Non-human animals expressing exogenous terminal deoxynucleotidyltransferase

Abstract

Provision of non-human animals expressing exogenous terminal deoxynucleotidyltransferase. [Solution] This specification provides methods and compositions relating to non-human animals expressing exogenous terminal deoxynucleotidyltransferase (TdT). In certain embodiments, what is provided herein are genetically modified non-human animals whose genomes contain exogenous nucleic acids encoding terminal deoxynucleotidyltransferase (TdT), as well as methods for producing and using such non-human animals. In some embodiments, the exogenous TdT is human TdT. In some embodiments, the exogenous TdT is of endogenous species origin (for example, in mice, the exogenous TdT has a mouse sequence).

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 345,524, filed on 3 June 2016, which is incorporated in its entirety by reference herein. [Background technology]

[0002] Non-human animals, particularly mice and rats, have proven to be a valuable source of therapeutic antibodies and may potentially serve as a source of other antigen-binding molecules. High levels of antigen receptor diversity in such non-human animals increase the likelihood of generating antigen-binding molecules with desired therapeutic properties after immunization. Therefore, genetically engineered non-human animals with increased antigen receptor diversity are needed to improve the production of therapeutic antigen-binding molecules. [Overview of the Initiative] [Means for solving the problem]

[0003] In certain embodiments, provided herein are genetically modified non-human animals whose genomes contain an exogenous nucleic acid encoding terminal deoxynucleotidyltransferase (TdT), as well as methods for constructing and using such non-human animals. In some embodiments, the exogenous TdT is human TdT. In some embodiments, the exogenous TdT is of endogenous species origin (e.g., in mice, the exogenous TdT has a mouse sequence). In some embodiments, the non-human animals provided herein express TdT encoded by the exogenous nucleic acid during B cell development, for example, in pro-B cells and / or pre-B cells. In some embodiments, the non-human animals provided herein express TdT encoded by the exogenous nucleic acid during T cell development, for example, in double-negative (DN) thymocytes and / or double-positive (DP) thymocytes. In some embodiments, the genetically modified non-human animals contain multiple copies (e.g., at least 2, 3, 4, 5, 6, 7, or 8 copies) of the exogenous nucleic acid encoding TdT. In some embodiments, the genetically modified non-human animal is a mammal such as a rodent (e.g., a mouse or rat).

[0004] In some embodiments, the genetically modified non-human animal includes an immunoglobulin variable region in its genome, which comprises an unreorganized human immunoglobulin variable region gene segment (e.g., heavy chain gene segment, κ chain gene segment, λ chain gene segment) operably linked to an immunoglobulin constant region gene (e.g., heavy chain constant region gene, κ chain constant region gene, λ chain constant region gene). In some embodiments, the constant region gene is a human constant region gene, a mouse constant region gene, or a rat constant region gene. In some embodiments, the constant region gene is of endogenous species origin. In some embodiments, the variable region and constant region gene are located at endogenous immunoglobulin loci (e.g., heavy chain locus, κ locus, λ locus). In some embodiments, the genetically modified non-human organism expresses an antibody comprising a human immunoglobulin variable domain derived from the immunoglobulin variable region and an immunoglobulin constant domain encoded by an immunoglobulin constant region gene. In some embodiments, provided herein are methods for using such a genetically modified non-human animal to produce an antibody, B cells, hybridoma, or nucleic acid encoding a human immunoglobulin variable domain.

[0005] In certain embodiments, the genetically modified non-human animal includes a T cell receptor (TCR) variable region in its genome, which contains an unreorganized human TCR variable region gene segment (e.g., TCRα gene segment, TCRβ gene segment, TCRγ gene segment, TCRδ gene segment) operably linked to TCR constant region genes (e.g., TCRα constant region gene, TCRβ constant region gene, TCRγ constant region gene, TCRδ constant region gene). In some embodiments, the constant region genes are human constant region genes, mouse constant region genes, or rat constant region genes. In some embodiments, the constant region genes are of endogenous species origin. In some embodiments, the variable region and constant region genes are located at endogenous TCR loci (e.g., TCRα locus, TCRβ locus, TCRγ locus, TCRδ locus). In some embodiments, the genetically modified non-human organism expresses a TCR that includes a human TCR variable domain derived from the TCR variable region and a TCR constant domain encoded by the TCR constant region gene. In some embodiments, provided herein are methods for using such genetically modified non-human animals to produce TCRs, T cells, T cell hybridomas, or nucleic acids that encode human TCR variable domains.

[0006] In some embodiments, the genetically modified non-human animal includes an immunoglobulin variable region in its genome, which comprises an unreorganized human immunoglobulin variable region gene segment (e.g., heavy chain gene segment, κ chain gene segment, λ chain gene segment) operably linked to a TCR constant region gene (e.g., TCRα constant region gene, TCRβ constant region gene, TCRγ constant region gene, TCRδ constant region gene). In some embodiments, the constant region gene is a human constant region gene, a mouse constant region gene, or a rat constant region gene. In some embodiments, the constant region gene is of endogenous species origin. In some embodiments, the variable region and constant region gene are located at endogenous TCR loci (e.g., TCRα locus, TCRβ locus, TCRγ locus, TCRδ locus). In some embodiments, the genetically modified non-human organism expresses a chimeric antigen receptor (CAR) which comprises a human immunoglobulin variable domain derived from the immunoglobulin variable region and a TCR constant domain encoded by a TCR constant region gene. In some embodiments, provided herein are methods for using such genetically modified non-human animals to produce CARs, T cells, T cell hybridomas, or nucleic acids encoding human immunoglobulin variable domains.

