a conserved amino acid sequence comprising TSDPSYG, or at least one S, one P, one G, one D, one Y, or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ4 comprises QGSYDQQN, or at least one Q, one G, one D, one Y, one N, or one S. In one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of CDR3 from human Vδ4 comprises a conserved start and stop sequence. For the start sequence, the first three amino acids of CDR3 of Vδ4 are AMR, ASP, AMS, AMT, AMG, AMI, AIR, AKR, ATR, EMR, VMR, PMR, and ALP. In one aspect, the preferred amino acids for the first three amino acids of the CDR3 of Vδ4 are AMRE, wherein the fourth position can be any of G, C, D, V, N, A, L, V, T, or R. For the termination sequence, two of the last five amino acids of the CDR3 of Vδ4 are DK, LI, KL, TD, DT, PI, TA, TR, QL, PD, HI, KI, or SS. In some aspects, the preferred amino acids for the last four amino acids of the CDR3 of Vδ4 are DKLI or TRQM.

[0044] CDRs of human Vδ6

[0045] In one aspect, in a non-human animal having human or humanized γ / δT cells, the peptide sequence of the CDR1 from human Vδ6 comprises a conserved amino acid sequence comprising NTAFDY, or at least one N, one T, one A, one F, one D, or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ6 comprises IRPDVSE, or at least one I, one R, or one P, one D, one V, one S, or one E within the CDR2 sequence. The peptide sequence of the CDR3 from human Vδ6 comprises a conserved start and stop sequence. For the start sequence, the amino acids at the first and second positions of the CDR3 of Vδ6 are AA. In some aspects, the preferred amino acids for the first three amino acids of the CDR3 of Vδ6 are AAS or AAR. The further preferred amino acids for the first four amino acids are AASP, wherein the 4th P can be any one of T, G, M, V, L, or R. In some other aspects, the preferred amino acids for the first two amino acids of CDR3 of Vδ6 are QQ, EA, TA, QH, AV, SK, VA, ES, DA, SA, EG or EP. For the termination sequence, two consecutive amino acids in the last five amino acids of CDR3 of Vδ6 are DK, LI, KL, TD, DT, PI, TA, TR, PD, HI, HT, KI or SS. In some aspects, the preferred amino acids for the last three amino acids of CDR3 of Vδ6 are TRQ, KLI or KLN.

[0046] CDRs of human Vγ9

[0047] In one aspect, the peptide sequence of the CDR1 from human Vγ9 comprises a conserved amino acid sequence comprising GITISATS, or at least one G, one A, one I, one T, or one S within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vγ9 comprises ISYDGTV, or at least one I, one S, one Y, one G, one T, or one D. In one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vγ9 comprises a conserved start and stop sequence. For the start sequence, the amino acids at the first four positions of the CDR3 of Vγ9 are ALWE, ALWG, ASWE, ALCE, ALLE, ALRE, PCGR, AWWE, DLWE, ASWE, or AMWE. In some aspects, the first five amino acids of the CDR3 of Vγ9 are ALWEV, wherein the fifth position can be any one of A, E, or M. In some aspects, in the start sequence of ALWG, the fourth position can be any one of D, V, R, or K. In other aspects, the first four amino acids of the CDR3 of Vγ9 are TLWE, wherein the first position can be any one of G, V, S, P or A. In one aspect, the fourth position of CDR3 is E. For the termination sequence, two consecutive amino acids of the last five amino acids of the CDR3 of Vγ9 are KK. In some aspects, the last two amino acids of the CDR3 of Vγ9 are KTL, KEL, EKL, KNL, IKV, KSR, RNS, KNS, MKL, KRL, RKL, FKI or KNQG.

[0048] CDRs of human Vγ10

[0049] In one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR1 from human Vγ10 comprises a conserved amino acid sequence, the conserved amino acid sequence comprising STRFETDV, or at least one R, one F, one T, one E, one D, one V or one S within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vγ10 comprises IVSTKSAA, or at least one I, one S, one V, one K, one T or one A. In some aspects, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vγ10 comprises a conserved start and stop sequence. For the start sequence, the amino acids at the first three positions of the CDR3 of Vγ10 are AAW. In some aspects, the first four amino acids of the CDR3 of Vγ10 are AAWF, wherein the fourth position can be any one of F, L, A, R, G, C or V. In some other aspects, the second position is A and the first position can be any one of S, Y, V, E, G, D, T or P. In some aspects, the second position is E and the first position can be any one of S, D or A. In some aspects, the first and second positions are PR or SS. In some aspects, the first position is A, and the second position is any one of V, T, S, G or E. In some aspects, the first two positions are any one of VS, VT or CE. For the termination sequence, in some aspects, the last three amino acids of the CDR3 of Vγ10 are FKI. In one aspect, two consecutive amino acids in the last three amino acids of the CDR3 of Vγ10 are KK. In one aspect, the second of the last two amino acids of the CDR3 of Vγ10 is K. In other aspects, the last amino acid of the CDR3 of Vγ10 is I. In one aspect, the last amino acid of the CDR3 of Vγ10 is R. In other aspects, the second of the last two amino acids of the CDR3 of V(γ)10 is T.

[0050] In one aspect, a method for preparing a nucleic acid sequence encoding a human γ / δTCR variable domain that binds an antigen of interest, the method comprising: exposing a non-human animal as described herein to an antigen of interest or stimulating expression of an internal antigen; and maintaining the non-human animal to produce a humanized γ / δTCR against a specific antigen.

[0051] In some aspects, methods of introducing exogenous and / or endogenous antigens into non-human gamma / delta animals are provided. In one aspect, the antigens are from biological and / or chemical sources. In another aspect, the antigens include microorganisms or extracts thereof (e.g., E. coli, Staphylococcus extracts), and the mammalian organism includes mouse cancers (e.g., B16 melanoma) and / or human cancers (e.g., Daudi cells).

[0052] In other aspects, after the antigen is introduced into the human γ / δ T cell mouse, a specific human γ / δ T cell population is expanded.

[0053] In other aspects, the CDR3 sequences of human γ / δ T cells before and after expansion are sequenced and analyzed.

[0054] In one embodiment, the antigen binding protein comprises a TCR variable domain comprising a human TCR gamma variable domain and / or a human TCR delta variable domain.

[0055] In one aspect, there is provided use of a non-human as described herein for preparing a non-human cell expressing a humanized T cell receptor on its surface.

[0056] A genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unarrayed human T cell receptor (TCR) Vδ gene segment, at least one unarrayed human TCR Dδ gene segment, and at least one unarrayed human TCR Jδ gene segment, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are operably linked to a functional non-human TCRδ constant gene sequence.

[0057] The animal of the preceding embodiment, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are inserted into an endogenous TCRδ variable gene locus.

[0058] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replace at least one gene segment in the unarrayed endogenous TCR Vδ gene segment library, and / or at least one gene segment in the unarrayed endogenous TCR Dδ gene segment library and / or at least one gene segment in the unarrayed endogenous TCR Jδ gene segment library.

[0059] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replace at least one nucleotide of a gene segment in an unarrayed endogenous TCR Vδ gene segment library, and / or at least one nucleotide of a gene segment in an unarrayed endogenous TCR Dδ gene segment library and / or at least one nucleotide of a gene segment in an unarrayed endogenous TCR Jδ gene segment library.

[0060] The animal of any of the preceding embodiments, wherein the animal is heterozygous for TCRδ.

[0061] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replaces the complete library of unarrayed endogenous TCR Vδ, Dδ, and Jδ gene segments.

[0062] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment is selected from an unarrayed library of human TCR Vδ gene segments Vδ1, Vδ2, Vδ3, Vδ4, Vδ5, Vδ6, Vδ7 and Vδ8.

[0063] An animal as described in any of the preceding embodiments, wherein the amino acid sequence of at least one unaligned human TCR Vδ segment has at least 90% sequence identity with SEQ ID NO:64, 65, 66, 67, 68, 69, 70, 71 and 72.

[0064] An animal as described in any of the preceding embodiments, wherein the at least one unarranged human TCR Vδ gene segment, the at least one unarranged human TCR Dδ gene segment, and the at least one unarranged human TCR Jδ gene segment are capable of rearranging to form a rearranged human VDJδ sequence.

[0065] The animal of any of the preceding embodiments, wherein the animal expresses a humanized TCRδ variable region comprising the rearranged human VDJδ sequence on the surface of a γδ T cell population.

[0066] A genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unarranged human TCR Vγ gene segment and at least one human TCR Jγ gene segment, wherein the at least one unarranged human TCR Vγ gene segment and the at least one human TCR Jγ gene segment are operably linked to a functional non-human TCRγ constant gene.

[0067] The animal of the preceding embodiment, wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment are inserted into an endogenous TCRγ variable gene locus.

[0068] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment replace at least one gene segment in the unarrayed endogenous TCR Vγ gene segment library and / or at least one gene segment in the unarrayed endogenous TCR Jγ gene segment library.

[0069] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment replace at least one nucleotide of a gene segment in an unarrayed endogenous TCR Vγ gene segment library and / or at least one nucleotide of a gene segment in an unarrayed endogenous TCR Jγ gene segment library.

[0070] The animal of any of the preceding embodiments, wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment replace the complete repertoire of unarrayed endogenous TCR Vγ and Jγ gene segments.

[0071] The animal of any of the preceding embodiments, wherein the animal is heterozygous for TCRγ.

[0072] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vγ gene segment is selected from an unarrayed library of human TCR Vγ gene segments Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10 and Vγ11, preferably Vγ9, Vγ10 and Vγ11.

[0073] The animal of any of the preceding embodiments, wherein the amino acid sequence of at least one unaligned human TCR Vγ segment has at least 90% sequence identity to SEQ ID NO:62, 63 or 64.

[0074] The animal of any of the preceding embodiments, wherein the at least one unarranged human TCR Vγ gene segment and the at least one unarranged human TCR Jγ gene segment are capable of rearranging to form a rearranged human VJγ sequence.

[0075] The animal of any of the preceding embodiments, wherein the animal expresses a humanized TCRγ variable region comprising a rearranged human VJγ sequence on the surface of a population of γδ T cells.

[0076] A genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unarrayed human T cell receptor (TCR) Vδ gene segment, at least one unarrayed human TCR Dδ gene segment, and at least one unarrayed human TCR Jδ gene segment; and at least one unarrayed human TCR Vγ gene segment and at least one human TCR Jγ gene segment, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are operably linked to a functional non-human TCRδ constant gene, and wherein the at least one unarrayed human TCR Vγ gene segment and the at least one human TCR Jγ gene segment are operably linked to a functional non-human TCRγ constant gene.

[0077] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replace at least one gene segment in the unarrayed endogenous TCR Vδ gene segment library, and / or at least one gene segment in the unarrayed endogenous TCR Dδ gene segment library and / or at least one gene segment in the unarrayed endogenous TCR Jδ gene segment library; and wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment replace at least one gene segment in the unarrayed endogenous TCR Vγ gene segment library and / or at least one gene segment in the unarrayed endogenous TCR Jγ gene segment library.

[0078] An animal as described in any of the preceding embodiments, wherein the at least one unaligned human TCR Vδ gene segment, the at least one unaligned human TCR Dδ gene segment, and the at least one unaligned human TCR Jδ gene segment replace at least one nucleotide of a gene segment in the unaligned endogenous TCR Vδ gene segment library, and / or at least one nucleotide of a gene segment in the unaligned endogenous TCR Dδ gene segment library and / or at least one nucleotide of a gene segment in the unaligned endogenous TCR Jδ gene segment library; and wherein the at least one unaligned human TCR Vγ gene segment and the at least one unaligned human TCR Jγ gene segment replace at least one nucleotide of a gene segment in the unaligned endogenous TCR Vγ gene segment library and / or at least one nucleotide of a gene segment in the unaligned endogenous TCR Jγ gene segment library.

[0079] An animal as described in any of the preceding embodiments, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are inserted into an endogenous TCRδ variable gene locus, and wherein the at least one unarrayed human TCR Rvγ gene segment and the at least one unarrayed human TCR Jγ gene segment are inserted into an endogenous TCRγ variable gene locus.

[0080] An animal as described in any of the preceding embodiments, wherein the at least one unarranged human TCR Vδ gene segment, the at least one unarranged human TCR Dδ gene segment, and the at least one unarranged human TCR Jδ gene segment replace the complete library of unarranged endogenous TCR Vδ, Dδ, and Jδ gene segments, and wherein the at least one unarranged human TCR Vγ gene segment and the at least one unarranged human TCR Jγ gene segment replace the complete library of unarranged endogenous TCR Vγ and Jγ gene segments.

[0081] An animal as described in any of the preceding embodiments, wherein the at least one unarranged human TCR Vδ gene segment is selected from an unarranged library of human TCR Vδ gene segments Vδ1, Vδ2, Vδ3, Vδ4, Vδ5, Vδ6, Vδ7 and Vδ8, and wherein the at least one unarranged human TCR Vγ gene segment is selected from an unarranged library of human TCR Vγ gene segments Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10 and Vγ11.

[0082] An animal as described in any of the preceding embodiments, wherein the at least one unarranged human TCR Vδ gene segment, the at least one unarranged human TCR Dδ gene segment, and the at least one unarranged human TCR Jδ gene segment are capable of rearranging to form a rearranged human VDJδ sequence, and wherein the at least one unarranged human TCR R Vγ gene segment and the at least one unarranged human TCR Jγ gene segment are capable of rearranging to form a rearranged human VJγ sequence.

[0083] The animal of any one of the preceding embodiments, wherein the animal expresses a humanized TCRγδ comprising the rearranged human VDJδ sequence and the rearranged human VJγ sequence on the surface of a γδ T cell population.

[0084] The animal of any of the preceding embodiments, wherein Vγ and Vδ have at least 90% sequence identity with human Vγ and Vδ.

[0085] The animal of any of the preceding embodiments, wherein Dδ has at least 90% sequence identity to human Dδ.

[0086] The animal of any of the preceding embodiments, wherein said Jγ and said Jδ have at least 90% sequence identity to human Jγ and Jδ.

[0087] The animal of any of the preceding embodiments, wherein the humanized TCR is expressed on the surface of the γδ T cell population together with at least one mouse CD3.

[0088] The animal of any of the preceding embodiments, wherein the animal produces central and effector memory γ / δ T cell populations to internal or external antigens.

[0089] The animal of any of the preceding embodiments, wherein the animal is heterozygous for TCRδ and / or TCRγ.

[0090] A method for producing a humanized TCR, the method comprising: administering a target antigen to the genetically modified animal as described in any one of the preceding embodiments; and obtaining a humanized TCR that recognizes the target antigen.

[0091] A method for determining and / or analyzing the TCR repertoire of humanized TCRs produced according to the previous embodiment by next generation sequencing.

[0092] A method of establishing cancer, the method comprising: administering cancer cells to the genetically modified animal of any of the preceding embodiments, and determining cancer growth.

[0093] A method of establishing infection, the method comprising: administering an antigen-derived pathogen to a genetically modified animal as described in any of the preceding embodiments; and determining the TCR amplified repertoire by sequencing.

[0094] A method for preparing a genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unarrayed human T cell receptor (TCR) Vδ gene segment, at least one unarrayed human TCR Dδ gene segment, and at least one unarrayed human TCR Jδ gene segment; and at least one unarrayed human TCR Vγ gene segment and at least one human TCR Jγ gene segment, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are operably linked to a functional non-human TCRδ constant gene, and wherein the at least one unarrayed human TCR Vγ gene segment and the at least one human TCR Jγ gene segment are operably linked to a functional non-human TCRγ constant gene.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the usual meanings understood by those skilled in the art. Unless otherwise defined, any of the aspects and embodiments herein may be used in combination with each other. Methods and materials for the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods and examples in the following specific embodiments do not limit the claimed invention. The accompanying drawings are for illustration only and are not intended to be limiting. In the event of a conflict, the present specification (including definitions) shall prevail. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 A strategy for replacing one of the mouse T cell gamma loci with one of the human T cell gamma loci is shown. The figure shows the scale shown by the scale bar on the lower left of the figure. The upper panel shows the overall organization of endogenous mouse Vγ1, mouse Jγ4, mouse gamma constant region C4, and adjacent 5'upstream and 3'downstream mouse sequences on chromosome 13. The middle panel depicts a targeting vector for replacing the mouse gamma region. The targeting vector has the following segments: a) 5'upstream mouse homology arm, including the 5'UTR of mouse Vγ1; b) human Vγ9, human Vγ10, human Vγ11, human JγP1, human JγP, and human Jγ1; c) selection box; d) 3'downstream mouse homology arm, including mouse constant region C4 (from exon 1 to exon 4). Exons are represented by vertical bars. The lower panel depicts the target event. The targeting vector with human sequences replaces mouse Vγ1 and mouse Jγ4 at the mouse endogenous locus on chromosome 13.

[0097] Figure 2 A strategy for replacing a portion of the mouse T cell delta locus with a portion of the human T cell delta locus is shown. The figure shows the scale indicated by the scale bar on the lower left of the figure. The upper panel shows the overall organization of endogenous mouse Vδ4, mouse Dδ1, Dδ2, Jδ1, Jδ2 and mouse delta constant region C and adjacent 5' upstream and 3' downstream mouse sequences on chromosome 14 (including mouse Vδ5). The middle panel depicts the targeting vector used to replace the mouse delta region. The targeting vector has the following segments: a) 5' upstream mouse homology arm; b) human Vδ8 with Vδ rabbit promoter 2; c) human Vδ7 with Vδ rabbit promoter 1; d) human Vδ3 with Vδ bovine promoter 1; e) human Vδ5 with its own human promoter; f) human Vδ4 with its own human promoter; g) human Vδ6 with its own human promoter; h) human Vδ1 with its own human promoter; i) human Vδ2 with its own human promoter; j) human Dδ1-3; k) human Jδ1-4; l) selection cassette; m) 3' downstream mouse sequence, including the mouse δC region (from exon 1 to exon 4). Exons are represented by vertical bars. The lower panel depicts the target event. The targeting vector with human sequences replaces mouse Vδ4, mouse Dδ1, Dδ2, Jδ1, and Jδ2 at the mouse endogenous locus on chromosome 1.