[0007] In some embodiments, what is provided herein is a method for producing a non-human animal disclosed herein, comprising manipulating a non-human animal to include the genetic modification described herein in its germline. In some embodiments, what is provided herein is a non-human ES cell containing the genetic modification described herein. The present invention provides, for example, the following items: (Item 1) A genetically modified non-human animal whose genome contains: A nucleic acid sequence encoding an exogenous terminal deoxynucleotidyltransferase (TdT) operably linked to a transcriptional regulatory element, A genetically modified non-human animal comprising an immunoglobulin variable region containing an unreorganized human immunoglobulin variable region gene segment operably linked to an immunoglobulin constant region gene. (Item 2) The genetically modified non-human animal according to item 1, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding exogenous TdT in pro-B cells and / or pre-B cells. (Item 3) The genetically modified non-human animal according to item 2, wherein the transcriptional regulatory element is selected from the group consisting of a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, an immunoglobulin heavy chain transcriptional regulatory element, an immunoglobulin κ light chain transcriptional regulatory element, and / or an immunoglobulin λ light chain transcriptional regulatory element. (Item 4) A genetically modified non-human animal according to any one of items 1 to 3, wherein the nucleic acid sequence encoding the exogenous TdT is located at the immunoglobulin κ light chain locus, immunoglobulin λ light chain locus, immunoglobulin heavy chain locus, RAG1 locus, or RAG2 locus. (Item 5) A genetically modified non-human animal according to any one of items 1 to 4, wherein at least 10% of the VJ immunoglobulin light chain links in the animal include non-template addition. (Item 6) A genetically modified non-human animal according to any one of items 1 to 5, wherein the human immunoglobulin variable region gene segment is a human heavy chain variable region gene segment. (Item 7) The genetically modified non-human animal described in item 6, wherein the constant region gene is a heavy chain constant region gene. (Item 8) The genetically modified non-human animal described in item 7, wherein the heavy chain constant region gene is derived from an endogenous species. (Item 9) A genetically modified non-human animal according to any one of items 6 to 8, wherein the immunoglobulin variable region and the immunoglobulin constant region gene are located at the endogenous immunoglobulin heavy chain gene locus. (Item 10) A genetically modified non-human animal according to any one of items 6-9, further comprising an immunoglobulin variable region containing an unreorganized human light chain variable region gene segment operably linked to a second immunoglobulin constant region gene in the genome. (Item 11) The genetically modified non-human animal described in item 10, wherein the immunoglobulin variable region operably linked to the second immunoglobulin constant region gene is located at the endogenous immunoglobulin light chain locus. (Item 12) The genetically modified non-human animal according to item 11, wherein the human immunoglobulin variable region gene segment operably linked to the second immunoglobulin constant region gene is a human κ-chain variable region gene segment. (Item 13) The genetically modified non-human animal according to item 11, wherein the human immunoglobulin variable region gene segment operably linked to the second immunoglobulin constant region gene is a human λ-chain variable region gene segment. (Item 14) A genetically modified non-human animal as described in any one of items 11 to 13, wherein the second constant region gene is a κ constant region gene. (Item 15) A genetically modified non-human animal as described in any one of items 11 to 13, wherein the second constant region gene is a λ constant region gene. (Item 16) A genetically modified non-human animal as described in item 14, wherein the κ constant region gene is derived from an endogenous species. (Item 17) A genetically modified non-human animal as described in item 15, wherein the λ constant region gene is derived from an endogenous species. (Item 18) The genetically modified non-human animal described in item 16, wherein the endogenous immunoglobulin light chain locus is the immunoglobulin κ locus. (Item 19) The genetically modified non-human animal described in item 17, wherein the endogenous immunoglobulin light chain locus is the immunoglobulin λ locus. (Item 20) A genetically modified non-human animal according to any one of items 1 to 19, wherein the unreorganized human immunoglobulin variable region gene segment is rearranged during B cell development to produce the rearranged variable region gene in the B cells of the non-human animal. (Item 21) A genetically modified non-human animal as described in item 20, wherein at least 10% of the rearranged variable region genes include non-template addition. (Item 22) A genetically modified non-human animal whose genome contains: A nucleic acid sequence encoding an exogenous terminal deoxynucleotidyltransferase (TdT) operably linked to a transcriptional regulatory element, A genetically modified non-human animal comprising a T cell receptor (TCR) variable region containing an unreorganized human TCR variable region gene segment operably linked to a TCR constant region gene. (Item 23) The genetically modified non-human animal described in item 22, wherein the human TCR variable region gene segment is the human TCRα variable region gene segment. (Item 24) The genetically modified non-human animal described in item 22, wherein the human TCR variable region gene segment is the human TCRβ variable region gene segment. (Item 25) A genetically modified non-human animal according to any one of items 22 to 24, wherein the unreorganized human TCR variable region gene segment is rearranged during T cell development to produce the rearranged TCR variable region gene in the T cells of the non-human animal. (Item 26) A genetically modified non-human animal as described in item 25, wherein at least 10% of the rearranged variable region genes include non-template addition. (Item 27) A genetically modified non-human animal whose genome contains: A nucleic acid sequence encoding an exogenous terminal deoxynucleotidyltransferase (TdT) operably linked to a transcriptional regulatory element, A genetically modified non-human animal comprising an immunoglobulin variable region containing an unreorganized human immunoglobulin variable region gene segment operably linked to a TCR constant region gene. (Item 28) The genetically modified non-human animal described in item 27, wherein the human immunoglobulin variable region gene segment is a heavy chain gene segment. (Item 29) The genetically modified non-human animal described in item 27, wherein the human immunoglobulin variable region gene segment is a κ chain gene segment. (Item 30) The genetically modified non-human animal described in item 27, wherein the human immunoglobulin variable region gene segment is a λ-chain gene segment. (Item 31) A genetically modified non-human animal as described in item 28 or 29, wherein the TCR constant region gene is a TCRα constant region gene. (Item 32) The genetically modified non-human animal described in item 28, wherein the TCR constant region gene is the TCRβ constant region gene. (Item 33) A genetically modified non-human animal according to any one of items 22-32, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding exogenous TdT in CD4 / CD8 double-negative (DN) thymocytes and / or CD4 / CD8 double-positive (DP) thymocytes. (Item 34) The genetically modified non-human animal according to item 33, wherein the transcriptional regulatory element is the RAG1 transcriptional regulatory element, the RAG2 transcriptional regulatory element, the TCRα transcriptional regulatory element, the TCRβ transcriptional regulatory element, the TCRγ transcriptional regulatory element, and / or the TCRδ transcriptional regulatory element. (Item 35) A genetically modified non-human animal according to any one of items 22 to 34, wherein the nucleic acid sequence encoding the exogenous TdT is located at the RAG1 locus, RAG2 locus, TCRα chain locus, TCRβ chain locus, TCRγ chain locus, and / or TCRδ chain locus. (Item 36) The aforementioned non-human animal is a mammal, and is a genetically modified non-human animal as described in any one of items 1 to 35. (Item 37) The aforementioned mammal is a rodent, a genetically modified non-human animal as described in item 36. (Item 38) The aforementioned rodent is a rat or mouse, a genetically modified non-human animal as described in item 37. (Item 39) A method for inducing the expression of an antibody containing a human variable domain, comprising exposing a genetically modified non-human animal described in any one of items 1 to 21 to an antigen, thereby causing the genetically modified non-human animal to produce an antibody containing a human variable domain specific to the antigen. (Item 40) A method for producing T cells that express a T cell receptor (TCR) containing a human variable domain specific to a peptide presented on MHC, (a) Exposure of a genetically modified non-human animal as described in any one of items 22-26 to an antigen containing a peptide or a nucleic acid encoding an antigen containing a peptide, wherein the peptide is presented on the MHC in the non-human animal; (b) A method comprising obtaining T cells expressing a TCR specific to the peptide presented on the MHC from the genetically modified non-human animal of (a) according to (a). (Item 41) A method for producing T cells that express a chimeric antigen receptor (CAR) specific to a peptide presented on MHC, which includes a human immunoglobulin variable domain and immunoglobulin constant, (a) Exposure of a genetically modified non-human animal as described in any one of items 27-32 to an antigen containing a peptide or a nucleic acid encoding an antigen containing a peptide, wherein the peptide is presented on the MHC in the non-human animal; (b) A method comprising obtaining T cells expressing a CAR specific to the peptide presented on the MHC from the genetically modified non-human animal of (a) in (a). (Item 42) Genetically modified non-human animal ES cells, in which their genome contains: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), Genetically modified non-human animal ES cells comprising an immunoglobulin variable region containing an unreorganized human immunoglobulin variable region gene segment operably linked to an immunoglobulin constant region gene. (Item 43) Genetically modified non-human animal ES cells, in which their genome contains: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), Genetically modified non-human animal ES cells comprising a T cell receptor (TCR) variable region containing an unreorganized human TCR variable region gene segment operably linked to a TCR constant region gene. (Item 44) Genetically modified non-human animal ES cells, in which their genome contains: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), Genetically modified non-human animal ES cells comprising an immunoglobulin variable region containing an unreorganized human immunoglobulin variable region gene segment operably linked to a TCR constant region gene. (Item 45) A method for producing a genetically modified non-human animal, comprising using genetically modified non-human animal ES cells as described in any one of items 42-44. (Item 46) A method for producing a non-human animal that includes genetic modification, wherein the non-human animal has a germline: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), A method comprising manipulating an immunoglobulin variable region to include an immunoglobulin variable region comprising an unreorganized human immunoglobulin variable region gene segment operably linked to an immunoglobulin constant region gene. (Item 47) A method for producing a non-human animal that includes genetic modification, wherein the non-human animal has a germline: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), A method comprising manipulating a T cell receptor (TCR) variable region to include an unreorganized human TCR variable region gene segment operably linked to a TCR constant region gene. (Item 48) A method for producing a non-human animal that includes genetic modification, wherein the non-human animal has a germline: Nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT), A method comprising manipulating an immunoglobulin variable region to include an immunoglobulin variable region comprising an unreorganized human immunoglobulin variable region gene segment operably linked to a TCR constant region gene. (Item 49) A genetically modified non-human animal, non-human animal ES cell, or method according to any one of items 1 to 48, wherein the terminal deoxynucleotidyltransferase (TdT) is human TdT. (Item 50) A genetically modified non-human animal, non-human animal ES cell, or method according to any one of items 1 to 49, wherein the terminal deoxynucleotidyltransferase (TdT) is a short isoform of TdT (TdTS). [Brief explanation of the drawing]

[0008] [Figure 1]A diagram (not to exact scale) of an exemplary targeting vector is shown, with a portion of the mouse Rag2 gene replaced by a DNA sequence encoding a short isoform human TdT (hTdTs). In the exemplary embodiment, the vector is randomly integrated into the genome. Unless otherwise indicated by the labeling in the diagram (e.g., with respect to selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. E1, E2, etc., represent exons of specific exemplary genes, GFP is green fluorescent protein, CM is the chloramphenicol resistance gene, and neo is the neomycin resistance gene. Conjugates 1-4 correspond to the conjugates shown in Table 1.

[0009] [Figure 2] A diagram (not to exact scale) of an exemplary targeting vector in which a portion of the mouse Rag2 gene is replaced with a DNA sequence encoding a short isoform human TdT (hTdTs) is shown. In the illustrated embodiment, the vector is used to insert hTdT, driven by the mouse RAG2 promoter, into the Igκ locus. Unless otherwise indicated by the labeling in the diagram (e.g., with respect to selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. E1, E2, etc., represent exons of specific exemplary genes, GFP is green fluorescent protein, CM is the chloramphenicol resistance gene, and hyg is the hygromycin resistance gene. Conjugates 1-7 correspond to conjugates in Table 2.

[0010] [Figure 3]A diagram (not to exact scale) of an exemplary targeting vector used to insert DNA sequences encoding human TdT (hTdTs) driven by the VH1-72 promoter and Eμ enhancer into the immunoglobulin κ locus is shown. Unless otherwise indicated by the labeling in the diagram (e.g., regarding the selection cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. E1, E2, etc., represent exons of specific exemplary genes, GFP is green fluorescent protein, CM is the chloramphenicol resistance gene, and hyg is the hygromycin resistance gene. Conjugates 1-4 correspond to conjugates in Table 3.

[0011] [Figure 4] This shows the expression of hTdT mRNA in lymphocytes of VELOCIMMUNE® TdT mice compared to VELOCIMMUNE® control mice. VELOCIMMUNE® mice as used herein are mice containing a diverse repertoire of unreorganized human heavy chain and κ-light chain variable (V(D)J) gene segments. Het indicates a heterozygous mouse, and HO indicates a homozygous mouse.

[0012] [Figure 5] This graph shows the hIgκ sequence diversity (number of unique light chain CDR3 sequences per 10,000 hIgκ sequencing reads) in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0013] [Figure 6] This graph shows the distribution of hIgκ non-template addition in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "Het" indicates heterozygous mice, and "HO" indicates homozygous mice. "NT" represents a nucleotide.

[0014] [Figure 7-1] The panel consists of two panels. Panel (A) shows a graph illustrating the distribution of hIgκ CDR3 length in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "AA" represents an amino acid. Panel (B) shows a graph illustrating the frequency of exonuclease deletion length in the 5' region of the JK segment in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice. [Figure 7-2] The panel consists of two panels. Panel (A) shows a graph illustrating the distribution of hIgκ CDR3 length in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "AA" represents an amino acid. Panel (B) shows a graph illustrating the frequency of exonuclease deletion length in the 5' region of the JK segment in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0015] [Figure 8-1] The panel consists of two panels. Panel (A) shows a graph illustrating Vκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Panel (B) shows a graph illustrating Jκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice. [Figure 8-2]The panel consists of two panels. Panel (A) shows a graph illustrating Vκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Panel (B) shows a graph illustrating Jκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0016] [Figure 9] This graph shows the mIgλ sequence diversity (number of unique light chain CDR3 sequences per 10,000 Igλ sequencing reads) in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0017] [Figure 10] This graph shows the distribution of mIgλ non-template addition in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "Het" indicates heterozygous mice, and "HO" indicates homozygous mice. "NT" represents a nucleotide.