[0098] Figure 3 Detailed illustration (not to scale) of the progressive strategy for combining human segments together as targeting vectors. Human variable region gene segments were sequentially added to partially deleted human BACs, generating an additional 7 human Vδ at the original human Vδ2 locus.

[0099] Figure 4 Figure 1 is a flow cytometry analysis of humanized γδT cells from mouse spleen. In the spleen of humanized γδT cells mice, most γ / δT cells are humanized Vδ1 / humanized Vγ9 with an abundance of 61%, while the abundance of humanized Vδ2 / humanized Vγ9 is 7.6%.

[0100] Figure 5 Development of B16 melanoma cancer in the lungs of wild-type and humanized γδ T cell mice

[0101] Figure 6 Further humanization of the mouse VγC1 region is shown. Human Vγ1-8 was inserted into this region.

[0102] Figure 7 The sequence of the PCR product of human γTCR sequenced by Sanger method is shown

[0103] Figure 8The sequence of the PCR product of human γTCR sequenced by Sanger method is shown

[0104] Fig. 9A -E provides the sequence of CDR3 of Vδ1–DV1

[0105] Fig. 10A -D provides the sequence of CDR3 of Vδ3–DV3

[0106] Fig.11A -K provides the sequence of CDR3 of Vδ2–DV2

[0107] Fig. 12A -H provides the sequence of CDR3 of Vδ6–DV6

[0108] Fig.13A -L provides the sequence of CDR3 of Vδ4–DV4

[0109] Fig.14A -B provides the sequence of CDR3 of Vγ9–G32G9

[0110] Fig.15A -C provides the sequence of CDR3 of Vγ9–G33G9

[0111] Fig.16A -G provides the sequence of CDR3 of Vγ10–G35G10

[0112] Fig.17A -E provides the sequence of CDR3 of Vγ10–G37G10

[0113] Fig.18A -J provides the sequence of CDR3 of Vγ10–G36G10

[0114] Fig.19A -B provides the sequence of CDR3 of Vδ3–M4DV3

[0115] Fig. 20A -V provides the sequence of CDR3 of Vδ1–M4DV1

[0116] Fig.21A -B provides the sequence of CDR3 of Vδ3–M7DV3

[0117] Fig.22A -K provides the sequence of CDR3 of Vδ6–M4DV6

[0118] Fig.23A -L provides the sequence of CDR3 of Vδ6–M7DV6

[0119] Fig.24A-U provides the sequence of CDR3 of Vδ1–M7DV1 DETAILED DESCRIPTION

[0120] Some embodiments of the present invention are discussed in detail below and in the accompanying drawings. When describing the embodiments, specific terms are used for the purpose of clarity. However, the present invention is not intended to be limited to the selected specific terms. The present invention is also not intended to be limited to the embodiments depicted in the accompanying drawings. Those skilled in the relevant art will recognize that other equivalent components and other methods can be adopted without departing from the broad concepts of the present invention. All references cited anywhere in this specification (including background technology and specific embodiments) are incorporated by reference, just as if each was incorporated separately.

[0121] The definitions included herein are for purposes of understanding the present subject matter and the appended patent claims and drawings. The abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0122] Although various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Without departing from the present invention, those skilled in the art will appreciate that many variations, changes and alternatives will be appreciated. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to implement the present invention.

[0123] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as generally understood by those of ordinary skill in the art.See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd edition, J.Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any method, device and material similar or equivalent to those described herein may be used to practice the present invention. The following definitions are provided to help understand certain terms frequently used herein, but are not intended to limit the scope of the present disclosure.

[0124] As used herein, "a", "an" or "the" may mean one or more than one. For example, "a" cell may mean a single cell or a plurality of cells.

[0125] As used herein, unless specifically indicated otherwise, the word "or" is used in its "inclusive" sense of "and / or" rather than in its "exclusive" sense of "either / or."

[0126] As used herein, with respect to a recombinant protein, the term "modification" means any insertion, deletion or substitution of amino acid residues in the recombinant sequence relative to a reference sequence (eg, a wild-type or native sequence).

[0127] As used herein, the term "homologous recombination" or "HR" refers to a natural cellular process in which double-stranded DNA breaks are repaired using homologous DNA sequences as repair templates (see, e.g., Cahill et al. (2006), From. Biosci. 11: 1958-1976). The homologous DNA sequence can be an endogenous chromosomal sequence or an exogenous nucleic acid delivered to the cell.

[0128] The terms "recombinant DNA construct", "recombinant construct", "expression cassette", "expression construct", "chimeric construct", "construct" and "recombinant DNA fragment" are used interchangeably herein and are single-stranded polynucleotides or double-stranded polynucleotides. Recombinant constructs comprise artificial combinations of single-stranded polynucleotides or double-stranded polynucleotides, including but not limited to regulatory sequences and coding sequences that do not co-exist in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with a vector.

[0129] As used herein, the term "human TCR" or "humanized TCR" refers to a partial or complete TCR derived from a human sequence.

[0130] The TCR CDR3 region was defined according to the International Immunogenetics (IMGT) nomenclature and TCR numbering system. Similarly, the gene names of the V and J regions were assigned according to the IMGT nomenclature for human or mouse T cell receptors.

[0131] The present disclosure relates to genetically modified non-human animals having humanized gamma and / or delta T cell receptors.

[0132] When the terms "transgenic and gene-targeted non-human animals" are used to describe genetically modified animals (e.g., mice or rats), those skilled in the art will understand that transgenics is achieved by randomly integrating exogenous DNA sequences into any chromosome, and gene targeting is achieved by integrating exogenous DNA sequences into a designed position on a specific chromosome. Non-human targeted animals can better control gene expression and protein translation. But transgenic non-human animals with the correct sequence can also produce the correct protein. Therefore, by adding or removing several genetic segments to appropriately modify the core targeting vector, a transgenic vector that can be randomly integrated into the genome of a non-human animal and achieve correct protein expression can be produced. Those skilled in the art will understand that DNA sequences that play a role in gene targeting can also play a role in a transgenic manner.

[0133] The term "conservative" is used to describe that an amino acid can be replaced by another amino acid residue with a side chain R group with similar chemical properties (e.g., charge or hydrophobicity) without significantly changing the biological properties of the protein or peptide. Examples of amino acid groups with side chains with similar chemical properties include acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., asparagine, cysteine, glycine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine), aromatic side chains (e.g., histidine, phenylalanine, tyrosine, tryptophan) and β-branched side chains (e.g., isoleucine, threonine, valine). Sequence similarity can be measured by two sequences being identical or different but having similar physicochemical properties. In many cases, the homology percentage is greater than the identity percentage. In some embodiments, alanine scanning mutagenesis can be used to replace any native residue in a protein.

[0134] The term "conservatively identical" means that an amino acid has similar biophysical properties as another amino acid, and replacing the amino acid with a "conservatively identical" amino acid will not cause a deleterious change in the function of the protein in which the amino acid is located.

[0135] Those skilled in the art will appreciate that the degeneracy of the genetic code can encode the polypeptides of the present invention. Humanized γ / δ TCR polypeptides described elsewhere may differ from those described herein.

[0136] Sequence identity is the ratio of the number of identical amino acids between two aligned sequences to the length of the alignment, expressed as a percentage. Two amino acid sequences having "100% amino acid sequence" means that the amino acid residues of the two amino acid sequences are identical and are conservatively identical when aligned to achieve maximum correspondence. Sequence comparisons can be performed using standard software programs, such as those included in the LASERGENE bioinformatics computing suite (DNASTAR. Madison, Wis.). Other methods for comparing two nucleotide or amino acid sequences by determining the best alignment are well known to those skilled in the art. (See, e.g., Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Bishop (ed.), Guide to Human Genome Computing (2nd Edition, Academic Press, Inc. 1998).) If two amino acid sequences have at least 80%, at least 85%, at least 90%, or at least 95% and greater sequence identity relative to each other, the two sequences are considered to have "substantial sequence similarity or homology".

[0137] The term "C region" refers to the constant region of the gamma / delta T cell receptor that is common to all receptors. The constant region domain has separate gene segments of the hinge region, transmembrane region, and cytoplasmic region that provide signal transduction after the gamma / delta T cell receptor binds to the antigen.

[0138] The term "functionally compatible C region" refers to a constant region from an endogenously different species but which retains all or part of the correct signal transduction function after the γ / δ T cell receptor binds to an antigen, including sequences with 70%, 75%, 80%, 85%, 90%, 95% and 100% similarity to the original C region of the species.

[0139] The term "synthetic C region" refers to a sequence that forms a new C region by simulation, replacement or change, which sequence still retains all or part of the correct signal transduction function after the γ / δ T cell receptor binds to the antigen, for example, replaced by a non-γ / δ C region (e.g., α / β C region) or other receptor components.

[0140] In the present invention, the phrase "functionally compatible C segment" encompasses the term "synthetic C segment" and vice versa.

[0141] "Polypeptide" or "polypeptide chain" is a polymer of amino acid residues linked by peptide bonds, which polymer may be naturally or synthetically produced. Peptide generally refers to a polypeptide of less than about 10 amino acid residues.

[0142] In the non-human animal models of the present invention, "humanized γ / δ TCRs" are largely interchangeable with human γ / δ TCRs unless specifically stated otherwise.

[0143] A "humanized gamma / delta TCR" is a TCR that comprises one or both of a humanized gamma domain and a humanized delta domain, and constant regions need not be present, but if present, they are all or substantially derived from human immunoglobulin constant regions.

[0144] The present invention relates to amino acids substituted with conservative amino acids based on functional human or humanized γ / δ CRD3. In some embodiments, conservative amino acid substitutions may be in the V region, D region, J region, C region and CDR3 region.

[0145] In some aspects, CDR can be from non-human species, and CDR herein can be engineered to be similar to human CDR. In this regard, when at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the corresponding residues (defined by Kabat (or IMGT)) between these similar human forms CDR (including amino acids in their conservative groups) are the same, the identity of each CDR is "substantially homologous" to the corresponding CDR in human CDR. In the specific variation of humanized TCRγ or TCRδ, wherein CDR is substantially homologous to human γ / δTCR, relative to the corresponding human γ / δTCR, the CDR of γ / δTCR or δTCR has no more than six (e.g., no more than five, no more than four, no more than three, no more than two or no more than one) amino acid substitutions (including conservative substitutions) in all three CDRs. The framework sequences of the humanized TCR gamma or TCR delta variable regions are "substantially homologous" to the human framework sequences of at least about 80%, at least 85%, at least 90%, at least 95%, or 100% of the corresponding residues defined by the Kabat numbering convention. The sequences of the humanized gamma / delta TCR constant regions are homologous when at least about 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the corresponding residues defined by the Kabat numbering convention (including conserved amino acids) are homologous to rodent (e.g., mouse) constant regions.

[0146] All parts of the humanized γ / δ TCR except the CDRs are derived entirely or substantially from the corresponding parts of the native human γ / δ TCR sequence.

[0147] The percentage of sequence identity is determined by the Kabat numbering convention by the γ and / or δ sequence of the maximum comparison. The subject TCR region (e.g., the entire variable domain of CDR1, CDR2, CDR3 or γ / δTCR) is compared with the same region of the reference human TCR, and the percentage of sequence identity between the subject and reference TCR regions is calculated in the following manner: the number of positions occupied by the same amino acids in both the subject and reference TCR regions is divided by the total number of aligned positions in the two regions (not calculating gaps) and then multiplied by 100 to convert to a percentage. In some aspects, conservative amino acids (e.g., the same polar group, or the same non-polar group) can be considered to be identical or replaceable.

[0148] The term "homology" with respect to sequences (e.g., nucleotide or amino acid sequences) means that the two sequences are identical or substantially identical in at least about 75% of the nucleotides or amino acids, at least about 80% of the nucleotides or amino acids, at least about 90%-95% of the nucleotides or amino acids (e.g., greater than 97% of the nucleotides or amino acids), after comparison based on optimal alignment.

[0149] Those skilled in the art will appreciate that gene targeting is based on homologous recombination between the targeting construct and the targeted endogenous sequence through homology arms, which are two sequences on the targeting vector that are homologous to the endogenous DNA sequence.

[0150] The term "operably linked" refers to at least two genetic or protein elements that are linked together in a manner that enables them to perform the desired function. In the present invention, portions of humanized proteins can be operably linked to maintain the correct folding, processing, transport, expression and other functional properties of proteins in cells. In addition, the nucleic acid sequence encoding the protein can be operably linked to a DNA sequence (e.g., a promoter, enhancer, silencer, insulator, etc.) to maintain correct transcription.

[0151] The term "replacement" refers to the process of placing exogenous genetic material at the endogenous gene locus, thereby removing all, part or no endogenous genes, and inserting an orthologous or homologous nucleic acid sequence at the position where the endogenous gene is removed. In the present invention, the endogenous non-human gene segment is replaced with the corresponding human gene segment. As shown in the following examples, the nucleotide sequence of the endogenous non-human TCRγ and δ variable gene loci is replaced by the nucleotide sequence corresponding to the human TCRγ and δ variable gene loci.

[0152] As used herein, "functional", for example, with respect to a functional protein, refers to a protein that retains at least one biological activity normally associated with a native protein (e.g., binding to an antigen, modulating the TCR on the surface of a T cell, activating or inactivating downstream signaling, inducing cell proliferation, infected cell recognition and / or cancer cell recognition).

[0153] TCRγ / δ locus refers to genomic DNA containing TCRγ / δ coding region (including unrearranged V(D)J sequence), enhancer, silencer, insulator, constant domain sequence and upstream or downstream (e.g., 5' and 3'UTR, regulatory region, etc.) or intermediate DNA sequence (e.g., intron, etc.) or RNA with regulatory function. TCR variable locus (e.g., TCRγ variable gene locus or TCRδ variable gene locus) refers to genomic DNA containing a region containing TCR variable region segments (V(D)J region) but not containing TCR constant region sequences. V region does not narrowly refer to only the V element of V(D)J region, on the contrary, in the following description, V region may mean the entire V(D)J region.

[0154] As used herein, the term "chimeric protein" refers to a protein in which two or more portions of the protein are derived from different species. In the present invention, a portion of the chimeric protein is derived from a human source.

[0155] V(D)J recombination refers to a mechanism that contributes to the diversity of γ / δTCRs in the vertebrate immune system. The mechanism requires cutting DNA boundaries (e.g., DNA of the V and J segments of TCRγ and the V, D, and J segments of TCRδ) and then reconnecting specific pairs of the resulting ends. The inaccuracy of the ligation reaction significantly increases the variability of the functional genes produced. The signal sequences around the V, D, and J segments can be different, but can still be recognized and processed by one or both of the two nucleases RAG1 and / or RAG2, which cut the DNA at the signal sequence boundaries.

[0156] In vivo and in vitro systems for human gamma / delta T cells are provided, the systems comprising humanized rodent cells, wherein the rodent cells express one or more humanized immune system molecules.

[0157] Also provided are unrearranged humanized γ / δTCR rodent loci encoding humanized γ / δTCR proteins.Also provided are non-human animals, e.g., rodents, comprising non-human cells expressing humanized molecules that function in cellular immune responses.

[0158] Immunotherapy is centered on identifying targets that are abnormally expressed on the surface of infected or cancerous cells. To date, most established and successful targets are proteins or peptides. Protein targets are suitable for antibody intervention, and peptide targets are recognized by MHC-dependent α-βT cell receptors. As newly discovered targets are becoming increasingly scarce, the development of next-generation immunotherapy requires a new type of non-protein, non-peptide target. With the growing interest in finding new categories of targets, γ / δT cell receptors can bind to non-MHC-dependent targets, such as phospholipids, glycolipids, haptens, or small molecules (Willcox., Nat Immunol. (2019) 20: 121-128), while αβT cell receptors do not bind to these targets. γ / δT cells and their receptors have been retained in jawed vertebrates (e.g., fish, mice, and humans) for 450 million years. In humans, γ / δT cell receptors are able to bind to the MHC-independent antigens mentioned above and are naturally biased against microbially infected human cells and human malignant cells, but the molecular and biological interactions between them are unclear, partly due to the lack of human and human-like γ / δT cell receptor animal models. Primates (e.g., monkeys, chimpanzees) do have human-like γ / δT cells, but availability and ethical issues limit their use in understanding human γ / δT cell development, infection and cancer intervention, and ultimately γ / δTCR-based immunotherapy.

[0159] The γ / δ T cell receptor consists of a γ polypeptide chain and a δ polypeptide chain, which are bound together by disulfide bridges. The γ chain contains a leader signal peptide, variable regions (Vγ and Jγ), constant domains (including hinge domains), transmembrane domains, and cytoplasmic tail domains. The δ chain contains its leader signal peptide, variable regions (Vδ, Dδ, and Jδ), constant domains (including hinge domains), transmembrane domains, and cytoplasmic tail domains. γδ T cells use V(D)J gene rearrangement to potentially generate a set of highly diverse receptors capable of recognizing antigens. This diversity is generated in the CDR1, CDR2, and CDR3 regions, but primarily in the complementarity determining region 3 (CDR3) of the T cell receptor (TCR) through combinatorial and functional diversity.

[0160] In humans, γ / δ T cells (including receptors) are present in many tissues such as blood, lung, liver, spleen, thymus, intestine and reproductive tract. Four human γδ T cell populations can be easily identified by TCR Vδ expression (Vδ1, Vδ2, Vδ3 and Vδ5) (Zhao., J Transl Med (2018) 16(1):3). Vδ1, Vδ2, Vδ3 and Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9 and Vγ11 are the most frequently used gene segments in δ chain and γ chain rearrangement, respectively (Adams., Cell Immunol (2015) 296(1):31–40). In addition, four γδ T cell subsets (Vδ4, Vδ6, Vδ7 and Vδ8) were detected in the peripheral blood of patients with B cell non-Hodgkin's lymphoma, suggesting that they may be involved in the development of human non-Hodgkin's lymphoma. γδT cells expressing Vδ1 or Vδ3 TCR chains can pair with various Vγ chains (Thedrez., Immunol Rev (2007) 215: 123–35), and they are mainly found in epithelial tissues of the skin, lungs, intestines and reproductive tract (Carding., Nat Rev Immunol (2002) 2(5): 336–45), liver, spleen and thymus (Bonneville., Nat Rev Immunol (2010) 10(7): 467–78). In humans, most peripheral blood γδT cells express the Vδ2 TCR chain paired with the Vγ9 chain (Braza., Haematologica (2011) 96(3): 400–7).

[0161] In wild-type mice, the TCRγ locus spans 205kb and consists of four variable element clusters, a connecting element, and a constant element. According to the IMGT annotation (www.igmt.org), the first cluster contains Vγ4-7, Jγ1, and Cγ1. The second cluster contains Vγ3, Jγ3, and Cγ3. The third cluster contains Vγ2, Jγ2, and Cγ2. The fourth cluster contains Vγ1, Jγ4, and Cγ4. The Cγ4 gene differs from the other Cγ genes in that it has an additional exon encoding a hinge. Enhancer elements are located downstream of the Cγ gene and are responsible for regulating the production of mouse γTCRs.

[0162] In contrast, the human γ locus spans 175 kb and consists of only one gene cluster and gene segment on chromosome 7. Within this cluster, it has a sequence segment of Vγ1-11 and two sets of Jγ and Cγ elements downstream. According to IMGT annotation, the first set contains JPγ1, JPγ, Jγ1, and Cγ1 constant regions, and the second set contains JPγ2, Jγ2, and Cγ2 constant regions. Enhancer elements are located downstream of the Cγ1 and Cγ2 genes and are responsible for regulating the production of human γTCRs.

[0163] Very different from the γ locus, both the mouse and human δ loci are located within the αTCR locus, but have their own Vδ, Dδ, Jδ regions, their own Cδ regions and downstream enhancers that are solely responsible for regulating the production of the δTCR. During the maturation of αβT cells, the γ / δ loci are completely lost, so there is no γ / δTCR in mature αβT cells.

[0164] In wild-type mice, according to IMGT annotation, the δ locus spans 2100 kb and consists of the Vδ1-9 variable elements, two D elements (Dδ1 and Dδ2), two joining elements (Jδ1 and Jδ2), a constant element and an enhancer (located on mouse chromosome 14).

[0165] The human δ locus has a similar structure to that of the mouse, but with more D and J. According to the IMGT annotation, the human δ locus spans 1000 kb and is composed of Vδ1-8 variable elements, three D elements (Dδ1, Dδ2, and Dδ3), four joining elements (Jδ1, Jδ2, Jδ3, and Jδ4), and a constant element (Cδ) and enhancer (located on human chromosome 14).

[0166] The γδTCR complex consists of γδTCR and various CD3 chains in the following stoichiometry: TCRγδCD3δ2γδζ2 in humans and TCRγδCD3δ2γ2ζ2 in mice (Siegers., J Exp Med (2007) 204: 2537–44). The γ / δ T cell receptor interacts with a variety of signaling proteins through its constant region, thereby initiating phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of CD3 by Src-family kinases (SFKs) Lck and Fyn (Kuhns., Immunol Rev (2012) 250: 120–43), thereby triggering the recruitment, phosphorylation and activation of Zap70, which in turn promotes the phosphorylation of the scaffold proteins SLP-76 and LAT. These cascade events trigger the formation of supramolecular signalosomes, which recruit the phospholipase PLCγ1 and initiate the propagation of downstream signal transduction events (Smith-Garvin., Annu Rev Immunol (2009) 27: 591–619). Unlike αβT cells, the γ / δT cell TCR complex is independent of CD4 and CD8 co-receptors (Kuhns., Immunol Rev (2012) 250: 120–43), and the development of γ / δT cells is not blocked by mutations in the binding site of PLCγ1 on LAT. In contrast, mutations in the binding site of PLCγ1 on αβ T cells, LAT, lead to severe blockage of mouse αβ thymocyte development (Hayes., Immunol Rev (2003) 191:28–37 and Sullivan., J Immunol (2014) 192:2865–74), indicating that the C regions of γ / δ and αβ are very different, suggesting that the γ / δ T cell receptor C region plays a unique and specialized role in the γ / δ T cell-related cascade signaling.

[0167] The present invention provides a genetically modified non-human animal (e.g., a rodent, e.g., a mouse, a rat), wherein the genetically modified non-human animal comprises an unrearranged human or humanized γTCR locus in its genome, wherein the unrearranged human or humanized γTCR locus also comprises human Vγ and Jγ gene segments.

[0168] The present invention also provides a genetically modified non-human animal (e.g., a rodent, e.g., a mouse, a rat), comprising an unrearranged human or humanized δTCR locus in its genome, wherein the unrearranged human or humanized δTCR locus further comprises human Vδ, Dδ and Jδ gene segments.

[0169] The present invention also provides a genetically modified non-human animal (e.g., a rodent, e.g., a mouse, a rat), comprising in its genome an unrearranged human or humanized γ / δTCR locus, wherein the unrearranged human or humanized γ / δTCR locus further comprises human Vγ, Jγ, Vδ, Dδ and Jδ gene segments.

[0170] After an enzymatic reaction (e.g., Rag1, Rag2 or other suitable recombinases), the unrearranged human or humanized γ / δTCR can be rearranged to produce a nucleotide sequence encoding a γ / δTCR protein sequence comprising a V region, a D region (for δ only) and a J region and non-human Cγ and δ regions. The generated γ / δTCR comprises variable CDR1, CDR2 and CDR3. The present invention also provides non-human animals that are capable of producing a variety of libraries of human or humanized γ / δTCRs before and after antigen stimulation, including but not limited to (e.g., E. coli, Staphylococcus aureus extracts).

[0171] In one embodiment, the DNA fragments of human Vγ and Jγ replace the endogenous non-human Vγ and Jγ at their endogenous Vγ and Jγ loci, thereby generating a humanized γTCR locus capable of generating a humanized γTCR with a non-human Cγ region. In other embodiments, the non-human Cγ region can be derived from a different non-human Cγ region other than the endogenous Cγ region, as long as these Cγ regions are functionally compatible, for example, including but not limited to replacing the endogenous Cγ region (rat) with a rabbit Cγ region, replacing the endogenous Cγ region (mouse) with a primate Cγ region, without significantly hindering the function of γTCR signaling.

[0172] In another embodiment, the DNA fragment comprising human Vγ, Jγ and non-human Cγ regions is at a site other than the endogenous non-human γTCR locus in the genome. The non-human animal produced is a transgenic form. The non-human Cγ region can be from a functionally compatible Cγ region, not limited to, for example, primates, rabbits, cows, mice, rats, a mixture of different non-humans, or a synthetic Cγ region.

[0173] In one embodiment, the present invention provides a kind of genetically modified non-human animal (for example, rodent, for example, rat or mouse), the genetically modified non-human animal comprises human or humanized unrearranged Vγ and Jγ in the germline of non-human animal, and the human or humanized unrearranged Vγ and Jγ are operably connected to the compatible Cγ region at endogenous non-human TCRγ locus. Non-human animal comprises a single copy of human or humanized unrearranged Vγ and Jγ. In some embodiments, non-human animal comprises two copies of human or humanized unrearranged Vγ and Jγ. In some embodiments, non-human animal is a heterozygote or homozygote of human or humanized unrearranged Vγ and Jγ.

[0174] In one embodiment, human or humanized unrearranged Vγ and Jγ replace the corresponding endogenous V and J regions of the non-human animal. In some embodiments, human or humanized unrearranged Vγ and Jγ are inserted, but do not replace the corresponding endogenous V and J regions of the non-human animal.

[0175] In one embodiment, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises a further genetically modified genome comprising a non-human Vγ and / or Jγ in a non-human region that is partially or completely removed. In other embodiments, the non-human animal cannot rearrange endogenous Vγ and / or Jγ, but can rearrange human or humanized unrearranged Vγ and Jγ.

[0176] In some embodiments, the Vγ of a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a promoter from an endogenous animal Vγ. In other embodiments, the Vγ of a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a promoter from a Vγ promoter derived from a non-human animal other than an endogenous animal.

[0177] Similar to γTCR, at the TCRδTCR locus, in one embodiment, the DNA fragments of human Vδ, Dδ and Jδ replace the endogenous non-human Vδ, Dδ and Jδ at the endogenous Vδ, Dδ and Jδ loci, thereby generating a humanized δTCR locus capable of generating a humanized δTCR with a non-human Cδ region. In other embodiments, the non-human Cδ region can be derived from a different non-human Cδ region other than the endogenous C region, as long as these Cδ regions are functionally compatible, for example, including but not limited to replacing the endogenous Cδ region (rat) with the rabbit Cδ region, replacing the endogenous Cδ region (mouse) with the primate Cδ region, without significantly impairing the function of δTCR signaling.

[0178] In another embodiment, the DNA fragment comprising human Vδ, Dδ, Jδ and non-human Cδ regions is at a site in the genome other than the endogenous non-human TCRδ locus. The resulting non-human animal is transgenic. The non-human Cδ region can be from a functionally compatible Cδ region, not limited to, for example, primates, rabbits, cows, mice, rats, a hybrid of different non-humans, or a synthetic Cδ region.

[0179] In some embodiments, the present invention provides a kind of genetically modified non-human animal (for example, rodent, for example, rat or mouse), the genetically modified non-human animal is included in the germline of non-human animal, wherein the Vδ, Dδ and Jδ of human or humanization unrearranged are operably connected to the compatible Cδ region at endogenous non-human TCRδ locus. Non-human animal comprises a single copy of Vδ, Dδ and Jδ of human or humanization unrearranged. In some embodiments, non-human animal comprises two copies of Vδ, Dδ and Jδ of human or humanization unrearranged. In some embodiments, non-human animal is a heterozygote or homozygote of Vδ, Dδ and Jδ of human or humanization unrearranged.

[0180] In one embodiment, human or humanized unrearranged Vδ, Dδ and Jδ

[0181] Replace the corresponding endogenous Vδ, Dδ and Jδ regions of the non-human animal. In some embodiments, the human or humanized unrearranged Vδ, Dδ and Jδ inserts, but do not replace or partially replace the corresponding endogenous Vδ, Dδ and Jδ regions of the non-human animal.

[0182] In one embodiment, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises a further genetically modified genome comprising Vδ, Dδ and / or Jδ of a non-human region that is partially or completely removed. In some embodiments, the non-human animal cannot rearrange endogenous Vδ, Dδ and / or Jδ, but can rearrange human or humanized unrearranged Vδ, Dδ and Jδ.

[0183] In one embodiment, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a continuous human sequence comprising human Vδ, Dδ, and Jδ. In some embodiments, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a gene segment of a human sequence comprising human Vδ, Dδ, and Jδ. In some embodiments, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a mixture of small fragments comprising human Vδ arranged in an order that is not naturally present in a human Vδ locus. In some embodiments, the Vδ of a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a promoter from an endogenous animal Vδ. In other embodiments, the Vδ of a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a promoter derived from a non-human animal other than an endogenous animal Vδ promoter.

[0184] In one embodiment, the genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises an inverted human Vδ that is oriented opposite to endogenous Cδ. In another embodiment, the inverted human Vδ is genetically modified to be oriented in the same direction as endogenous Cδ.

[0185] In one embodiment, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises an unrearranged human TCRγ (e.g., human Vγ and Jγ) with an endogenous Cγ region or a functionally compatible Cγ region and a TCRδ (e.g., human Vδ, Dδ and Jδ) with an endogenous C region or a functionally compatible Cδ region. Human TCRγ and TCRδ are located at the corresponding TCRγ and TCRδ loci of their non-human animals. In some embodiments, non-human animals are able to rearrange human Vγ and Vδ with their D or J before and after antigen stimulation to produce a γ / δTCR library, including but not limited to, for example, Escherichia coli and Staphylococcus extracts.

[0186] In one embodiment, genetically modified non-human animals (e.g., rodents, e.g., rats or mice) include unrearranged human TCRγ (e.g., human Vγ and Jγ) with endogenous C regions or functionally compatible C regions and TCRδ (e.g., human Vδ, Dδ and Jδ) with endogenous C regions or functionally compatible Cδ regions. Human TCRγ and TCRδ are not located as transgenics at the corresponding TCRγ and TCRδ loci of their non-human animals. In some embodiments, non-human animals can use their D or J to rearrange human Vγ and Vδ to produce γ / δTCR libraries before and after antigen stimulation, including but not limited to, e.g., Escherichia coli and staphylococcal extracts. Non-human animals with human TCRγ and TCRδ can be double homozygotes, one homozygote / one heterozygote, and both are heterozygotes.

[0187] In one embodiment, the genetically modified non-human animal (eg, a rodent, eg, a rat or mouse) comprises a genetic modification in the gamma and / or delta TCR loci.

[0188] In some embodiments, in a genetically modified non-human animal (eg, a rodent, eg, a rat or mouse), the human Vγ and / or Vδ can have an opposite orientation of Cγ and / or Cδ.

[0189] In some embodiments, in a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse), the sequence between two human Vγ and / or Vδ can be shortened, and in some embodiments, the sequence between two Vγ and / or Vδ can be lengthened.

[0190] In some embodiments, human Vγ and / or Vδ can follow the natural order in their loci (e.g., toward the human Cδ region, follow the order of Vδ4, Vδ1, Vδ5, Vδ7, Vδ8, Vδ3). In some embodiments, human Vγ and / or Vδ do not follow the natural order in their loci (e.g., the order of Vδ4, Vδ1, Vδ5, Vδ7, Vδ8, Vδ3). Thus, a new order of human Vγ and / or Vδ is generated, for example, including but not limited to Vδ7, Vδ8, Vδ5, Vδ4, Vδ3, Vδ1, and Vδ2.

[0191] In some embodiments, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises as many human Vγ and / or δ as in the human γ / δ TCR locus. In some embodiments, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) may have more repeats of, for example, including but not limited to, Vγ and / or δ than in humans.

[0192] In some embodiments, the introduced Vγ and Jγ, and / or Vδ, Dδ and Jδ sequences are highly homologous to human Vγ and Jγ, and / or Vδ, Dδ and Jδ (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identical to the amino acid sequences of human Vγ and Jγ and / or Vδ, Dδ and Jδ (including the conserved amino acids described in the definitions)).

[0193] In some embodiments, the genetically modified non-human animal (eg, rodent, eg, rat or mouse) comprises less human Vγ and / or δ than in the human Vγ / δ locus.

[0194] In some embodiments, the genetically modified non-human animal (eg, a rodent, eg, a rat or mouse) comprises at least one human Vγ and / or Jγ.

[0195] In some embodiments, the genetically modified non-human animal (eg, a rodent, eg, a rat or mouse) comprises at least one human Vγ and / or Jγ operably linked to a non-human Cγ region.

[0196] In some other embodiments, the genetically modified non-human animal (eg, a rodent, eg, a rat or mouse) comprises at least one human Vδ, Dδ, and / or Jδ.

[0197] In some other embodiments, the genetically modified non-human animal (eg, a rodent, eg, a rat or mouse) comprises at least one human Vδ, Dδ, and / or Jδ operably linked to a non-human Cδ region.

[0198] In some embodiments, in a genetically modified non-human animal known to those skilled in the art (e.g., a rodent, e.g., a rat or a mouse), a Vγ and / or δ pseudogene, e.g., including but not limited to human Vγ5P, human Vγ6, human Vγ7, or human Vγ11, can be converted to a mature protein by replacing a stop codon with a non-stop codon.

[0199] In some embodiments, endogenous Vγ and / or δ can be removed in the genome of a genetically modified non-human animal (eg, a rodent, eg, a rat or mouse), as known to those skilled in the art.

[0200] In some embodiments, in a genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse), the human or humanized γ / δ TCR locus can comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or 100% human Vγ and / or Vδ.

[0201] In one embodiment, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises a continuous human sequence of human Vγ9, human Vγ10, human Vγ11, human JPγ1, human JPγ and human Jγ1. In other embodiments, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises a non-adjacent human sequence of Vγ9, human Vγ10, human Vγ11, human JPγ1, human JPγ and human Jγ1, and is operably connected to an endogenous Cγ4 region.

[0202] In one embodiment, the genetically modified non-human animal (e.g., a rodent, e.g., a rat or mouse) comprises a continuous human sequence of human Vδ8 and Vδ rabbit promoter 2, human Vδ7 and Vδ rabbit promoter, human Vδ3 and Vδ bovine promoter 1, human Vδ5 and its human promoter, human Vδ4 and its human promoter, continuous human sequence of human Vδ6 and Vδ1, human Vδ2, human Dδ, and continuous human sequence of human Jδ1-4.

[0203] In one embodiment, the genetically modified non-human animal (e.g., rodent, e.g., rat or mouse) comprises human Vδ8 and Vδ rabbit promoter 2, human Vδ7 and Vδ rabbit promoter, human Vδ3 and Vδ cattle promoter 1, human Vδ5 and its natural human promoter, human Vδ4 and its natural human promoter continuous human sequence, human Vδ6 and Vδ1 continuous human sequence, human Vδ2, human Dδ, human Jδ1-4 continuous human sequence, and is operably linked to the endogenous Cδ region. In one aspect, human Vδ3 is engineered to be positioned in the same direction as the endogenous Cδ region.