[0018] [Figure 11] This graph shows the distribution of mIgλ CDR3 length in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "Het" indicates heterozygous mice, and "HO" indicates homozygous mice. "AA" represents an amino acid.

[0019] [Figure 12] This graph shows Vλ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0020] [Figure 13] The graph shows the hIgκ sequence diversity (number of unique light chain CDR3 sequences per 10,000 Igκ sequencing reads) in double light chain mice (DLC, mice containing two unreorganized human Vk gene segments and five unreorganized human Jk gene segments, as well as a diverse repertoire of unreorganized human heavy chain V, D, and J gene segments) expressing hTdT (left panel, hTdT gene present as shown) compared with VELOCIMMUNE® mice expressing hTdT (left panel, hTdT gene present as shown) and DLC and VELOCIMMUNE® control mice that do not express hTdT. Het indicates heterozygous mice for hTdT, and HO indicates homozygous mice for hTdT.

[0021] [Figure 14] This graph shows the distribution of hIgκ non-template addition in mice expressing hTdT compared to DLC control mice (DLC) that do not express hTdT. "Het" indicates heterozygous hTdT mice, and "HO" indicates homozygous hTdT mice. "NT" represents a nucleotide.

[0022] [Figure 15] This graph shows the distribution of hIgκ CDR3 length in DLC mice expressing hTdT compared to DLC control mice that do not express hTdT. Het represents heterozygous mice expressing hTdT, and HO represents homozygous mice expressing hTdT.

[0023] [Figure 16] The graphs show Vκ and Jκ usage in DLC mice expressing hTdT compared to DLC control mice that do not express hTdT. "Het" indicates heterozygous hTdT mice, and "HO" indicates homozygous hTdT mice. Only two distinct Rag TdT tg(HO)DLC mice, shown separately, are used. [Modes for carrying out the invention]

[0024] general This specification provides methods and compositions relating to non-human animals that contain exogenous nucleic acids encoding TdT (e.g., human, mouse, or rat TdT) in their genomes. In some embodiments, the genetically modified non-human animals are mammals such as rodents (e.g., mice or rats). In certain embodiments, the genome of the non-human animal includes further modifications to express antigen-binding molecules (e.g., antibodies, TCRs, and / or CARs) having human variable domains.

[0025] TdT is a DNA polymerase that catalyzes template-independent nucleotide addition (N addition) during conjugation in V(D)J recombination, resulting in increased antigen-receptor diversity in B lymphocytes and T lymphocytes. In some embodiments, non-human animals provided herein express increased levels of TdT during B cell development and / or T cell development compared to corresponding non-human animals (i.e., non-human animals of the same species and strain) that do not have the exogenous nucleic acid encoding TdT in their genome. In some embodiments, non-human animals provided herein express TdT during B cell development and / or T cell development (e.g., during the pre-B cell stage), where corresponding non-human animals that do not have the exogenous nucleic acid encoding TdT in their genome do not express TdT. In some embodiments, the genetically modified non-human animals described herein have increased antigen-receptor diversity (e.g., antibody diversity, TCR diversity, and / or CAR diversity) compared to corresponding non-human animals that do not have the exogenous nucleic acid encoding TdT in their genome.

[0026] definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "a certain element" means one or more elements.

[0027] The term "amino acid" is intended to encompass any natural or synthetic molecule that contains both amino and acidic functional groups and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids, their analogs, derivatives, and homologs, amino acid analogs with variant side chains, and all stereoisomers of any of the above.

[0028] As used herein, the term “antibody” may refer to both an intact antibody and its antigen-binding fragment. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (V in this specification). H Each light chain includes a light chain variable region (V in this specification). L (Abbreviated as ) and includes the light chain constant region. V H and V L The region can be further subdivided into hyper-variable regions called complementary determination regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "antibody" also includes single-domain antibodies, antibodies consisting only of a heavy chain, and antibodies having a light chain variable gene segment on the heavy chain.

[0029] As used herein, the terms “antigen-binding fragment” and “antigen-binding portion” refer to one or more fragments of an antigen-binding molecule (e.g., an antibody, a T-cell receptor (TCR), or a chimeric antigen receptor (CAR)) that retain the ability to bind to an antigen. An antigen-binding fragment may include any antibody, TCR, or CAR fragment that retains at least a portion of the variable region of an intact antigen-binding molecule and is capable of binding to an antigen. Examples of binding fragments encompassed within the term “antigen-binding fragment” include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-bound Fv, Fd, single-chain antibodies, soluble TCRs, single-chain TCRs, soluble CARs, single-chain CARs, isolated CDRH3 (antibody or TCR), and other antigen-binding fragments that retain at least a portion of the variable region of an intact antigen-binding molecule. These antigen-binding fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0030] The term “corresponding” in relation to non-human animals is used to describe a control non-human animal of the same species that contains the same genetic modification as the non-human animal of interest, except that the non-human animal of interest expresses exogenous TdT, while the corresponding non-human animal does not.

[0031] As used herein, “chimeric antigen receptor” or “CAR” refers to an antigen-binding protein that contains an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain or a portion thereof. As used herein, the “constant domain” of a TCR polypeptide includes the membrane-proximal TCR constant domain and may also include the TCR transmembrane domain and / or the TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer comprising a first polypeptide containing an immunoglobulin heavy chain variable domain linked to the TCRβ constant domain and a second polypeptide containing an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to the TCRα constant domain.

[0032] When used in reference to a rearranged variable region gene or variable domain that "derives" from an unrearranged variable region and / or an unrearranged variable region gene segment, the phrase "derives from" refers to the ability to trace the sequence of the rearranged variable region gene or variable domain back to the set of unrearranged variable region gene segments that were rearranged to form the rearranged variable region gene that expresses the variable domain (taking into account splice differences and somatic mutations, where applicable). For example, a rearranged variable region gene that has undergone a somatic mutation does not change the fact that it originates from an unrearranged variable region gene segment.