[0204] In some embodiments, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a human TCRγ variable segment library. A genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) is capable of using an enzymatic activity, e.g., including but not limited to Rag1 and / or Rag2 and / or other suitable enzymatic activities to remove the DNA sequence between Vγ and Jγ, and remove (similar to splicing) the DNA sequence between Jγ and the endogenous Cγ region to produce a TCR encoding library. In one aspect, the order of removing the DNA sequence between the segments can be: first remove the sequence between Jγ and the endogenous Cγ region, and then remove the sequence between Vγ and Jγ. In other aspects, the endogenous Cγ region can be replaced by a functionally compatible Cγ derived from a non-endogenous source other than a non-human animal.

[0205] In similar embodiments, a genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) comprises a human TCRδ variable segment library. A genetically modified non-human animal (e.g., a rodent, e.g., a rat or a mouse) can use an enzymatic activity, e.g., including but not limited to Rag1 and / or Rag2 and / or other suitable enzymatic activities to remove the DNA sequence between Vδ, Dδ and Jδ, and remove the DNA sequence between Jδ and the endogenous Cδ region to produce a TCR encoding library. In one aspect, the order of removing the DNA sequence between the segments can vary, e.g., first removing the sequence between Jδ and the endogenous C region, and then removing the sequence between Vδ and Dδ. In other aspects, the endogenous Cδ region can be replaced by a functionally compatible Cδ derived from a non-endogenous source other than a non-human animal.

[0206] In some embodiments, in cells of a genetically modified non-human animal (eg, a rodent, eg, a rat or mouse), endogenous γ / δ T cells express a human or humanized γ / δ T cell receptor on their surface.

[0207] In some embodiments, antigens can be introduced into genetically modified non-human animals (e.g., rodents, e.g., rats or mice) by various approaches, including but not limited to, for example, injection, spraying, skin absorption and implantation, eye drops or implantation. In some embodiments, antigens can come from various sources, including but not limited to, for example, single or mixtures of lipids, pyrophosphates, proteins, sugars, or artificial molecules or materials. In some embodiments, antigens can be living or non-living forms. In some embodiments, antigens can be viruses. In some embodiments, antigens can be on the surface or inside of an object, including but not limited to, for example, oil droplets or lipid droplets, liposomes, viruses, cells (including but not limited to, for example, prokaryotic cells, eukaryotic cells, bacteria, yeast, or fungi). In some embodiments, antigens can be infected cells or uninfected cells. In some embodiments, antigens can be cancerous cells or non-cancerous cells. In some embodiments, antigens can be DNA or RNA.

[0208] CDRs of human Vδ1

[0209] According to IgBlast TCRCDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ) (Ye et al., "IgBLAST: an immunoglobulin variabledomain sequence analysis tool," NucleicAcids Research 41 (Web Server issue): W34-W40, 2013), in some aspects, in non-human animals with human or humanized γ / δT cells, the peptide sequence of CDR1 from human Vδ1 contains a conserved amino acid sequence, and the conserved amino acid sequence contains TSWWSYY, or at least one S, one W, one Y or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ1 contains QGS, or at least one Q, one G or one S.

[0210] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vδ1 contains conserved start and stop sequences. For the start sequence, the amino acids at the second and third positions of the CDR3 of Vδ1 are L and G, respectively. In other aspects, the preferred amino acids for the first three amino acids of the CDR3 of Vδ1 are ALG. Further preferred amino acids for the first four amino acids are ALGE, wherein position E can be any one of G, A, D, V, E or R. For the termination sequence, two of the last five amino acids of the CDR3 of Vδ1 are DK, LI, KL, TD, DT, PI, TA, TR, QL or WD. In some aspects, the preferred amino acids for the last three amino acids of the CDR3 of Vδ1 are KLI.

[0211] CDRs of human Vδ2

[0212] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of CDR1 from human Vδ2 comprises a conserved amino acid sequence comprising GEAIGNYY, or at least one G, one E, one A, one I, one N, one Y within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ2 comprises EKD, or at least one E, one K, or one D.

[0213] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vδ2 contains a conserved start and stop sequence. For the start sequence, the amino acids at the first three positions of the CDR3 of Vδ2 are ACD, ACG, ACE, ARD, or ASD. In some aspects, the first two amino acids of the CDR3 of Vδ2 are AW, PV, or DC. Further preferred amino acids for the first four amino acids are ACDS, wherein the fourth position can be any one of T, K, I, P, Y, C, L, M, or R. For the termination sequence, two of the last five amino acids of the CDR3 of Vδ2 are DK, LI, KL, TD, DT, PI, TA, TR, QL, SS, HI, TH, or PD. In some aspects, the preferred amino acids for the last three amino acids of the CDR3 of Vδ2 are KLI.

[0214] CDRs of human Vδ3

[0215] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal having human or humanized γ / δ T cells, the peptide sequence of CDR1 from human Vδ3 comprises a conserved amino acid sequence comprising TVYSNPD, or at least one T, one V, one Y, one S, one N, one P or one D within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal V(δ)3 comprises GDNSR, or at least one G, one D, one N, one S or one R.

[0216] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δ T cells, the peptide sequence of the CDR3 from human Vδ3 comprises a conserved start sequence. The first two starting amino acid sequences of the CDR3 of Vδ3 are AF. In some aspects, the preferred amino acids for the first three amino acids of the CDR3 of Vδ3 are AL, AS, AC, AY, AI or AV. Further preferred amino acids for the first two amino acids are PF, TF, LF, GF or GC.

[0217] CDRs of human Vδ4

[0218] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal having human or humanized γ / δT cells, the peptide sequence of CDR1 from human Vδ4 comprises a conserved amino acid sequence comprising TSDPSYG, or at least one S, one P, one G, one D, one Y or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ4 comprises QGSYDQQN, or at least one Q, one G, one D, one Y, one N or one S.

[0219] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vδ4 contains conserved start and stop sequences. For the start sequence, the first three amino acids of the CDR3 of Vδ4 are AMR, ASP, AMS, AMT, AMG, AMI, AIR, AKR, ATR, EMR, VMR, PMR and ALP. In one aspect, the preferred amino acids for the first three amino acids of the CDR3 of Vδ4 are AMRE, wherein the fourth position can be any one of G, C, D, V, N, A, L, V, T or R. For the stop sequence, two of the last five amino acids of the CDR3 of Vδ4 are DK, LI, KL, TD, DT, PI, TA, TR, QL, PD, HI, KI or SS. In some aspects, the preferred amino acids for the last four amino acids of CDR3 of Vδ4 are DKLI or TRQM.

[0220] CDRs of human Vδ6

[0221] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal having human or humanized γ / δT cells, the peptide sequence of CDR1 from human Vδ6 comprises a conserved amino acid sequence comprising NTAFDY, or at least one N, one T, one A, one F, one D or one T within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vδ6 comprises IRPDVSE, or at least one I, one R or one P, one D, one V, one S or one E within the CDR2 sequence.

[0222] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vδ6 contains a conserved start and stop sequence. For the start sequence, the amino acids at the first and second positions of the CDR3 of Vδ6 are AA. In some aspects, the preferred amino acids for the first three amino acids of the CDR3 of Vδ6 are AAS or AAR. Further preferred amino acids for the first four amino acids are AASP, wherein the 4th P can be any one of T, G, M, V, L or R. In some other aspects, the preferred amino acids for the first two amino acids of the CDR3 of Vδ6 are QQ, EA, TA, QH, AV, SK, VA, ES, DA, SA, EG or EP. For the termination sequence, two consecutive amino acids in the last five amino acids of the CDR3 of Vδ6 are DK, LI, KL, TD, DT, PI, TA, TR, PD, HI, HT, KI or SS. In some aspects, the preferred amino acids for the last three amino acids of CDR3 of Vδ6 are TRQ, KLI, or KLN.

[0223] CDRs of human Vγ9

[0224] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δ T cells, the peptide sequence of CDR1 from human Vγ9 comprises a conserved amino acid sequence comprising GITISATS, or at least one G, one A, one I, one T, or one S within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vγ9 comprises ISYDGTV, or at least one I, one S, one Y, one G, one T, or one D.

[0225] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vγ9 contains a conserved start and stop sequence. For the starting sequence, the amino acids at the first four positions of the CDR3 of Vγ9 are ALWE, ALWG, ASWE, ALCE, ALLE, ALRE, PCGR, AWWE, DLWE, ASWE, or AMWE. In some aspects, the first five amino acids of the CDR3 of Vγ9 are ALWEV, wherein the fifth position can be any one of A, E, or M. In some aspects, in the starting sequence of ALWG, the fourth position can be any one of D, V, R, or K. In other aspects, the first four amino acids of the CDR3 of Vγ9 are TLWE, wherein the first position can be any one of G, V, S, P, or A. In one aspect, the fourth position of CDR3 is E. For the termination sequence, two consecutive amino acids in the last five amino acids of the CDR3 of Vγ9 are KK. In some aspects, the last few amino acids of the CDR3 of Vγ9 are KTL, KEL, EKL, KNL, IKV, KSR, RNS, KNS, MKL, KRL, RKL, FKI or KNQG.

[0226] CDRs of human Vγ10

[0227] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in one aspect, in a non-human animal with human or humanized γ / δ T cells, the peptide sequence of CDR1 from human Vγ10 comprises a conserved amino acid sequence comprising STRFETDV, or at least one R, one F, one T, one E, one D, one V, or one S within the CDR1 sequence. In other aspects, the CDR2 sequence of non-human animal Vγ10 comprises IVSTKSAA, or at least one I, one S, one V, one K, one T, or one A.

[0228] According to IgBlast TCR CDR3 calculation (https: / / www.ncbi.nlm.nih.gov / igblast / ), in some aspects, in non-human animals with human or humanized γ / δT cells, the peptide sequence of the CDR3 from human Vγ10 contains a conservative start and stop sequence. For the start sequence, the amino acid at the first three positions of the CDR3 of Vγ10 is AAW. In some aspects, the first four amino acids of the CDR3 of Vγ10 are AAWF, wherein the fourth position can be any one of F, L, A, R, G, C or V. In some other aspects, the second position is A and the first position can be any one of S, Y, V, E, G, D, T or P. In some aspects, the second position is E and the first position can be any one of S, D or A. In some aspects, the first and second positions are PR or SS. In some aspects, the first position is A, and the second position is any one of V, T, S, G or E. In some aspects, the first two positions are any one of VS, VT or CE. For the termination sequence, in some aspects, the last three amino acids of the CDR3 of Vγ10 are FKI. In one aspect, two consecutive amino acids of the last three amino acids of the CDR3 of Vγ10 are KK. In one aspect, the second of the last two amino acids of the CDR3 of Vγ10 is K. In other aspects, the last amino acid of the CDR3 of Vγ10 is I. In one aspect, the last amino acid of the CDR3 of Vγ10 is R. In other aspects, the second of the last two amino acids of the CDR3 of V(γ)10 is T.

[0229] Embodiments of the present invention provide a TCR comprising two polypeptides (i.e., polypeptide chains), such as a gamma chain of a TCR, a delta chain of a TCR, or a combination thereof. The polypeptide of the TCR of the present invention may comprise any amino acid sequence, as long as the TCR has antigen binding specificity.

[0230] In one embodiment of the invention, the TCR comprises two polypeptide chains, each polypeptide chain comprising a variable region comprising the complementarity determining regions CDR1, CDR2 and CDR3 of a γ / δ TCR.

[0231] The first group of TCRs contained eight human or humanized delta chains.

[0232] The TCR of the present invention also comprises a constant region from a non-human animal (eg, rodent, rat - mouse). The TCR of the present invention may additionally comprise a constant region derived from any suitable species (eg, rabbit or goat).

[0233] In one embodiment of the invention, the TCR also comprises rabbit, cow and possibly human constant regions.

[0234] In one embodiment of the invention, the TCR comprises a murine constant region. For example, the TCR can be a chimeric TCR comprising a human variable region and a murine constant region. The murine constant region of the TCR δ chain comprises SEQ ID (amino acid) NO: 73 (constant region of the murine δTCR chain); the murine constant region of the TCR γ chain comprises SEQ ID (amino acid) NO: 74 (constant region of the murine γTCR chain); there is more than one γ constant region.

[0235] In some aspects, the constant region of TCRδ comprises one or more of the conserved amino acid sequences: PSVF, MKNG, GTNVACL, SAVKLGQ, SVTCSV, KVNMMSL, VLGLR, LFAK and / or NFLL.

[0236] In some aspects, the constant region of TCRγ comprises one or more conserved amino acid sequences: PKPT, LCLL, KTKD, MKFSWLT, TSAYY and / or LLLLLKS.

[0237] It will be appreciated by those skilled in the art that one or more amino acid substitutions in the murine or non-human animal constant region of the γ and / or δ chain can still produce a protein with a functional γ and / or δ chain.

[0238] Genetically modified non-human animals

[0239] As used herein, the term "genetically modified non-human animal" refers to a non-human animal having exogenous DNA in at least one chromosome of the animal genome, in some aspects, the exogenous DNA is present in the form of extrachromosomal DNA fragments (e.g., plasmids, mitochondrial DNA). In some embodiments, at least one or more cells of a genetically modified non-human animal, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 30%, 40% or 50% of the cells have exogenous DNA in their genome. Cells with exogenous DNA include various types of cells, for example, somatic cells, germ cells (e.g., sperm, eggs, blastocysts), immune cells (e.g., T cells, B cells, natural killer T cells, mast cells), antigen presenting cells (e.g., macrophages, dendritic cells) or endogenous tumor cells or brain cells. In some embodiments, genetically modified non-human cells can also be introduced into species with or without the same genetically modified genes.

[0240] In some embodiments, a genetically modified non-human animal comprising a modified endogenous γ / δTCR locus is provided, the modified endogenous γ / δTCR locus comprising an exogenous sequence (e.g., a human sequence), for example, an exogenous sequence is inserted, or one or more non-human sequences are replaced with one or more human sequences by homologous recombination (HR) in mouse ES cells. The animal is generally capable of passing this modification to offspring, i.e., through germline transmission.

[0241] Genetic modification of humanized γ / d TCR can

[0242] Produced in other animals (e.g., rats, rabbits, pigs, cattle, deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys)) by several techniques known in the art, including, e.g., nonhomologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs) (reviewed in Durai et al. (2005), Nucleic Acids Res 33, 5978), transcription activator-like effector nucleases (TALENs) (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013). Nat Methods 10:957-63)). A variety of other methods are also provided that can be used for genome editing, such as microinjecting a genetically modified cell nucleus into an enucleated oocyte or fusing an enucleated oocyte with another genetically modified cell, in other aspects, transferring the modified genome to a suitable cell, such as an oocyte, and gestating the modified cell (e.g., a modified oocyte) in a non-human animal under suitable conditions to form an embryo.

[0243] In one aspect, the animal is a mammal, for example, a Dipodoidea or Muroidea mammal. In some embodiments, the genetically modified animal is a rodent. Rodents include mice, rats and / or hamsters. In some embodiments, the genetically modified animal is from a family selected from: Platacanthomyidae, Spalacidae and Eumuroidae, which further include Calomyscidae (e.g., Calomyscidae), Cricetidae (e.g., hamsters, New World rats and New World mice, voles), Muridae (Muridae) (true mice and true rats, gerbils, agouti, crowned mice), Nesomyidae (climbing mice, rock mice, tailed rats, Madagascar rats and Madagascar mice). In some embodiments, the genetically modified rodent is selected from true mice or true rats (Muridae), gerbils, agouti and crowned mice. In some embodiments, the non-human animal is a mouse. In some embodiments, the mouse, rat and / or hamster has at least 80%, 90%, 95% homology to the C57BL strain mouse at the nucleotide sequence level.

[0244] In some embodiments, the animal is a C57BL strain mouse selected from the group consisting of C57BL / 6, C57BL / 6ByJ, C57BL / 6J, C57BL / 10, C57BL / 6NJ, C57BL / 6NIH, B6NTac, C57BL / A, C57BL / KaLwN, C57BL / GrFa, C57BL / 10Cr, C57BL / 10ScSn, C57BL / An, and C57BL / Ola.

[0245] In some embodiments, the mouse is selected from the group consisting of 129 strains, including 129P1, 129P2, 129P3, 129X1, 129S1, 129S1 / SV, 129S1 / SvIm, 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6, 129 / SvEvTac, 129S7, 129S8, 129T1, 129T2. These mice are described in whole or in part in, for example, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in their entirety. In some embodiments, as described above, the genetically modified mouse is a mixture of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a mixture of the 129 strain or a mixture of the BL / 6 strain. In some embodiments, the mouse is a BALB strain, for example, a BALB / c strain. In some embodiments, the mouse is a mixture of the BALB strain and another strain. In some embodiments, the mouse is from a mixed strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0246] In some embodiments, the animal is a mouse. The rat can be selected from Wistar rats, Long-Evans Agouti strains, Sprague Dawley strains, Fischer strains, F344, F6 and Dark Agouti strains. In some embodiments, the rat strain is a mixture of two or more strains selected from the aforementioned rat groups.

[0247] Use of genetically modified human or humanized γ / δ T cell animals

[0248] There is growing interest in γ / δ T cell-based therapies in the field because γ / δ TCRs recognize stress-induced self-antigens (Groh V, Science (1998) 279: 1737-40 and Uldrich AR, Nat Immunol (2013) 14: 1137-45), lipids or pyrophosphates secreted by microorganisms or overproduced in tumor cells (Hayday AJ Immunol. (2019), 203 (2): 311-320)

[0249] People are particularly interested in Vγ9 / Vδ2T cells because it is the main subpopulation of γ / δT cells in human blood, accounting for 2%-10% of all T cells. γ / δTCR recognizes self or exogenous non-peptide phosphorylated molecules (called phosphoantigens) (Espinosa, JBC (2001) 276: 18337-44). There is evidence that Vγ9 / Vδ2T cells may exhibit pathogen specificity (Worku, (2001) J. Infect. Dis 184: 525-532) and immune memory (Shen, Science (2002) 295: 2255-58), which are two characteristics of adaptive immunity. After microbial exposure, infants' γ / δT cells undergo polyclonal expansion as well as Vδ1 expansion (Ravens, PNAS (2020) 117: 18649-60), indicating that TCR can recognize specific pathogen antigens. Bioinformatics analysis of large-scale metagenomic data sets determined that the relative abundance of γ / δT cells in tumors was significantly correlated with patient outcomes. Tumor-infiltrating γ / δT cells are present in all tumor entities. It has been confirmed that there is a correlation between the relative abundance of γ / δTILs and the good response to immune checkpoint therapy in various cancers (Gentles, Nat. Med (2015) 1-12). Vγ9 / Vδ2T cells have been shown to be able to recognize various stress markers (Dai, JBC (2012) 287: 16812-19 and Gober, J. Exp. Med (2003) 197: 163-8). γ / δ cells are also stimulated by certain tumor cells, such as Daudi B lymphoma (Fisch, Eur. J. Immunol (1997) 27: 3368-79).

[0250] However, Vγ9 / Vδ2T cells and other human or humanized γ / δ subclasses do not exist in most experimental animals, including but not limited to rodents, such as mice, rats, hamsters, cats, dogs, sheep, cows, and chickens. The unavailability of human or humanized VγVγ9 / Vδ2T cells has hampered research and development in many fields, including but not limited to vaccine development, cancer therapy development, infection disease prevention, and aging.

[0251] More specifically, human or humanized γ / δTCR mice can be good models for human vaccine development. Small animals (such as mice) play an important role in vaccine development because they are low-cost, easy to produce in large quantities, and can generate adaptive immune responses. However, their value is limited because clinical trials for a large number of infectious diseases have failed, and the results vary greatly between individuals, even though most candidate vaccines have been tested and evaluated in these small animals (Dantzler, Clinical & Translational Immunology 2019; e1072). The adaptive immune response generated by such animals is the result of a long process, especially the bridge between innate and adaptive responses, which is not well understood in both humans and animals. Among various immune cells, the γδT cell is unique. It has tens of thousands of TCR repertoires, both public and private, representing both groups and individuals. It has been well documented that γδT cells play a role in both innate (clonal expansion, clonal focusing, cytokine release, and immune cell recognition) and adaptive (memory cell formation). In fact, mice lacking γδT cells have significantly delayed recovery from a variety of bacterial and yeast infections.

[0252] The rationale for replacing mouse γδTCR with human γδTCR to make a better vaccine testing model is based on the following reasons: 1) Mouse and human γδTCR are not very different. A particular vaccine can generate mouse γδTCR, but the same vaccine may not generate human γδTCR. Therefore, subsequent clinical trials are completely based on mouse γδTCR studies and the results of innate, bridging and adaptive reactivation triggered by mouse γδTCR. In fact, γ9δ2 (one of the most studied γδTCRs in humans) does not even exist in mice. Using wild-type animals for vaccine studies completely ignores the immune response triggered by Vγ9Vδ2 or other human Vδ. 2) Unlike α / β, γδTCR recognizes non-protein antigens, such as phosphoantigens, which can be common to many animals including humans. γδTCRs cloned from humanized mice can directly interact with infected human cells, and vice versa, infected human cells can stimulate the expansion and focusing of human γδT cells, which makes in vivo testing very feasible. 3) Humanized mice provide further translational value, as sequences obtained from expanded and memory γδ T cells can be directly compared to a large existing human γδ repertoire. In fact, in the preliminary data presented in this application, many of the γ or δ TCRs are identical to human γδ TCRs found in previous human studies. 4) The formation of memory γδ T cells is one of the hallmarks of vaccine development. This human γδ T cell mouse can be repeatedly challenged and the process of γδ memory T cell formation is closely monitored, which cannot be done in any other animal. In summary, the proposed humanized γδ T cell model provides us with a unique platform to study human or human-like innate, bridging, and adaptive immune responses for vaccine development.

[0253] In various embodiments, the present invention solves this problem by providing genetically modified non-human animals comprising human or humanized γδT cell receptors, and the expression of these TCRs, especially human or humanized Vγ9Vδ2T cells. In non-human animals, a variety of γδTCR libraries have been established, and these γδTCR libraries encode human or humanized γδT cell receptors. Antigen stimulation includes, but is not limited to, aqueous suspensions of, for example, Escherichia coli and Staphylococcus aureus. Non-human animals undergo clonal expansion and clonal focusing, which indicates that non-human animals are able to use their internal mechanisms to process introduced human genomic DNA to produce functional γδT cell receptors. Since γδT cells have been shown to have a wide range of applications in adoptive cellular immunotherapy, antigen-specific sequences of human or humanized receptors can be cloned and expressed on selected cells.

[0254] In some embodiments, the selected cells may include, but are not limited to, non-immune cells, immune cells, or cell lines.

[0255] In some embodiments, cancer cells from human and non-human sources are introduced into non-human animals, and tumor growth is monitored and provided in the present invention. Cancer cell lines include, but are not limited to, mouse cancer cell lines, e.g., B16 mouse melanoma cancer cell line, and human cell lines, e.g., lymphoma Daudi B cell line.

[0256] A major advantage of γδT cells over αβT cells is that, due to the great diversity of HLA between individuals, the αβT cell immune system cannot be transferred unless all HLA molecules are precisely matched. In contrast, due to the presence of common antigens, there are fewer restrictions on the transfer of γδT cell immune systems between individuals, and γδT cells from normal volunteers can be used as "universal donors". Therefore, in principle, well-characterized γδT cells and their receptors can become "off-the-shelf" products, providing T cell products for a larger population from one donor, thereby reducing development costs and time.

[0257] In some embodiments, the human or humanized γδT cell comprises one human or humanized γTCR and one human or humanized δTCR. In other aspects, the human or humanized γδT cell comprises two human or humanized γTCRs or two human or humanized δTCRs. In other aspects, the human or humanized γδT cell comprises more than two human or humanized γTCRs or more than two human or humanized δTCRs.

[0258] In other aspects, the human or humanized γδT cells comprise one or more human or humanized γTCRs in combination with other receptors to form a receptor complex. In other aspects, the human or humanized γδT cells comprise one or more human or humanized δTCRs in combination with other cell surface receptors to form a receptor complex.

[0259] In some embodiments, a human or humanized δTCR can be fused to a second entity via a molecular linker to provide biochemical or cytotoxicity to kill infected or cancerous cells. The second entity includes, but is not limited to, for example, small molecules, radioisotopes, toxins, photoactivated drugs, antibodies, prodrugs, target activating molecules, enzyme-based drug release molecules, chemokines, cytokines, immune activation and inhibition molecules, αTCR, βTCR, IgG heavy chain or IgG light chain.

[0260] In humans, γδNKT cells develop in the neonatal thymus and migrate to peripheral sites such as the spleen, lymph nodes, and most notably the liver (Azuara, V, 1997, Eur. J. Immunol. 27: 544–553). γδNKT cells are considered to be an innate-like lineage based on the expression of the innate signature transcription factor PLZF (Kreslavsky, T, 2009, Proc. Natl. Acad. Sci. USA 106: 12453–12458.). The mature γδNKT cell phenotype is CD24 low CD44 high NK1.1+, and can secrete various cytokines after TCR stimulation, including IFN-γ, IL-4, and IL-13 (Alonzo, E, Curr. Opin. Immunol. 23: 220–227.). These innate properties make γδ NKT cells important in immune protection (Belles, C. 1996, J. Immunol. 156: 4280–4289.) and immune conditions such as Sjögren's syndrome (Belle, I. 2014 Cytokine 69: 226–233.), dermatitis (Kreslavsky, T, 2009, Proc. Natl. Acad. Sci. USA 106: 12453–12458.) and asthma (Felices, M., Proc. Natl. Acad. Sci. USA 106: 8308–8313.)). γδNKT cells are also found in mice, and the size of the γδNKT cell population is strictly regulated during thymic development, usually accounting for 10% of the total γδT cells in C57BL / 6 mice (Verykokakis, M., 2010. PLoS One 5: e9303), so the human or humanized γδT mice in the present invention can also include this type of NKT cells with human γδTCR. If this mouse NKT based on human γδTCR can recognize human infected or cancerous cells, then the human TCR can be cloned and expressed on various cell types to fight these degenerated cells.

[0261] Example

[0262] The following examples will further describe the present invention, but these examples do not limit the scope of the present invention. Conventional methods known in the art are not described in detail in the examples, such as PCR, molecular cloning techniques, bacterial BAC recombineering, embryonic stem cell culture, microinjection, animal hybridization, etc. The CDR3 clone numbers are all from actual experiments. It is understood in the art that experimental data may vary according to different mice, different PCR and NGS programs, different library analysis software, and different data presentation methods. The data shown herein are intended to support the claims, but do not limit the claims.

[0263] Example 1

[0264] Generation of human or humanized TCRγ mice (introduction of human Vγ9-11)

[0265] In this embodiment, non-human animals (such as mice) are modified so that the non-human animals contain nucleic acid sequences comprising human TCRγ and also comprising human TCR Vγ and Jγ. Specifically, the endogenous mouse sequence containing mouse TCR Vγ and Jγ is replaced with a sequence containing human TCR Vγ and Jγ, and is operably connected to the mouse TCR Cγ region.

[0266] More specifically, the 2680bp mouse sequence containing mouse TCR Vγ1 and mouse Jγ4 was replaced with a 48732bp human sequence corresponding to 3 human Vγs (Vγ9, Vγ10, and Vγ11) and 3 human Jγs (JPγ1, JPγ, and Jγ1). The human sequence ends at the 5' upstream of the mouse endogenous Cγ4 region, so human γV and J are genetically connected to the mouse Cγ4 region. The humanization strategy is summarized in Figure 1 The junction nucleic acid sequences within the targeting vector between mouse, human and prokaryotic / eukaryotic selection cassettes are summarized in Table 1 and provided in the sequence listing (SEQ ID NOs: 1-5).

[0267] Table 1

[0268]

[0269] The first step in generating genetically modified mice with human TCRγ is to construct a TCRγ targeting vector (vector ID NO: 1). The targeting vector (vector ID NO: 1) has four DNA segments: a 5' mouse homology arm containing the mouse Vγ1 promoter (DNA segment NO: 2), a human sequence with human Vγ9, Vγ10 and Vγ11 and JPγ1, JPγ and Jγ1 (DNA segment NO: 6), an antibiotic box for eukaryotic selection flanked by Frt sites (DNA segment NO: 7), and a 3' mouse homology arm containing mouse Cγ4 exons 1-3 (DNA segment NO: 4).

[0270] Two BAC clones were used to construct TCRγ targeting vectors (vector ID NO: 1) (mouse RP23-475C6 and human CTD-2563O9, Invitrogen). The 5' mouse homology arm containing the mouse Vγ1 promoter (DNA segment NO: 2) had a DNA sequence of 7052bp in length. The 3' mouse homology arm containing mouse Cγ4 exon 1-3 (DNA segment NO: 4) had a DNA sequence of 8868bp in length. Both the 5' and 3' homology arms were derived from mouse BAC clone RP23-475C6 and modified by bacterial BAC recombineering. A human replacement sequence (DNA segment NO: 6) with a DNA sequence of 48732bp in length was cloned from human BAC clone CTD-2563O9 and modified by bacterial BAC recombineering in addition. The antibiotic box (DNA segment NO:7) for eukaryotic selection has a ubiquitin promoter that drives the Neo selection box flanked by the Frt site. Five DNA segments (DNA segments NO:2, 6, 7, 4) were combined with a BAC-based vector (vector ID NO:1) using homology arms with an average length of 130bp (50-200bp not equal) by DNA connection and bacterial homologous recombination. Bacterial Spec and Kan selection boxes were used to help bacterial homologous recombination. During the construction of the targeting vector, a unique (not cut into DNA segments NO:2, 6, 7, 4) restriction site AscI was introduced to linearize the targeting vector (vector ID NO:1). The linearized targeting vector (vector ID NO:1) was electroporated into a hybrid mouse embryonic stem (ES) cell line derived from a hybrid strain (produced by hybridization of two different inbred strains (B6 and 129) mice). ES cells with correct targeted clones are identified and confirmed by a combination of various methods known in the art (e.g., PCR-based assays, long-arm PCR, long-distance PCR, Southern and / or human displacement PCR). Targeted ES clones are amplified and microinjected into mouse blastocysts to produce chimeric mice with human target gene segments (DNA segment NO:6). Germline mice are obtained by further mating of chimeras with B6 inbred strains, and additional PCR confirmation is performed. The Neo box is removed by the activity of Flp recombinase ES cells or mouse tissues.

[0271] Example 2

[0272] Generation of human or humanized TCRδ mice

[0273] In this embodiment, a non-human animal (such as a mouse) is modified so that the non-human animal contains a nucleic acid sequence comprising human TCRδ and also comprising human TCRδV, D and J. Specifically, the endogenous mouse sequence containing mouse TCRδV, D and J is replaced with a sequence containing human TCRδV, D and J, and is operably linked to the mouse TCR Cδ region ( Figure 2 ).

[0274] More specifically, a 67562 bp mouse DNA sequence (DNA segment ID NO: 10) containing mouse TCR Trdv4 (mouse Vδ1) corresponding to mDV4, mouse TCR TrDδ1, 2 and Trdj 1, 2 corresponding to mD1, 2 and mJ1, 2 (respectively) was replaced with a total of 97,129 bp of hybrid DNA sequences (82.4% human sequences) containing 8 human V (hVδ1-8), 3 human D (hDδ1-3) and 4 human J (hδJ1-4). The hybrid sequence ends at 5' upstream of the mouse endogenous Cγ region, so human δV, D and J are genetically connected to the mouse Cδ region. The humanization strategy is summarized in Figure 2 The junction nucleic acid sequences within the targeting vector between rabbit, bovine, mouse, human and the prokaryotic / eukaryotic selection cassette are summarized in Table 2 and provided in the sequence listing (SEQ ID NOs: 6-16).

[0275] Table 2

[0276]

[0277]

[0278] The first step in generating genetically modified mice with human TCRδ is to construct a TCRδ targeting vector (vector ID NO: 7) having ten DNA segments (DNA segments ID NO: 9, 13-20, 11), as described in detail in Table 3.

[0279] Table 3

[0280]

[0281]

[0282] The BAC clone was used to construct a TCRδ targeting vector (vector ID NO: 7) containing mouse RP23-6A14 and human CTD-3112B11, CH17-153K15, CTG-3064K1, CTD-2012J23, CTD-2521H9, CTD-2124P16, CTD-3012C18, CTD-3112B11, CTD-2382D16, CTD2382D16 (Invitrogen). Prior to completing the TCRδ targeting vector (vector ID NO: 7), five mini DNA vectors flanked by bacterial homology arms were constructed by DNA ligation and bacterial homologous recombination so that they all shared the same 5' arm and the 3' arm had a sequence in common with the DNA sequence at the 5' end of the next insertion site ( Figure 3 ). Detailed descriptions of the five mini-DNA vectors are shown in Table 4. In order to complete the TCRδ targeting vector (vector ID NO: 7), based on bacterial homologous recombination and PCR confirmation, alternative antibiotic selection strategies (e.g., first Spec selection, second Zeo selection, third Spec selection, fourth Zeo selection, etc.) are used to introduce and connect different DNA segments to achieve a linear sequence consisting of DNA segments 9, 13-19. Based on bacterial homologous recombination, DNA segments 20 and 11 are also connected to DNA segment 19 to complete the TCRδ targeting vector (vector ID NO: 7). During the construction of the targeting vector, a unique (not cut into DNA segments NO: 9, 13-20, 11) restriction site AscI is introduced to linearize the targeting vector (vector ID NO: 7). The linearized targeting vector (vector ID NO: 7) is electroporated into a hybrid mouse embryonic stem (ES) cell line derived from a hybrid strain (produced by hybridization of two different inbred strains (B6 and 129) mice). ES cells with correct targeted clones are identified and confirmed by a combination of various methods known in the art (e.g., PCR-based assays, long-arm PCR, long-distance PCR, Southern and / or human displacement PCR). Targeted ES clones are amplified and microinjected into mouse blastocysts to produce chimeric mice with human target gene segments (DNA segments NO: 13-19). Germline mice are obtained by further mating of chimeras with B6 inbred strains, and additional PCR confirmation is performed. The Neo box is removed by the activity of the Flp recombinase in ES cells or mouse tissues.

[0283] Table 4

[0284]

[0285] Example 3

[0286] Generation of double homozygotes for humanized γδ TCR

[0287] The humanized γ and δ heterozygous mice obtained in Example 1 and Example 2 were hybridized. The offspring obtained by PCR amplification of the tail DNA of these mice included wild-type, homozygous human γTCR / heterozygous δTCR mice, homozygous human δTCR / heterozygous γTCR mice, heterozygous human γTCR, heterozygous δTCR mice, and double homozygous human γδTCR mice. The disappearance of the wild-type PCR band at the replacement region of the TCRγ and δ loci indicated that the human transgene was homozygous.

[0288] Example 4

[0289] Analysis of TCRδ expression in humanized TCRδ mice

[0290] The diversity of the TCRδCDR3 repertoire reflects the development, maturation, clonal composition, potential antigen recognition spectrum, and number of available T cell responses of TCRδ T cells. Humanized TCRδ mice are able to use the endogenous mouse VDJ recombinase system to recombine human Vδ, human Dδ, and human Jδ to form human TCRδCDR3s that are completely derived from human genomic sequences introduced by targeted replacement of mouse endogenous sequences at the TCRδ locus.