[0033] As used herein, the term “locus” refers to a region on a chromosome that contains a set of associated gene elements (e.g., genes, gene segments, regulatory elements). For example, an unreorganized immunoglobulin locus may include an immunoglobulin variable region gene segment that induces V(D)J recombination and immunoglobulin expression, one or more immunoglobulin constant region genes, and associated regulatory elements (e.g., promoters, enhancers, switch elements), while an unreorganized TCR locus may include a TCR variable region gene segment that induces V(D)J recombination and TCR expression, a TCR constant region gene, and associated regulatory elements (e.g., promoters, enhancers). Similarly, an unreorganized CAR locus may include an immunoglobulin variable region gene segment that induces V(D)J recombination and CAR expression, a TCR constant region gene, and associated regulatory elements (e.g., promoters, enhancers). Loci can be endogenous or non-endogenous. The term “endogenous locus” refers to a location on a chromosome where a particular gene element is found naturally.

[0034] An unreorganized variable region gene segment is "operably ligated" to an adjacent constant region gene if it can be rearranged to form a rearranged variable region gene that is expressed together with a constant region gene as a polypeptide chain of an antigen-binding protein.

[0035] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to nucleotides in polymeric form of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function. Coding or non-coding regions of genes or gene fragments, loci (plural) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers are non-limiting examples of polynucleotides. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure can be imparted either before or after assembly of the polymer. A polynucleotide can be further modified, for example, by conjugation to a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0036] As used herein, "specific binding" and "antigen specificity" refer to the ability of an antigen-binding molecule (e.g., an antibody, TCR, CAR) to bind to a given target, e.g., a given antigen. Typically, an antigen-binding molecule specifically binds to its given target with an affinity corresponding to a K -7 of about 10 D M or less and binds to its given target with an affinity at least 10-fold lower, at least 100-fold lower, or at least 1000-fold lower than its K D for non-specific and irrelevant targets (e.g., BSA, casein). In some embodiments, an antigen-binding molecule specifically binds to its given target with an affinity corresponding to a K D of about 10 -8 M or less, 10 -9 M or less, or 10 -10 M or less. D

[0037] As used herein, “T cell receptor” or “TCR” refers to an antigen-binding protein that contains both a TCR antigen-binding domain (e.g., a TCR variable domain) and at least a portion of a TCR constant domain. As used herein, the “constant domain” of a TCR polypeptide includes a membrane-proximal TCR constant domain and may also include a TCR transmembrane domain and / or a TCR cytoplasmic tail. In certain embodiments, the TCR is a soluble TCR and does not include a TCR transmembrane domain or a TCR cytoplasmic tail. For example, in some embodiments, the TCR is a dimer comprising a first polypeptide containing a TCRβ variable domain linked to a TCRβ constant domain (or a fragment thereof) and a second polypeptide containing TCRα linked to a TCRα constant domain (or a fragment thereof).

[0038] The term “unreorganized” includes states of immunoglobulin, TCR, or CAR variable region loci or variable region gene segments in which the V gene segment and J gene segment (and, for the duplicate or TCRβ variable region, the D gene segment as well) are maintained separately but can be linked to form a rearranged V(D)J gene ("variable region gene") containing a single V, (D), J of the V(D)J repertoire.

[0039] Genetically modified non-human animals and ES cells In certain embodiments, the Specified Provision provides non-human animals and ES cells having exogenous nucleic acids encoding TdT (e.g., human, mouse, or rat TdT) in their genomes. In certain embodiments, the genomes of non-human animals and ES cells include further modifications, such as modifications resulting in the expression of antigen-binding molecules (e.g., antibodies, TCRs, and / or CARs) having human variable domains.

[0040] The genetically modified non-human animals and ES cells provided herein can be prepared using any suitable method known in the art. For example, non-human animal ES cells containing targeted genetic modifications can be prepared using the VELOCIGENE® technology described in U.S. Patents 6,586,251, 6,596,541, 7,105,348, and Valenzuela et al. (2003) “High-throughput engineering of the mouse genome coupled with high-resolution expression analysis” Nat. Biotech. 21(6):652-659, each of which is incorporated herein by reference, as well as in U.S. Patent Application Publication No. 2014 / 0310828. Targeted modifications can also be made, for example, using the CRISPR / Cas systems described in U.S. Patent No. 9,228,208, and U.S. Patent Publications 2015 / 0159174A1, 2016 / 0060657A1, 2015 / 0376650A1, 2015 / 0376651A1, 2016 / 0046960A1, 2015 / 0376628A1, and 2016 / 0115486A1, each of which is incorporated herein by reference. Targeted modifications can also be made, for example, using the meganucleases described in U.S. Patent Nos. 8,703,485, 8,530,214, and 8,624,000, each of which is incorporated herein by reference in their entirety. Non-targeted gene modifications can be carried out, for example, using the standard methods described in U.S. Patents 6,150,584, 6,114,598, 5,633,425, 7,501,552, 6,235,883, 6,998,514, and 5,776,773, each of which is incorporated herein by reference in whole.

[0041] Using the ES cells described herein, non-human animals can be produced using methods known in the art. For example, the mouse non-human animal ES cells described herein can be used to produce genetically modified mice using VELOCIMOUSE®, as described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each incorporated herein by reference. Using rat ES cells, genetically modified rats can be produced, for example, using the method described in U.S. Patent Application Publication No. 2014 / 0310828, incorporated herein by reference. The resulting mice or rats can be bred into homozygotes. Multiple different modifications can be combined in a single genetically modified organism, either by breeding separately modified animals or by introducing further modifications into already modified ES cells (for example, using the methods described herein).

[0042] In some embodiments, the non-human animal can be any non-human animal. In some embodiments, the non-human animal is a vertebrate. In some embodiments, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animals described herein may be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals in which suitable genetically modifiable ES cells are not readily available, non-human animals containing the genetic modifications described herein can be produced by other methods. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblasts or induced pluripotent cells), transferring the modified genome into a suitable cell such as an oocyte using nuclear transfer, and fertilizing the modified cell (e.g., modified oocyte) under conditions suitable for embryo formation in a non-human animal.

[0043] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is, for example, a small mammal of the superfamily Dipodoidea or Muroidea. In some embodiments, the non-human animal is a rodent. In certain embodiments, the rodent is a mouse, rat, or hamster. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the non-human animal is derived from a family selected from Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rat and mouse, field vole), Muridae (e.g., true mouse and rat, gerbil, spiny mouse, crested rat), Nesomyidae (e.g., climbing mouse, rock mouse, white-tailed rat, Madagascar rat and mouse), Platacanthomyidae (e.g., spiny mouse), and Spalacidae (e.g., mole rat, bamboo rat, and zocol). In some embodiments, rodents are selected from true mice and rats (Muridae family), gerbils, spiny mice, and crested rats. In some embodiments, mice are derived from members of the Muridae family. In some embodiments, non-human animals are rodents. In some embodiments, rodents are selected from mice and rats. In some embodiments, non-human animals are mice.