[0291] Specifically, blood and various tissues were collected from homozygous human Vδ mice. Blood was first treated with an erythrocyte lysis buffer (0.8 g NH4Cl and 0.11 g NaHCO3 in 100 ml of aqueous solution) to remove erythrocytes, and then white blood cells including T cells were collected by centrifugation and processed in the same manner as other tissues. Tissues including 1-peritoneal fluid, 2-brain (olfactory), 3-ear (middle ear), 4-nose, 5-ligament, 6-lymph node, 7-throat, 8-lung, 9-spleen, 10-bone marrow, 11-eye (eyeball), 12, 13, 14 small intestine (front, middle, back three parts), 15-uterine tube, 16-kidney, 17-thymus, 18-muscle, 19-heart, 20-liver, 21-skin, 22-blood were collected, and total RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer's recommendations. Total RNA was translated into cDNA using random primed oligonucleotides provided by the transcript first-strand cDNA synthesis kit (Roche) in a total volume of 15 μl. One microliter of the resulting cDNA was used as a template for the first round (round 1) of PCR reactions (40 μl in total), with primers covering human Vδ to mouse C region (including CRD3 region). For each VδCDR3 detection, one primer was located at the 5' end of Vδ, and the second primer was designed and located inside exon 1 of the mouse C region, also as a common antisense primer for all Vδ1-8PCR reactions (SEQ ID NO: 18). For example, as shown in Table 5, the first round of PCR for the CDR3 of human Vδ1 was amplified between primers (SEQ ID NO: 19) and common Cδ primers (SEQ ID NO: 18). The first round of PCR for the CDR3 of human Vδ2 was amplified between primers (SEQ ID NO: 20) and common Cδ region primers (SEQ ID NO: 18), and continued in the same manner. Since the total RNA is impure, a second round of PCR is generally required.

[0292] Table 5

[0293]

[0294] For the second round of PCR, two primers were designed within the previous primers described in the first round of PCR. Typically, the primer is 50-100bp downstream of the first round primer of Δ1, and the second common primer (SEQ ID NO: 27) is 50-100bp upstream of the first round common primer (SEQ ID NO: 18). One microliter of the PCR product of the first round is introduced as a template for the second round of PCR, with a total volume of 40 μl. For example, as shown in Table 6, the second round of PCR of the CDR3 of human Δ1 is amplified between primers (SEQ ID NO: 28) and common C region primers (SEQ ID NO: 27). The second round of PCR of the CDR3 of human Vδ2 is amplified between primers (SEQ ID NO: 29) and common C region primers (SEQ ID NO: 27), and continues in the same manner (Table 7). The final PCR products of Vδ1-8 from various tissues were Sanger sequenced, and the diversity of human TCR was estimated by checking the height on the DNA sequencing chromatogram and the reduction of the normal peak. H, M and L represent high, medium and low diversity of human δTCR, respectively. As shown in Table 8, RNA of human TCR Vδ1-8 is expressed in most tissues examined, and many tissues show highly diverse TCRδ. In particular, human Vδ1-4 and 6 are highly diversely expressed in tissues including peritoneal fluid, spleen, bone marrow, small intestine and thymus, among which γδT cells are also highly present in natural humans (Silva-Santos, B., 2021.naturereviewsimmunology 21:221-232 and Hayday, A., 2019.203:311-320). In short, the mouse model with the human δ locus can reproduce VDJ recombination like natural humans.

[0295] Table 6

[0296]

[0297] Table 7

[0298]

[0299] 1-peritoneal fluid, 2-brain (olfactory sense), 3-ear (middle ear), 4-nose, 5-tongue, 6-lymph nodes, 7-throat, 8-lung, 9-spleen, 10-bone marrow, 11-eye (eyeball), 12, 13, 14 small intestine (front, middle, and back parts), 15-uterine tube, 16-kidney, 17-thymus, 18-muscle, 19-heart, 20-liver, 21-skin, 22-blood. The positive sign represents the expression of human δCDR3

[0300] Table 8

[0301]

[0302] 1-peritoneal fluid, 2-brain (olfactory), 3-ear (middle ear), 4-nose, 5-tongue, 6-lymph nodes, 7-throat, 8-lung, 9-spleen, 10-bone marrow, 11-eye (eyeball), 12, 13, 14 small intestine (front, middle, and back parts), 15-uterine tube, 16-kidney, 17-thymus, 18-muscle, 19-heart, 20-liver, 21-skin, 22-blood. Human δTCR H-high diversity, M-medium diversity, L-low diversity.

[0303] Example 5

[0304] TCRδ Repertoire Analysis of Humanized TCRδ Mice

[0305] The diversity of the TCRδCDR3 library was confirmed by PCR (Example 3), and then the sequence containing CDR3 was obtained by next generation sequencing (NGS). (Figures 9-24)

[0306] Specifically, the PCR products of the second round of PCR using human TCRδ specific primers and mouse Cδ region primers were purified and submitted for next generation sequencing (Genewiz). Because the sequence data was too large, only the first 100,000 nucleotide sequences were analyzed for library analysis, and the boundaries and actual amino acid sequences of δCDR3 were calculated using the Analyze T Cell Receptor (TR) Sequence function of IgBlast (https: / / www.ncbi.nlm.nih.gov / igblast / ). The table lists the amino acid sequences of various TCRδCDR3s and their respective percentages of abundance in tissues of human TCRδ mice and their unique sequence IDs (other TCR CDR3 analysis platforms also exist and will produce CDR3 sequences with slightly different beginnings and ends of the boundary sequences. They can be re-analyzed by IgBlast to have sequences comparable to the invention described herein.

[0307] CDR3 was generated by two experiments performed on two different sets of human TCRδ mice.

[0308] In the first experiment, blood was collected from two homozygous human TCRδ mice. PCR products of human TCRδ were obtained for human Vδ1, 3, and 6. NGS data showed that in the blood, the CDR3 amino acid sequences were highly diverse.

[0309] In mouse number 1 (M4), only 100,000 DNA sequences from NGS-IgBlast analysis (NGS SEQ ID NO: 1-8576) ( Fig. 20A-V) there are 8576 unique human Vδ1CDR3, the first ten CDR3 are shown in Table 9. In the second mouse (M7) (Table 10), similarly, there are 8129 unique human Vδ1CDR3 (NGS SEQ ID NO: 8577-16705) ( Fig.24A -U). Vδ1 CDR3 is highly individual, as the two mice share very few common CDR3s. In fact, of the first twenty CDR3s in mouse number one (M4), only one CRD3 matched one of more than eight thousand unique sequences, indicating that the VDJ recombination is not a germline recombination, but a somatic recombination with hypermutations, including additions and deletions around the boundaries and within the D region. In addition, in mouse number one (M4) and mouse number two (M7), CDR3s are not focused until highly stimulatory antigens are encountered. It turns out that the first two Vδ1 CDR3s of mouse number one (M4) account for only 3.87% and 3.71%, respectively, with a total of 38 CDR3s, accounting for more than 1% of the total CRD3s, and 80.1% of the CDR3s appear only once to twice in the total CDR3s. Similarly, in mouse No. 2 (M7), the first two Vδ1CDR3s from mouse No. 1 (M4) accounted for only 4.44% and 3.94%, respectively, with a total of 40 CDR3s, accounting for more than 1% of CDR3s (Table 10), and 79.7% of CDR3s appeared only once or twice in the total CDR3s. CDR3 diversification is also reflected at the individual mouse level, because of the first twenty δCDR3s from mouse No. 1 (M4), only one matched a CDR3 sequence from mouse No. 2 (M7), and only two from M7 shared with M4. Individualized diversification has also been observed in humans (Chen, H., J Immunol Methods. 2017. 443: 9-17 and Ravens, S., Frontiers in Immunology 2018. https: / / doi.org / 10.3389 / fimmu.2018.00510 , and Davey, M., Trends in immunology 2018. 36:446-459), indicating that human TCRδ mice are able to largely recapitulate the phenomena occurring in humans.

[0310] Table 9

[0311]

[0312] Table 10

[0313]

[0314] For human TCRVδ3, CRD3 is less diverse, as shown in Tables 9 and 10. In mouse 1 (M4), there were only 504 unique Vδ3 CDR3s (out of 100,000 DNA sequences from NGS-IgBlast analysis (NGS SEQ ID NO: 16706-17209). Fig.19A -B). In mouse No. 2 (M7), similarly, there are 649 unique Vδ3 CDR3s (NGS SEQ ID NOs: 17210-17858) ( Fig.21A -B). The Vδ3 CDR3s are highly shared between the two mice. In fact, of the first twenty CDR3s in mouse one (M4), nine match those in mouse two (M7). The Vδ3 CDR3s are also focused, as the first CDR3 from mouse one (M4) accounts for 83% of the total CDR3s. Similarly, the first two CDR3s from mouse two (M7) account for 50% and 34%, respectively, reflecting possible early exposure to specific antigens. Comparative studies may be very limited because there is not much information available from humans.

[0315] Human TCR Vδ6 is also highly diverse, but to a lesser extent than TCR Vδ1. In mouse 1 (M4), there were 4263 unique Vδ6 CDR3s (out of only 100,000 DNA sequences from NGS-IgBlast analysis (NGS SEQ ID NO: 17859-22120). Fig.22A -K). Similarly, in mouse No. 2 (M7), there are 4478 unique Vδ6 CDR3s (NGS SEQID NOs: 22121-26597) ( Fig.23A-L). Vδ6 CDR3s are longer, some as long as 16 amino acids. There are many Vδ6 CDR3s shared between M4 and M7, as in the first twenty CDR3s of mouse number one (M4), 14 CDR3s match CDR3s from 8yyyy, although there are no shared CDR3s between the first 20 of the two mice. The first five CDR3s of human TCR Vδ6 in mouse number one (M4) have percentages of 21%, 20%, 15%, 11%, and 7%. The first five CDR3s in mouse number two (M7) have percentages of 15%, 15%, 13%, 11%, 10%, and 9%, indicating that this is a semi-focused library, yet a large number of low-abundance CDR3s are still retained. In fact, in M4, 84% of the CDR3s appear only once or twice in the total CDR3s, while in M7 it is 82%. The observations herein suggest that human Vδ6 plays a role in certain housekeeping functions, while maintaining its state of readiness to respond to the different internal and external challenges faced by each mouse. The observations herein also suggest that human TCRs can function with mouse-based antigens, many of which are shared (e.g., lipid-based and sugar-based), and therefore these human TCR mice can serve as models close to humans and can be used to identify new therapeutic targets and, ultimately, to identify meaningful human-based TCRs for human or animal therapy.

[0316] Example 6

[0317] Repertoire analysis of TCRδ1-4,6 in mouse spleen

[0318] In wild-type mice, γδT cells use the spleen as one of the main residence sites (van der Heyde., H. 2006. Infect Immun 75: 2717-2725). In the present invention, the next generation sequencing (NGS) method is used to further study human δTCR CDR3, including Vδ1, Vδ2, Vδ3, Vδ4 and Vδ6. NGS results show that all CDR3s are highly diverse. Similar to Example 4, IgBlast analysis was performed only on the first 100,000 sequence reads. Table 11 lists the top ten CDR3s for each δ subclass.

[0319] Table 11

[0320]

[0321] For human Vδ1 (NGS SEQ ID NOs: 26598-28541), there were a total of 1944 unique CRD3 sequences ( Fig. 9A-E). The top 2 CDR3s accounted for 46% and 32%, respectively. CDR3s that appeared only once and twice accounted for 83% of the total 1944 CDR3s, indicating that the library is relatively focused, which is very different from the results of Vδ1 from blood.

[0322] For human Vδ2 (NGS SEQ ID NOs: 28542-32583), there were a total of 4043 unique CDR3 sequences ( Fig.11A -K). The top 2 CDR3s accounted for 3.8% and 3.1%, respectively. CDR3s that appeared only once and twice accounted for 80% of the total 4043 CDR3s, indicating that the library was not relatively focused.

[0323] For human Vδ3 (NGS SEQ ID NOs: 32584-33868), there were a total of 1285 unique CRD3 sequences ( Fig. 10A -D). The top 2 CDR3s account for 1.1% and 0.8%, respectively. CDR3s that appear only once and twice account for 65% of the total 1285 CDR3s, indicating that the repertoire is not focused, in stark contrast to Vδ3 CDR3s in blood, where the top two account for 83% of all CDR3s.

[0324] The results for Vδ1 and Vδ3 also suggest that different organs can have their own repertoire of traits that are highly adaptable and flexible.

[0325] For human Vδ4 (NGS SEQ ID NOs: 33869-38496), there were a total of 4628 unique CRD3 sequences ( Fig.13A -L). The first two CRD3s accounted for 0.4% each. CDR3s that appeared only once and twice accounted for 75% of the total 4628 CDR3s, indicating that the library is not focused. The results of this paper also show that the diversity and quantity of Vδ4 are comparable to Vδ2, which provides new research opportunities, especially for the study of tissues with very limited access in humans.

[0326] For human Vδ6 (NGS SEQ ID NOs: 38497-41381), there were a total of 2885 unique CRD3 sequences ( Fig. 12A -H). The top 2 CDR3s account for 0.6% each. CDR3s that appear only once and twice account for 80% of the total 2885 CDR3s, indicating that the library is not focused.

[0327] By comparing the top 12 most abundant TCRδCDR3s present in human fetal cells at 10 weeks postpartum (Papadopoulou et al. PNAS 2020.117: 18638-18648) with the TCRVδ2CDR3s of the described 4043 unique CDR3 sequences, it is strongly demonstrated that these human TCRγ / δ mice can produce human or human-like TCRs. The comparison results showed that among the top 12 human CDR3s, 4 CDR3s were identical to the sequences listed in (NGSSEQ ID 28542-32583). As shown in Table 12, clone ACDTLGDTDKLI ranked second in mouse spleen with an abundance percentage of 3.1%. Another clone, ACDTVΓDTDKLI, used N addition during the formation of CDR3 by VDJ recombination, indicating that human sequences containing human V, D, and J are completely suitable for the mouse VDJ recombination system, which is unexpected because mice and humans have great genetic differences.

[0328] Table 12

[0329]

[0330] Example 7

[0331] Human Vδ functional testing by E. coli infection

[0332] Tagawa et al. demonstrated that mouse Vδ1 plays an important role in bacterial clearance. In mouse Vδ1 knockout mice, the clearance of intraperitoneally injected (IP) bacteria Escherichia coli was significantly delayed compared to wild-type mice (Tagawa., T. 2004 J Immuno 173: 5156-5164). The greatest difference in bacterial clearance was observed on day 3. The bacterial counts (CFU / mouse) of peritoneal fluid collected from infected mice in Vδ1 knockout mice were nearly 2 logs higher than those in wild-type mice.

[0333] In the human Vδ mouse of the present invention (Example 2), mouse Vδ1 (mouse TRDV4) is replaced by human Vδ1-8 to obtain a mouse Vδ1-deficient mouse model, but the model still expresses human Vδ. Basically according to the protocol provided in the article by Tagawa et al., 10*8 Escherichia coli (DH5α) in 100μl PBS were intraperitoneally injected into wild-type control, heterozygous and homozygous human Vδ mice. Three days later, 5ml PBS was injected into the peritoneal cavity of these mice, and the peritoneal fluid containing bacteria was subsequently collected and 1μl of the liquid was plated on an antibiotic blank LB plate. E. coli colonies were counted overnight. As shown in Table 13, there was no statistical significance between the three test groups, indicating that human Vδ functionally compensated for the loss of mouse Vδ1. The human δTCR cleared for E. coli can be cloned and sequenced, and the human δTCR can be transferred into human cells for therapeutic purposes.

[0334] Table 13

[0335]

[0336] No statistical significance was detected between any of the groups

[0337] Example 8

[0338] Human TCRγδ clonal expansion and focusing

[0339] In humans, TCRγδ can sense changes on the surface of infected and malignant cells, thereby expanding and focusing the well-studied γδ repertoire containing Vδ1, Vδ2 and Vδ3 (Hunter., S. 2018 Journal of Hepatology 69:654-665). Therefore, expansion and specific focusing are hallmarks of immune responses, and they can be used as biomarkers for many immune-related tests and developments, including cancer treatment, vaccine development, skin wound healing, lung and liver diseases, intestinal diseases, and brain dysfunction.

[0340] Humanized mice containing TCR Vδ (homozygous) and Vγ (heterozygous) were generated by the hybridization process detailed in Example 3. The pathogen was introduced by intraperitoneal injection, and blood was collected through the retro-orbital sinus after appropriate anesthesia procedures. Specifically, blood was collected 3 days before and after injection of 5×10*8 Escherichia coli (DH5α) in 50μl PBS. The PCR and library of Vδ1-4, 6 were analyzed using the procedure detailed in Example 6. Positive PCR bands were detected in Vδ1, 2, 3, 4 and 6 before injection. However, after injection, only Vδ1 and 4 were positive, indicating that the expression of Vδ2, 3 and 6 was downregulated to below the PCR detection level. Further library analysis was performed on Vδ1 and 4 before and after injection, and the results are listed in Table 14. The results clearly show that there is clonal focus in Vδ1 and 4.

[0341] More specifically, for Vδ1, before injection, the clone distribution was relatively uniform, as the top ten most common clones had percentages averaging between 2.0%-3.0%. In sharp contrast, after E. coli injection, the clones were highly focused, as a single amplified clone (the top-ranked clone) accounted for 62.4%. The top-ranked clone with the CDR3 sequence ALGFYWGTPYTDKL was only ranked 143rd among 10,038 unique Vδ1 CDR3s on the CDR3 list in the blood sample collected before E. coli injection, with a percentage of 0.053%. After E. coli injection, the number of unique clones in mouse blood also decreased, totaling only 3530, about 1 / 3 of that before injection.

[0342] Similarly, for Vδ4, before injection, the clone distribution was relatively uniform, as the top ten most common clones had a percentage average of 3.4%-5.1%. In sharp contrast, after E. coli injection, the clones were highly focused, as the three amplified clones (the top three clones) accounted for 41.0%, 36.4.0% and 13.5%, respectively. The top three clones with CDR3 sequences AMREGGVYDKLI, AMRPSYYKLI, and AMREGLPGGYARDKLI were only ranked 78th, 75th and 240th among 3284 unique Vδ4CDR3s on the CDR3 list in the blood sample collected before E. coli injection, with percentages of 0.043%, 0.050%, and 0.011%, respectively. After E. coli injection, the number of unique clones in mouse blood also decreased, with a total of only 880, which was about 1 / 4 of the number before injection.

[0343] The second pathogen was also tested in humanized mice containing Vδ (homozygous) and Vγ (heterozygous). Blood was collected 3 days before and after injection of 100 μl of S. aureus extract (Wood 46 strain, sigma S2014), which is protein A-deficient and spa-negative. It has 98% to 99% genome identity with S. aureus and shows low surface expression of cell wall-associated protein A. And it is a formalin-fixed crude cell suspension of substantially non-viable S. aureus (Wood 46 strain) in 0.05M potassium phosphate buffer (pH 7.5) containing 0.2% sodium azide, as described by the manufacturer.

[0344] The PCR and pools of Vδ1-4, 6 were analyzed using the procedures detailed in Example 6. Positive PCR bands were detected before injection in Vδ1, 2, 3, 4, and 6. However, after injection, all except Vδ3 were positive, indicating that the expression of Vδ3 was downregulated to below the level of PCR detection. Further pool analysis was performed on Vδ1, 2, 4, and 6 before and after injection, and the results are listed in Table 15. The results clearly showed that positive PCR products were present in all Vδs with clonal focus.

[0345] More specifically, for Vδ1, before staphylococcal injection, the clone distribution was relatively uniform, as the top ten most common clones had a percentage average of between 2.1% and 3.9%. In sharp contrast, after staphylococcal injection, the clones were highly focused, as the three expanded clones (the top three clones) accounted for 19.9%, 19.90%, and 15.6%, respectively. The top three clones with CDR3 sequences ALGELEGIRHKLI, ALGELLPGGYVΔKL, and ALGELFLLGDTDKL were only ranked 391st, 347th, and 646th among 10070 unique Vδ4 CDR3s on the CDR3 list in the blood sample collected before staphylococcal injection, with percentages of 0.016%, 0.018%, and 0.011%, respectively. After staphylococcal injection, the number of unique clones in mouse blood also decreased, with a total of only 5695, a little more than 1 / 2 of that before injection.

[0346] For Vδ2, some clones were already enriched before staphylococcal injection, as the top 4 clones accounted for 79.7% compared to spleen Vδ2, which were still evenly distributed as shown in Example 6. Highly enriched Vδ2 CDR3 was also observed in human blood (Hunter., S. 2018. Journal of Hepatology69:654-665). The top 4 CDR3s of Vδ2 in Table 13 are ACDTGGDYTDKLI, ACDTGGGYDTDKLI, ACDKYWGLYTDKLI, and ACDNTGAYTDKLI, accounting for 27.9%, 24.8%, 15.2%, and 11.8%, respectively. In sharp contrast, after staphylococcal injection, cloning is highly focused, as a single amplified clone (the top-ranked clone) accounts for 78.9%. The top-ranked clone with the CDR3 sequence ACELLGDΔDKLI ranks only 85th among 3161 unique Vδ2 CDR3s in the CDR3 list in the blood sample collected before staphylococcal injection, with a percentage of 0.06%. After staphylococcal injection, the number of unique clones in mouse blood also decreased, with a total of only 2460, about 3 / 4 of that before injection. Furthermore, in unstimulated blood, the sequences of CDR3 from staphylococcal injections predominantly started with ACE rather than ACD, suggesting that CDR3 conversion from ACD to ACE or other non-ACDs is a clear immune marker for many immune activation processes.

[0347] For Vδ4, before staphylococcal injection, the clone distribution was relatively uniform, as the top ten most common clones had percentages averaging between 3.0%-3.7%. In sharp contrast, after staphylococcal injection, the clones were highly focused, as only one clone (the top-ranked clone) was amplified and counted at 89.0%, while the second clone was only 0.6%. The top-ranked clone with the CDR3 sequence AMRERGSYTDKLI ranked 62nd out of 3198 unique Vδ4 CDR3s on the CDR3 list in the blood sample collected before staphylococcal injection, with a percentage of 0.03%. After staphylococcal injection, the number of unique clones in mouse blood also decreased, with a total of only 466, a little more than 1 / 6 of the number before injection, indicating that the reduction in unique TCR clones was relatively large.

[0348] Finally, for Vδ6, before staphylococcal injection, the clonal distribution was relatively uniform, but the cloning was slightly focused, as the top ten most common clones had percentages with averages between 4.1% and 8.0%. In stark contrast, after staphylococcal injection, the cloning was highly focused, as only two clones were amplified and counted, at 38.4% and 36.3%, respectively, and they had CDR3 sequences AEGDTDKLI and AALGSSWDTRQM. They were ranked 284th and 219th out of 7552 unique Vδ6 CDR3s on the list of CDR3s in the blood sample collected before staphylococcal injection, with percentages of 0.02% and 0.03%, respectively.

[0349] After staphylococcal injection, the number of unique clones in the mouse blood also decreased, totaling only 3020, a little more than 2 / 5 of the number before injection, indicating that the reduction in unique TCR clones was relatively large.

[0350] Example 9

[0351] Repertoire analysis of TCRγ9 and 10 in humanized TCRγ / δ mice

[0352] Humanized mice containing TCR Vδ (homozygous) and Vγ (heterozygous) were generated by the hybridization process detailed in Examples 3 and 8. TCRγ9 library analysis was first performed by PCR using the primer sets shown in Tables 16 and 17. Strong positive PCR bands were observed in the blood and thymus, but not in the lungs, intestines, kidneys, and spleen. Similar to the description in Example 5, the positive PCR bands were subjected to next generation sequencing, and the results are shown in Table 18 and NGS SEQ ID NOs: 41382-41899 and 41900-42761 ( Fig.14A -B, and Fig.15A -C). In the thymus, TCR Vγ9 CDR3 is slightly focused, with the first five CDR3s accounting for 87.2% of the total CDR3s, but the tail of the library is relatively long, with a total of 863 unique CDR3s. In sharp contrast, the mature CDR3s in the blood have a very focused library. The top-ranked CDR3 accounts for 91% of the total CDR3s, but is only ranked 10th in the library from the thymus, with a percentage of 0.3%. Focusing also reduced the degree of diversity in TCR Vγ9 to only 519 unique clones, down to 3 / 5 of that in the thymus. Unexpectedly, in the thymus, the second-ranked clone (20.9%) with the sequence ALWEVQELGKKIKV has the same sequence as Vγγ9 / Vδ2 in human blood in many studies (van Diest., E. 2021. J Immunother Cancer 9: e003850). In particular, in the paper published by Papadopoulou et al. (PNAS 2020.117:18638-18648), ALWEVQELGKKIKV was the major CDR3(γ9) in human blood from human Vγγ9 / Vδ2 cells, which strongly suggests that human TCR Vγγ9 / Vδ2 mice can produce clones that are very similar, or even in some cases, identical to clones from humans.

[0353] Table 16

[0354]

[0355] Table 17

[0356]

[0357] Table 18

[0358]

[0359] *The sequence ALWEVQELGKKIKV has been reported in many human blood studies

[0360] The limited access to human tissue has greatly hampered the study of human Vγ10. There are very few publications describing the sequence and function of human Vγ10. To investigate the possibility of establishing human Vγ10 in mice, tissues from humanized mice composed of TCR Vδ (homozygous) and Vγ (heterozygous) were collected and PCR, NGS, and data processing were performed similar to that described for Vγ9, but using different PCR primer sets (Tables 14 and 15) and NGS ID NOs: 42762-45490, 45491-49130, and 49131-50786 ( Fig.16A -G, Fig.17A -E and Fig.18A -J). The data showed that Vγ10 was expressed in all tissues tested, including lung, thymus, intestine, kidney, and spleen, and the top ten CDR3s for these tissues are listed in Table 19. CDR3s have different degrees of diversity in different tissues. For example, in descending order, the thymus has 3641 unique CDR3s, the lung has 2730 unique CDR3s, the intestine has 1657 unique CDR3s, the kidney has 1277 unique CDR3s, and the spleen has 689 unique CDR3s. This order of abundance is consistent with previous observations by others in human studies, that the thymus has the most individual clones because it is where these clones are first generated. The lungs and intestines are the two highest ranked tissues where human γδ T cells are expressed.

[0361] surface

[0362]

[0363] Table 15

[0364]

[0365] Table 19

[0366]

[0367] Example 10

[0368] Surface expression of human TCR Vγ9 / Vδ2 and Vγ9 / Vδ1 obtained by flow cytometry

[0369] The presence of the γδTCR revealed by the library study was further confirmed by flow cytometry analysis, which uses fluorescently labeled antibodies that specifically bind to T cell receptors on the surface of T cells in the form of proteins. Blood was collected through the retro-orbital sinus of human double homozygous γδTCR mice. The tissue was first treated with an erythrocyte lysis buffer (0.8g NH4Cl and 0.11g NaHCO3 in 100ml of aqueous solution) to remove red blood cells, and then white blood cells including T cells were collected by centrifugation and diluted to an appropriate concentration and then sent to a flow cytometer (Becton Dickinson LSRFortessa). The antibodies and their suppliers are listed in Table 20. The results of the flow cytometric analysis in Table 21 show that the spleen is the site with the highest ranking of γ / δT cells (about 17%), followed by the thymus (5%) and lungs (2.6%), which is very consistent with the results of human studies. In humans, the spleen is also the site with the highest ranking of γδT cells, with an abundance of 12.5% ​​± 8.1%, followed by the thymus with an abundance of 1.4% ± 0.5% (Inghirami, G., 1990 Am J Pathol 136: 357-367). The results of this article show that the γδT cells in human γδTCR mice are well developed and maintain the correct ratio among other CD3-positive cells. The data in this article also show that most of the γδT cells in the spleen come from the contribution of humanized Vδ1 and Vδ2, while the contribution from mouse Vδ is very small. Also in the spleen, most γ / δT cells are humanized Vδ1 / humanized Vγ9, with an abundance of 61%, while the abundance of humanized Vδ2 / humanized Vγ9 is 7.6% ( Figure 4 ). In all categories, the proposition of Vδ1 is higher than Vδ2, which is also consistent with the observations of others, because tissue-resident γδT cells are usually δ1T cells, while δ2 is mainly found in the blood (Mossow, G., 2021 Front. Immunol., https: / / doi.org / 10.3389 / fimmu.2021.741218 ). In summary, human γ / δTCR mice have normal development and maturation in multiple tissues, and the subsets of humanized γ and humanized δ T cells can maintain a ratio similar to that of humans.

[0370] Table 20

[0371]

[0372] Table 21

[0373]

[0374] Embodiment 11

[0375] Cancer development in human TCRγδ mice (B16 cancer)

[0376] One of the biggest advantages of developing a human TCRγδ mouse model is that the model has a complete mouse immune system, which provides researchers with a better model than immunodeficient mouse models (e.g., NSG and SCID) because immunodeficient mice lack B cells and T cells, which play a key role in immunotherapy. Therefore, the human TCRγδ model is suitable for adapting to the same mouse cancer line because many studies have demonstrated their importance in cancer research. In the art, the mouse B16 melanoma cell line has been isolated from B6 mice and has been shown to be able to grow stably in pure B6 and Balb / C mouse strains, but has poor growth in the 129 mouse strain. Human TCRγ / δ mice have a mixed background of B6 and 129 and are mainly produced through a littermate mating scheme. 500,000 B16 cells suspended in 250μl PBS were injected through the tail vein, and the results showed that B16 mainly accumulated and metastasized in the lungs. After fourteen days, the lungs were harvested and archived. As shown in Tables 22 and Figure 5 As shown, wild-type mice and mice with only one human TCRγ or TCRδ did not develop cancer at all. In addition to the doubly homozygous mice, there were a large number of B16 cancers in the homozygous γ mice and homozygous δ mice. The results show that the number of copies of TCRγ and / or δ plays a very important role in the development of melanoma cancer.

[0377] Table 22

[0378]

[0379] WT wild-type TCRγ / δ directly from mice. Homo-homozygous Hetero-heterozygous

[0380] Example 12

[0381] Reduction and elimination of human cancer cells (Daudi) in human γδ TCR mice

[0382] It has been demonstrated in the art that an important aspect of the antigen specificity of human γδTCR cells is that they can recognize and kill tumor targets. In the SCID animal model (immunocompromised mice), T cells expressing Vγ9 / Vδ2 heterodimers, i.e., the same TCR stimulated by bacterial phosphorylated metabolites, can recognize bone marrow-derived tumor cells (such as non-Hodgkin's B cell lymphoma line Daudi) in vitro (Fisch, P. et al., Science. 250: 1269-1273.) and in vivo (Malkovska, V. et al., 1992 Cancer Res. 52: 5610-5616.). Vyborova et al. further showed that only a small fraction of in vitro expanded Vγ9 / Vδ2 T cells were active against cancer cell lines, and that clonal frequency was not associated with the functional affinity of the Vγ9 / Vδ2 T cell receptor (J Clin Invest. 2020; 130: 4637-4651), suggesting that the nature of TCR-based human γ / δ T cells killing human cancers is functional but complex. In addition to the extensive work of the past three decades, human cancer studies have not been conducted in the context of immunocompetent mice with human γδ T cell receptors.

[0383] In the present invention, 6 million human non-Hodgkin's B-cell lymphoma line Daudi (ATCC) in 200 μl PBS were injected into wild-type and human TCRγ (heterogeneous) / δ (homologous) mice through the tail vein. On the 4th day, multiple organs including lungs, liver, spleen, intestines and kidneys were collected and treated with liberase (Roche) according to the manufacturer's recommendations. Cells from these tissues were labeled with PE-anti-human CD22 (Biolegend), a well-established marker for Daudi cells highly expressed on the surface (Pop L. et al., 2014 Cancer Res. 74: 263-271). Tissues with excessive blood were treated with the red blood cell lysis kit described in the previous example. CD22-labeled cells were sent to a flow cytometer (Becton Dickinson LSRFortessa). The results in Table 23 show that Daudi cells (expressed as CD22) were dramatically reduced in all tissues studied in human TCRγ(heterogeneous) / δ(syngeneic) mice compared to wild-type littermate controls. The lungs, livers, and spleens were reduced by 3.4-fold, 3-fold, and 6.8-fold, respectively. In particular, human Daudi cells were completely eliminated in the intestines and kidneys of human TCRγ(heterogeneous) / δ(syngeneic) mice, although very small amounts of Daudi cells were present in wild-type mice. (Figures 9-11)

[0384] Table 23

[0385]

[0386] *Ratios represent the ratio between wild-type and human TCRγ / δ mice. Homo-homozygous. Hetero-heterozygous

[0387] Embodiment 13

[0388] Further humanization of TCRγ mice (addition of human Vγ1-8)

[0389] In the present embodiment, humanization Vγ9-11 mouse ES cell line is further humanized by introducing mankind Vγ1-8 on the same allele of humanization Vγ9-11.Non-human animals (such as mice) are modified so that non-human animals contain the nucleotide sequence comprising mankind TCRγ and also comprising mankind TCR Vγ and Jγ.Specifically, the endogenous mouse sequence containing mouse TCR Vγ and Jγ is replaced with the sequence containing mankind TCR Vγ and Jγ, and is operably connected to mouse TCR Cγ1 district.

[0390] More specifically, the DNA construct consists of a 5' mouse homology arm (10875 bp, DNA segment NO: 21), a 48234 bp human sequence containing human TCRVγ1-8 (DNA segment NO: 22), an additional human sequence with human JP1, JP, JP2 and J2 (15465 bp, DNA segment NO: 23), an antibiotic selection box (DNA segment NO: 24, which is identical or substantially identical to DNA segment NO: 20) and a 3' mouse homology arm (8625 bp, DNA segment NO: 25). The human sequence ends at the 5' upstream of the mouse endogenous Cγ1 region, so that human Vγ and Jγ are genetically connected to the mouse Cγ1 region. The humanization strategy is summarized in Figure 6 The junction nucleic acid sequences within the targeting vector between mouse, human and the prokaryotic / eukaryotic selection cassette are summarized in Table 24 and provided in the sequence listing (SEQ ID NOs: 56-61).

[0391] Table 24

[0392]

[0393] Table 25

[0394]

[0395] Table 26

[0396]

[0397] Bacterial Spec and Kan selection boxes are used to help bacterial homologous recombination to prepare vectors (vector ID NO: 8). Linearized targeting vectors (vector ID NO: 8) are electroporated into ES cell lines containing humanized Vγ9-11, as described in Example 1. Targeted clones are selected as described in Example 1 and microinjected into mouse blastocysts to produce chimeric mice. Linkage analysis confirms events of human Vγ1-8 and human Vγ9-11 on the same allele, wherein all progeny are positive for both human Vγ1-8 and human Vγ9-11. There are no events in which human Vγ1-8 and human Vγ9-11 are present in different progeny.

[0398] Germline transmission of human Vγ1-11 mice was achieved, and the mice were mated with humanized Vδ mice to produce humanized γδTCR mice. Since human Vγ1, 6, 7 and 11 are pseudogenes, only tissues from these humanized mice were analyzed for Vγ2-10TCR expression. PCR primers are listed in Tables 25 and 26. Two rounds of PCR were performed, and the expression patterns are listed in Table 27. Table 27 shows that human Vγ2, 3, 4, 5, 8, 9 and 10 are expressed in various tissues of mice. Some PCR products were sequenced by Sanger sequencing. Figure 7 and Figure 8 It was also shown that human γTCRs are highly diverse, as DNA sequences over 230 nt are highly overlapping, suggesting that multiple TCRs can be amplified using the same pair of PCR primers.