[0044] In some embodiments, the non-human animal is a mouse of the C57BL strain. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the non-human animal is a mouse of the 129 strain. In some embodiments, strain 129 is selected from the group consisting of strains 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. In some embodiments, the genetically modified mouse is a mixture of strain 129 and C57BL strain. In some embodiments, the mouse is a mixture of strain 129 and / or C57BL / 6 strain. In some embodiments, the strain 129 in the mixture is strain 129S6 (129 / SvEvTac). In some embodiments, the mouse is strain BALB (e.g., BALB / c). In some embodiments, the mouse is a mixture of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the non-human animal provided herein may be a mouse derived from any combination of the aforementioned strains.

[0045] In some embodiments, the non-human animals provided herein are rats. In some embodiments, the rats are selected from Wistar rats, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strains are Wistar, LEA, Sprague It is a mixture of two or more strains selected from the group consisting of Dawley, Fischer, F344, F6, and Dark Agouti.

[0046] Non-human animals that express exogenous TdT In certain embodiments, genetically modified non-human animals and ES cells containing nucleic acid sequences encoding exogenous terminal deoxynucleotidyltransferase (TdT) in their germline and / or genome are provided herein. Deoxynucleotidyltransferase (TdT) is a DNA polymerase that catalyzes template-independent nucleotide addition (NP addition) during conjugation in V(D)J recombination, resulting in increased antigen-receptor diversity in B lymphocytes and T lymphocytes. Template-independent addition, non-template addition, and non-germline addition all refer to nucleotide addition catalyzed by TdT, and these terms are used interchangeably herein.

[0047] In certain embodiments, the exogenous TdT sequence in the genome of a genetically modified non-human animal may originate from any animal encoding TdT or a TdT orthologue. In some embodiments, TdT is a vertebrate TdT. In some embodiments, TdT is a mammalian TdT. In some embodiments, TdT originates from a mammal selected from the group consisting of mouse, rat, rabbit, pig, cattle (e.g., cow, bull, buffalo), deer, sheep, goat, llama, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), or human. In some embodiments, TdT is of endogenous species origin (i.e., the TdT sequence is of the same species as the genetically modified non-human animal). In some embodiments, TdT is human TdT, mouse TdT, or rat TdT. In some embodiments, the nucleic acid sequence is a genomic TdT sequence (i.e., including exons and introns). In some embodiments, the nucleic acid sequence encodes TdT mRNA / cDNA (i.e., exons of one or more TdT isoforms).

[0048] Human TdT (hTdT) is encoded by the DNTT gene located on human chromosome 10. An exemplary genomic DNA sequence of hTdT can be found at NCBI accession number NC_000010.11, positions 96304328–96338564, which is incorporated herein by reference. Exemplary mRNA sequences of hTdT isoforms are provided by NCBI accession numbers NM_001017520.1 and NM_004088.3, ​​each of which is incorporated herein by reference. The protein sequences encoded by these isoforms are provided by NCBI accession numbers NP_001017520.1 and NP_004079.3, each of which is incorporated herein by reference. Among the TdT isoforms are a short isoform (hTdTS) and two long isoforms (hTdTL1 and hTdTL2). The sequences of the three isoforms are provided, for example, in Thai and Kearney, Adv. Immunol. 86:113-36 (2005), which are incorporated herein by reference. In certain embodiments, the exogenous nucleic acid sequence encodes hTdTS. In some embodiments, the exogenous nucleic acid sequence encodes hTdTL1. In some embodiments, the exogenous nucleic acid sequence encodes hTdTL2. In certain embodiments, the non-human organism includes an exogenous nucleic acid sequence encoding multiple isoforms (e.g., both hTdTS and hTdTL2). In certain embodiments, the non-human organism includes an exogenous nucleic acid sequence encoding all three human isoforms (e.g., both hTdTS and hTdTL2).

[0049] Mouse TdT (mTdT) is encoded by the Dntt gene located on mouse chromosome 19. An exemplary genomic DNA sequence of mTdT can be found at NCBI accession number NC_000085.6, positions 41029275–41059525, which is incorporated herein by reference. Exemplary mRNA sequences of isoforms of mTdT are provided by NCBI accession numbers NM_001043228.1 and NM_009345.2, each of which is incorporated herein by reference. Protein sequences encoded by these isoforms are provided by NCBI accession numbers NP_001036693.1 and NP_033371.2, each of which is incorporated herein by reference.

[0050] Rat TdT (rTdT) is encoded by the Dntt gene located on rat chromosome 1. An exemplary genomic DNA sequence of rTdT can be found at NCBI accession number NC_005100.4, positions 260289626–260321174, which is incorporated herein by reference. An exemplary mRNA sequence of rTdT is provided by NCBI accession number NM_001012461.1, which is incorporated herein by reference. The protein sequence encoded by this mRNA is provided by NCBI accession number NP_001012479.1, which is incorporated herein by reference.

[0051] In some embodiments, the genome of a genetically modified non-human animal includes multiple copies of the nucleic acid sequence encoding exogenous TdT. In some embodiments, the genetically modified non-human animal includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies of the nucleic acid sequence encoding exogenous TdT. In some embodiments, the genetically modified non-human animal includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies of the nucleic acid sequence encoding exogenous TdT.

[0052] In some embodiments, the nucleic acid sequence encoding exogenous TdT is operably ligated to one or more transcriptional regulatory elements (e.g., promoters and / or enhancers). In some embodiments, the transcriptional regulatory element is a constitutive (i.e., ubiquitous) promoter. Examples of constitutive promoters, but not limited to these, include SV40, CMV promoter, adenovirus promoter, EF1 promoter, β-actin promoter, EGR1 promoter, eIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, GRP78 promoter, GRP94 promoter, HSP70 promoter, β-Kin promoter, PGK-1 promoter, ROSA promoter, and ubiquitin B promoter. In some embodiments, the nucleic acid sequence is not operably ligated to a constitutive promoter.

[0053] In some embodiments, the transcriptional regulatory element induces the expression of TdT encoded during B cell development. In some embodiments, the transcriptional regulatory element induces the expression of TdT in pro-B cells and / or pre-B cells. In some embodiments, the transcriptional regulatory element is a transcriptional regulatory element (e.g., promoter and / or enhancer) of a gene expressed during B cell development in pro-B cells and / or pre-B cells. In some embodiments, the transcriptional regulatory element is a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, an immunoglobulin heavy chain transcriptional regulatory element, an immunoglobulin κ light chain transcriptional regulatory element, and / or an immunoglobulin λ light chain transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is of endogenous species origin. In some embodiments, the transcriptional regulatory element is a mouse transcriptional regulatory element, a rat transcriptional regulatory element, or a human transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is an endogenous transcriptional regulatory element (for example, a nucleotide sequence encoding exogenous TdT is inserted into the genome of a non-human animal at a position such that the expression of exogenous TdT is at least partially controlled by the endogenous transcriptional regulatory element). In some embodiments, the transcriptional regulatory element is one that modulates the transcription of RAG1, RAG2, λ5, VpreB, CD34, CD45, AA4.1, CD45R, IL-7R, MHC class II, CD10, CD19, CD38, CD20, CD40, various immunoglobulin light chain and heavy chain V gene segment promoters and enhancers (see, for example, the list of various V gene segments enumerated on the International Immunogenetics Information System® website, IMGT, imgt.org, e.g., mouse V H Examples include promoters and other elements (1-72). Transcriptional regulatory elements may include those derived from humans, mice, rats, or other species.

[0054] In some embodiments, the transcriptional regulatory element induces the expression of TdT encoded during T cell development. In some embodiments, the transcriptional regulatory element induces the expression of TdT in CD4 / CD8 double-negative (DN) thymocytes and / or CD4 / CD8 double-positive (DP) thymocytes. In some embodiments, the transcriptional regulatory element is a transcriptional regulatory element (e.g., promoter and / or enhancer) of a gene expressed during T cell development in DN thymocytes and / or DP thymocytes. In some embodiments, the transcriptional regulatory element is the RAG1 transcriptional regulatory element, the RAG2 transcriptional regulatory element, the TCRα transcriptional regulatory element, the TCRβ transcriptional regulatory element, the TCRγ transcriptional regulatory element, and / or the TCRδ transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is of endogenous species origin. In some embodiments, the transcriptional regulatory element is a mouse transcriptional regulatory element, a rat transcriptional regulatory element, or a human transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is an endogenous transcriptional regulatory element (for example, a nucleotide sequence encoding exogenous TdT is inserted into the genome of a non-human animal at a position such that the expression of exogenous TdT is at least partially controlled by the endogenous transcriptional regulatory element). In some embodiments, the transcriptional regulatory elements may include those that regulate the transcription of RAG1, RAG2, Lck, ZAP-70, CD34, CD2, HSA, CD44, CD25, PTα, CD4, CD8, CD69, and various TCRα, TCRβ, TCRδ, and TCRγV gene segment promoters and enhancers (for example, see the list of various V gene segments enumerated on the International Immunogenetics Information System® website, IMGT, imgt.org). The transcriptional regulatory elements may be derived from humans, mice, rats, or other species.

[0055] In some embodiments, the nucleic acid encoding TdT is located in the genome of a non-human animal at a genomic locus or its proximal location (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kb) of a gene expressed during B cell development in pro-B cells and / or pre-B cells. In some embodiments, the nucleic acid sequence encoding TdT is located at the immunoglobulin κ light chain locus, immunoglobulin λ light chain locus, immunoglobulin heavy chain locus, RAG1 locus, or RAG2 locus or its proximal location.

[0056] In some embodiments, the nucleic acid encoding TdT is located in the genome of a non-human animal at or near a genomic locus of a gene expressed during T cell development in DN thymocytes and / or DP thymocytes (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kb). In some embodiments, the nucleic acid sequence encoding TdT is located at or near the TCRα, TCRβ, TCRγ, TCRδ, RAG1, or RAG2 locus.

[0057] In some embodiments, the non-human animals provided herein express elevated levels of TdT expression during one or more stages of T cell and / or B cell development (e.g., in pro-B cells, pre-B cells, DN thymocytes, and / or DP thymocytes) compared to the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the genetically modified non-human animals provided herein express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% more TdT during one or more stages of T cell and / or B cell expression than the corresponding non-human animals.

[0058] In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin κ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin κ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugations, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions than corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin κ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin κ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1700 unique immunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chain CDR3 sequences.

[0059] In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin λ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin λ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain junctions, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin λ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin λ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 unique immunoglobulin λ chain CDR3 sequences per 10,000 immunoglobulin λ chain CDR3 sequences.

[0060] In some embodiments, the non-human animals provided herein have a higher proportion of VD immunoglobulin heavy chain conjugations containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD immunoglobulin heavy chain conjugations containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain conjugations that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD immunoglobulin heavy chain conjugations in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VD immunoglobulin heavy chain conjugations containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD immunoglobulin heavy chain conjugations containing at least one N addition in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VD immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD immunoglobulin heavy chain conjugations, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VD immunoglobulin heavy chain conjugations containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD immunoglobulin heavy chain conjugations containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD immunoglobulin heavy chain conjugations containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD immunoglobulin heavy chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD immunoglobulin heavy chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD immunoglobulin heavy chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin heavy chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.

[0061] In some embodiments, the non-human animals provided herein have a higher proportion of DJ immunoglobulin heavy chain conjugations containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugations containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of DJ immunoglobulin heavy chain conjugations that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugates without non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of DJ immunoglobulin heavy chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ immunoglobulin heavy chain conjugations, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugations containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of DJ immunoglobulin heavy chain conjugations containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugations containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ immunoglobulin heavy chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ immunoglobulin heavy chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the DJ immunoglobulin heavy chain conjugates in the animals contain non-template additions.

[0062] In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ TCRα chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ TCRα chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ TCRα chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα chain conjugates containing at least four N additions in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ TCRα chain conjugates in animals include non-template additions. In some embodiments, non-human animals have a frequency of unique TCRα CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animals.

[0063] In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRβ chain conjugates including non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRβ chain conjugates including non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCRβ chain conjugates without non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD TCRβ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCRβ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRβ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VD TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRβ chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRβ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRβ chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRβ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRβ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRβ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRβ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is greater than the proportion of VD in the corresponding non-human animals. The proportion of unique TCRβ CDR3 sequences is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of TCRβ chain linkages. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of VD TCRβ chain linkages in animals include non-template additions. In some embodiments, non-human animals have a frequency of unique TCRβ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% greater than that of the corresponding non-human animals.