[0399] Table 27

[0400]

[0401] 1-peritoneal fluid, 2-brain (olfactory sense), 3-ear (middle ear), 4-nose, 5-tongue, 6-lymph nodes, 7-throat, 8-lung, 9-spleen, 10-bone marrow, 11-eye (eyeball), 12, 13, 14 small intestine (front, middle, back three parts), 15-uterine tube, 16-kidney, 17-thymus, 18-muscle, 19-heart, 20-liver, 21-skin, 22-blood. The positive sign represents the expression of human yCDR3.

[0402] Sequence 1-74

[0403] SEQ ID NO:1TAGGCTATCACAGACAGGGAGAATTTACATCCACCCCAACTTTATACTTCTAATTATCC

[0404] SEQ ID NO:2CCTGAAATTCCAGCTTGCAGAGCACTTCCTGCCTCCCTGTGAAGCAGCCTGACCTTGGGATGCTGTCACTGCTCCACACATCAACGCTGGCAGTCCTTGGGGCTCGTAAGTAGTTTT

[0405] SEQ ID NO:3TCTCCAGAGGATTCAGCTGTACTTCTAACTGCTCAAATGGAAGGTTATCAACATCAGCTCGTACGTTCGTGGGATTGTGTCCGTGTCGCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTC

[0406] SEQ ID NO:4 GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGTTGGTACGAACCCGGGTTTGGGCCCCTTCTCAGTGCCAGTGTCCCCTCAGTGAAAAGAGACCAGCTTGTGTACTGTCATGTAGA

[0407] SEQ ID NO:5 TGCTTCTACAGTGTCCTATTAATCCCACTGTGCTAGCACTGGGTTTCCATAGTCCATGT

[0408] SEQ ID NO:6 CTGCTTGCCATGCTCAGGAGCTGCAATAAGTTCAGCTCTTCGTGTCGCTTTATGCCTTC

[0409] SEQ ID NO:7 GAGTACCTGTCAAATATAAACTATGCTCAGCAGGTACCAGCGCCTCACTTTTATCCTTTACGCGCCATTTATCAACTCACACCTATGGGGACAGCTATTATCAGAAAAACAAAAGGCAACAAGTATTAGTAGGGATGTG

[0410] SEQ ID NO:8 TCAGATATTCTAAGGATCACTGCTATCCAGAGAATCTTAGAGCTTGACCTAAAGCTGGTACCGTACTCATGGCAACAGCATTACTGACCATTTGAATGTAGAAGACTCATGAATTCTCCCTTCCTAGAGAC

[0411] SEQ ID NO:9 CTCAGAGTCATATCGCTGCATTCACTACTTTCTCTGTGAGTTTTCTATGGGATTCAGCATAGCTAATATATCTTCCAACCTGAGTGTCAGCACATTCAGACTTGATCCAGCCAGAAGCTACATTTTTCAGAGTCAGAG

[0412] SEQ ID NO:10 AGAGAGATGGTAGTCCTGTGGTTGCTTCATCTGTGGTCTGGATCAGAAGATTTAATTCTAAATAAAAGCTCATATGTCAATCTAATTCTGTGTTGTTAAAGTAAAGATATCAAAGGAATAGCTCTACTCCCATGTTTCAG

[0413] SEQ ID NO:11 CTTCTCATTGAGGTTTATTCTGATTGATTTCCAGCTTTGCCCAAGGGACCCTGAGTTATCTTACCAGTTGGCAACCCAAAAACATCCTATAAAATCTCCAGCAGGCCTTTGTTTCCTTGCCATCAGCTCCTCTTCGGC

[0414] SEQ ID NO:12 CAATTTTGTAAGAGTATTGATGGAAAATCGTTAGGCATCTACATAGCCTTGATTTGGCTCCTTGGACCTCTGACCAATAAACACAGGAAAAGATGCTCTACATCAGGAGTCAACAAAATATGGCCCATGAGCCAAATTC

[0415] SEQ ID NO:13 TCTGAACCAGTCAGTAACCTTCCCATGGTAGTAATCATGTGAGACAGGATTCAGCAGGACTGGCTGAGATTATATCAAAGCTTTCATAGGGGTTCTATAACCACAGAATGTAGTTAGTTCCCTGCCAAGTAAAACTTA

[0416] SEQ ID NO:14 ATCCCTTTGGCTAAGCTAACTAGTATGCGTGCCTCCTTGCAAACTGAAAACAAGTGTCCTACAGCTTGGACGTACGGTACCAACGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATTCTACCGGGTAGGGGAGG

[0417] SEQ ID NO:15 CGCGGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGCGACACGGACACAATCCCACGAACGTACGCCTGCACATACCTTCGGCTGAGCTAACGTGTCTGCATGCCTCCTTACAGAGCACAAATCTCCTACAGCT

[0418] SEQ ID NO:16 AAACGAAACCAAGGTCTCAAGACTGACAGCCCAAAGTTACTGTCCCAGGGGCTCCCT

[0419] SEQ ID NO:56 CTGCTATTAACTCCTGATTCAGCCTTCTAGSEQ ID NO:57 AGGCTGGCACACTCTCACTGATAGATGGCTAAGCGATACAGAGAGCCCAGACAACCATTTAGAGGTGAGGAATTAGTAGGGATGTG

[0420] SEQ ID NO:58 TAGCCCCAGAATATCCCTCTGTCAAAACGTTCTGCTAGCTTTGTATTAACTTTAAATAATTGGCATTATTTAAAGTTAGCAGAAAGAGATAAATGCATAGTTAGTGCCTGACTCASEQ ID NO:59 TCTAGCTAAAGAAAGGGGCAAGAGACAATGAATGCTGTGGTACCGTACAGAAAATGTCGTACGGAAGTTCCTATACTATTTGAAGAATAGGAACTTCCAGAGTCAGAG

[0421] SEQ ID NO:60 ACGCTGTGATGCGTCTTATGCTGTGTATCGTGTCATGGTACCCAAGGACATCTTGTCTGAAAC AAACTGTAATCGAGCACTCGACCACGGCTGTCCTCTG

[0422] SEQ ID NO:61 ACTGGGTTGCATGCTTATTCAGAGAACTGGCTT

[0423] SEQ ID NO:62(Vγ9)CVYGAGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLVSISYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCALWEV

[0424] SEQ ID NO:63(Vγ10)MFIGNSPLLLTVΓLGLSKVEQFQLSISTEVKKSIDIPCKISSTRFETDVIHWYRQKPNQALEHLIYIVSTKSAARRSMGKTSNKVEARKNSQTLTSILTIKSVEKEDMAVYYCAAWDSEQ ID NO:64(Vγ11)LGQLEQPEISISRPANKSAHISWKASIQGFSSKIIHWYWQKPNKGLEYLLHVFLTISAQDCSGGKTKKLEVSKNAHTSTSTLKIKFLEKEDEVVYHCACWIRH

[0425] SEQ ID NO:65(Vδ1)SSVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGE

[0426] SEQ ID NO:66(Vδ2)VMSAIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDT

[0427] SEQ ID NO:67(Vδ3)RGTLCDKVTQSSPDQTVASGSEVVLLCTYDTVYSNPDLFWYRIRPDYSFQFVFYGDNSRSEGADFTQGRFSVKHILTQKAFHLVISPVRTEDSATYYCAF

[0428] SEQ ID NO:68(Vδ4)PGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRE

[0429] SEQ ID NO:69(Vδ5)VNSQQKNDΔQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAS

[0430] SEQ ID NO:70(Vδ6)VSGQQKEKSDQQQVKQSPQSLIVQKGGISIINCAYENTAFDYFPWYQQFPGKGPALLIAIRPDVSEKKEGRFTISFNKSAKQFSSHIMDSQPGDSATYFCAA

[0431] SEQ ID NO:71(Vδ7)VSSEDKVVQSPLSLVVHEGDTVTLNCSYEVTNFRSLLWYKQEKKAPTFLFMLTSSGIEKKSGRLSSILDKKELFSILNITATQTGDSAIYLCAVE

[0432] SEQ ID NO:72(Vδ8)FSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRS

[0433] SEQ ID NO:73 (Constant region of murine δ TCR chain) XSQPPAKPSVFIMKNGTNVACLVKDFYPKEVTISLRSSKKIVEFDPAIVISPSGKY SAVKLGQYGDSNSVTCSVQHNSETVHSTDFEPYANSFNNEKLPEPENDTQISEPCYGPRVTVHT EKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF

[0434] SEQ ID NO:74 (Constant region of murine γ TCR chain) XKRTDSDFSPKPTIFLPSAAETNLHKAGTYLCLLEKFFPEVIRVYWKEKDGEKILESQEGNTIKTNDRYMKFSWLTVTEDSMAKEHSCIVKHENNKRGVDQEILFPP IGKAFTTINVNPRDSVLRHENVNNATDLEDCMKGRKDMLQLQVTTTYAFYTYLILFFKSMVHLAFVVFCLFRRAAMSCDDQRS

Claims

1. A genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unaligned human T cell receptor (TCR) Vδ gene segment, at least one unaligned human TCR Dδ gene segment, and at least one unaligned human TCR Jδ gene segment, wherein the at least one unaligned human TCR Vδ gene segment, the at least one unaligned human TCR Dδ gene segment, and the at least one unaligned human TCR Jδ gene segment are operably linked to a functional non-human TCRδ constant gene sequence.

2. The animal of claim 1, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment are inserted into an endogenous TCRδ variable gene locus.

3. The animal of claim 1, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replace the complete repertoire of unarrayed endogenous TCR Vδ, Dδ, and Jδ gene segments.

4. The animal of claim 1, wherein the at least one unarrayed human TCR Vδ gene segment is selected from an unarrayed library of human TCR Vδ gene segments Vδ1, Vδ2, Vδ3, Vδ4, Vδ5, Vδ6, Vδ7, and Vδ8.

5. The animal of claim 1, wherein the amino acid sequence of at least one unaligned human TCR Vδ segment has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72.

6. The animal of claim 1, wherein the at least one unarranged human TCR Vδ gene segment, the at least one unarranged human TCR Dδ gene segment, and the at least one unarranged human TCR Jδ gene segment are capable of rearranging to form a rearranged human VDJδ sequence.

7. The animal of claim 6, wherein the animal expresses a humanized TCRδ variable region comprising the rearranged human VDJδ sequence on the surface of a γδ T cell population.

8. The animal of claim 7, wherein the sequence of the complementarity determining region 3 (CDR3) of Vδ1 starts with amino acids ALGE, wherein E can be replaced by any one of G, A, D, V or R; and / or ends with KLI or TRQM.

9. The animal of claim 7, wherein the CDR3 of Vδ2 begins with amino acids ACD, ACG, ACE, ARD, or ASD; and / or ends with KLI or TRQM.

10. The animal of claim 7, wherein the CDR3 of Vδ3 begins with amino acids AL, AS, AC, AY, AI or AV.

11. The animal of claim 7, wherein the first three amino acids of the CDR3 of Vδ4 are AMR, ASP, AMS, AMT, AMG, AMI, AIR, AKR, ATR, EMR, VMR, PMR or ALP; and / or the last few amino acids are KLI or TRQM.

12. The animal of claim 7, wherein the first three amino acids of the CDR3 of the Vδ6 are AAS or AAR; or the first four amino acids are AASP, wherein P can be replaced by T, G, M, V, L or R.

13. The animal of claim 12, wherein the last three amino acids of the CDR3 of the Vδ6 are TRQ, KLI or KLN.

14. A genetically modified non-human animal, the genome of which comprises: at least one unarranged human TCR Vγ gene segment and at least one human TCR Jγ gene segment, wherein the at least one unarranged human TCR Vγ gene segment and the at least one human TCR Jγ gene segment are operably linked to a functional non-human TCRγ constant gene.

15. The animal of claim 14, wherein the at least one unpermuted human TCR Vγ gene segment and the at least one unpermuted human TCR Jγ gene segment are inserted into an endogenous TCRγ variable gene locus.

16. The animal of claim 14, wherein the at least one unarrayed human TCR Vγ gene segment and the at least one unarrayed human TCR Jγ gene segment replace the complete repertoire of unarrayed endogenous TCR Vγ and Jγ gene segments.

17. An animal as described in claim 14, wherein the at least one unarrayed human TCR Vγ gene segment is selected from an unarrayed library of human TCR Vγ gene segments Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10 and Vγ11, preferably Vγ9, Vγ10 and Vγ11.

18. The animal of claim 17, wherein the amino acid sequence of the at least one unaligned human TCR Vγ segment has at least 90% sequence identity to SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:

64.

19. The animal of claim 14, wherein the at least one unarranged human TCR Vγ gene segment and the at least one unarranged human TCR Jγ gene segment are capable of rearranging to form a rearranged human VJγ sequence.

20. The animal of claim 19, wherein the animal expresses a humanized TCRγ variable region comprising the rearranged human VJγ sequence on the surface of a population of γδ T cells.

21. The animal of claim 20, wherein: The first four amino acids of the CDR3 of Vγ9 are selected from the group consisting of ALWE, ALWG, ASWE, ALCE, ALLE, ALRE, PCGR, AWWE, DLWE, ASWE and AMWE; or The last several amino acids of the CDR3 of Vγ9 are selected from the group consisting of KTL, KEL, EKL, KNL, IKV, KSR, RNS, KNS, MKL, KRL, RKL, FKI and KNQG; or The first three amino acids of the CDR3 of Vγ10 are AAW; or The first four amino acids of the CDR3 of the Vγ10 are AAWF, wherein the fourth position can be any one of F, L, A, R, G, C or V; or The last three amino acids of Vγ10 are FKI.

22. A genetically modified non-human animal, the genome of the genetically modified non-human animal comprising: at least one unarrayed human T cell receptor (TCR) Vδ gene segment, at least one unarrayed human TCR Dδ gene segment, and at least one unarrayed human TCR Jδ gene segment; and at least one unarrayed human TCR Vγ gene segment and at least one human TCR Jγ gene segment, wherein the at least one unpermuted human TCR Vδ gene segment, the at least one unpermuted human TCR Dδ gene segment, and the at least one unpermuted human TCR Jδ gene segment are operably linked to a functional non-human TCRδ constant gene, and Wherein the at least one unarranged human TCR Vγ gene segment and the at least one human TCR Jγ gene segment are operably linked to a functional non-human TCRγ constant gene.

23. The animal of claim 22, wherein the at least one unpermuted human TCR Vδ gene segment, the at least one unpermuted human TCR Dδ gene segment, and the at least one unpermuted human TCR Jδ gene segment are inserted into an endogenous TCRδ variable gene locus, and Wherein the at least one unarranged human TCR Vγ gene segment and the at least one unarranged human TCR Jγ gene segment are inserted into an endogenous TCRγ variable gene locus.

24. The animal of claim 22, wherein the at least one unarrayed human TCR Vδ gene segment, the at least one unarrayed human TCR Dδ gene segment, and the at least one unarrayed human TCR Jδ gene segment replace the complete repertoire of unarrayed endogenous TCR Vδ, Dδ, and Jδ gene segments, and wherein the at least one unaligned human TCR Vγ gene segment and the at least one unaligned human TCR Jγ gene segment replace the complete repertoire of unaligned endogenous TCR Vγ and Jγ gene segments.

25. The animal of claim 22, wherein the at least one unarrayed human TCR Vδ gene segment is selected from an unarrayed library of human TCR Vδ gene segments Vδ1, Vδ2, Vδ3, Vδ4, Vδ5, Vδ6, Vδ7, and Vδ8, and wherein the at least one unarrayed human TCR Vγ gene segment is selected from an unarrayed library of human TCR Vγ gene segments Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10 and Vγ11, preferably Vγ9, Vγ10 and Vγ11.

26. The animal of claim 25, wherein the at least one unarranged human TCR Vδ gene segment, the at least one unarranged human TCR Dδ gene segment, and the at least one unarranged human TCR Jδ gene segment are capable of rearranging to form a rearranged human VDJδ sequence, and Wherein the at least one unarranged human TCR Vγ gene segment and the at least one unarranged human TCRJγ gene segment are capable of rearranging to form a rearranged human VJγ sequence.

27. The animal of claim 26, wherein the animal expresses a humanized TCRγδ comprising the rearranged human VDJδ sequence and the rearranged human VJγ sequence on the surface of a γδ T cell population.

28. The animal of claim 22, wherein the amino acid sequence of at least one Vγ segment has at least 90% sequence identity with a human Vγ sequence selected from the group consisting of SEQ ID NOs: 62, 63, and 64, and wherein the amino acid sequence of at least one Vδ segment has at least 90% sequence identity with a human Vδ sequence selected from the group consisting of SEQ ID NOs: 64, 66, 67, 68, 69, 70, 71, and 72.

29. The animal of claim 22, wherein Dδ has at least 90% sequence identity to human Dδ.

30. The animal of claim 22, wherein the Jγ and the Jδ have at least 90% sequence identity to human Jγ and Jδ.

31. The animal of any one of claims 7, claim 20 and claim 27, wherein the humanized TCR is expressed on the surface of a population of γδ T cells together with at least one mouse CD3.

32. The genetically modified animal of any one of claims 1, 14, and 22, wherein the animal is a rodent.

33. The genetically modified animal of any one of claims 1, 14, and 22, wherein the animal is a mouse.

34. The genetically modified animal of any one of claims 1, 14, and 22, wherein the animal produces central and effector memory gamma / delta T cell populations to internal or external antigens.

35. A method for producing a humanized TCR, the method comprising: administering an antigen of interest to a genetically modified animal as described in any one of claims 1, 14, and 22; And obtain a humanized TCR that recognizes the target antigen.

36. A method for determining and / or analyzing the TCR repertoire of the humanized TCR produced as claimed in claim 35 by next generation sequencing.

37. A method of establishing cancer, the method comprising: Cancer cells are administered to the genetically modified animal of any one of claims 1, 14, and 22, and cancer growth is determined.

38. A method of establishing an infection, the method comprising: The antigen-derived pathogen is administered to The genetically modified animal of any one of claims 1, 14, and 22; and determining the TCR amplified repertoire by sequencing.

Images