[0064] In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRβ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRβ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of DJ TCRβ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free DJ TCRβ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of DJ TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of DJ TCRβ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRβ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of DJ TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRβ chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRβ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRβ chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRβ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRβ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRβ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRβ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRβ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is greater than the proportion of DJ in the corresponding non-human animals. The proportion of DJ TCRβ chain joinings is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of TCRβ chain joinings. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of DJ TCRβ chain joinings in animals include non-template additions.

[0065] In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ TCRγ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ TCRγ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ TCRγ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ TCRγ chain conjugates in the animals include non-template additions. In some embodiments, non-human animals have a frequency of unique TCRγ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animals.

[0066] In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRδ chain conjugates including non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRδ chain conjugates including non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCRδ chain conjugates without non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD TCRδ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCRδ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRδ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRδ chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRδ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRδ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRδ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCRδ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD TCRδ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD TCRδ chain conjugates in the animals include non-template additions. In some embodiments, non-human animals have a frequency of unique TCRδ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animals.

[0067] In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRδ chain conjugates including non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRδ chain conjugates including non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of DJ TCRδ chain conjugates without non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free DJ TCRδ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of DJ TCRδ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRδ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRδ chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRδ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRδ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRδ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of DJ TCRδ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of DJ TCRδ chain conjugates containing at least four N additions in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of DJ TCRδ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the DJ TCRδ chain conjugates in animals include non-template additions.

[0068] In some embodiments, the endogenous TdT locus in non-human organisms is intact. In some embodiments, the endogenous TdT locus is inactivated. For example, in some embodiments, the endogenous TdT locus is completely or partially deleted so that the non-human organism does not express endogenous TdT.

[0069] Non-human animals expressing human variable domain antibodies and exogenous TdT In certain embodiments, genetically modified non-human animals and non-human animal ES cells containing exogenous TdT as described herein also include immunoglobulin loci (exogenous or endogenous) in their germline and / or genome, which include an immunoglobulin variable region comprising an unreorganized human immunoglobulin variable region gene segment and an immunoglobulin constant region comprising an immunoglobulin constant region gene, the unreorganized human immunoglobulin variable region gene segment being operably linked to the immunoglobulin constant region gene. In some embodiments, non-human animals and non-human ES cells include multiple such immunoglobulin loci in their germline and / or genome. For example, in some embodiments, genetically modified non-human animals and non-human animal ES cells include at least one immunoglobulin locus in their germline and / or genome, which includes at least one immunoglobulin locus comprising an unreorganized human heavy chain variable region gene segment and at least one immunoglobulin locus comprising an unreorganized human light chain variable region gene segment (e.g., κ chain gene segment and / or λ chain gene segment). In some embodiments, genetically modified non-human animals and non-human animal ES cells include in their germline and / or genome at least one immunoglobulin locus containing an unreorganized human heavy chain variable region gene segment, at least one immunoglobulin locus containing an unreorganized human κ chain variable region gene segment, and at least one immunoglobulin locus containing an unreorganized human λ chain variable region gene segment. In some embodiments, genetically modified non-human animals, e.g., genetically modified mice or rats, include in their germline and / or genome genetically modified immunoglobulin locus (genetically modified rearranged or unreorganized immunoglobulin locus) so that the mouse can produce human, humanized, partially human, and reverse chimeric (human variable region and non-human constant region) antibodies.

[0070] Immunoglobulin loci containing human variable region gene segments are known in the art, for example, U.S. Patents 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, and 8,5 These can be seen in U.S. Patent Publications 02,018, 8,697,940, 8,703,485, 8,754,287, 8,791,323, 8,907,157, 9,035,128, 9,145,588, and 9,206,263, each of which is incorporated herein by reference in its entirety, and also in U.S. Patent Publications 2008 / 0098490 and 2010 / 0146 The entirety of these can be seen in issues 647, 2011 / 0195454, 2012 / 0167237, 2013 / 0145484, 2013 / 0167256, 2013 / 0219535, 2013 / 0326647, 2013 / 0096287, 2014 / 013275, 2014 / 093908, and 2015 / 0113668, each of which is the whole of these. These are incorporated herein by reference and can also be found in PCT Patent Application Publications WO2007 / 117410, WO2008 / 151081, WO2009 / 157771, WO2010 / 039900, WO2011 / 004192, WO2011 / 123708, and WO2014 / 093908, each of which is incorporated herein by reference in its entirety.

[0071] In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a heavy chain gene segment, and the immunoglobulin constant region gene is a heavy chain constant region gene. In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a light chain, e.g., a κ chain gene segment, and the immunoglobulin constant region gene is a heavy chain constant region gene.

[0072] In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a heavy chain gene segment, and the immunoglobulin constant region gene is a κ chain constant region gene. In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a κ chain gene segment, and the immunoglobulin constant region gene is a κ chain constant region gene. In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a λ chain gene segment, and the immunoglobulin constant region gene is a κ chain constant region gene. In some embodiments, the human unreorganized immunoglobulin variable region gene segment is a λ chain gene segment, and the immunoglobulin constant region gene is a λ chain constant region gene.

[0073] In certain embodiments, the immunoglobulin variable region includes an unreorganized human Ig heavy chain variable region gene segment. In some embodiments, the unreorganized human Ig variable region gene segment includes multiple human V H Segment, one or more human D H Segments, and one or more human J H The segment includes. In some embodiments, the unreorganized human Ig variable region gene segment contains at least three V H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H Gene segment, or at least 80 V H Includes a gene segment. In some embodiments, the unreorganized human Ig gene segment is human D H Includes all gene segments. In some embodiments, the unreorganized human Ig gene segment is human J HIncludes all gene segments. An example variable region including the Ig heavy chain gene segment is, for example, Macdonald et al., Proc. Natl. Acad. Sci. USA. This is described in 111:5147-52 and the supplementary information, which is incorporated herein by reference. In some embodiments, the non-human animals provided herein are (for example, as described in U.S. Patent Application Publication No. 2013 / 0096287, which is incorporated herein by reference) a single polymorphic human V. H gene segment, multiple D H gene segment, and multiple J H It has a restriction immunoglobulin heavy chain locus characterized by a gene segment. In some embodiments, V H The gene segment is either VH1-2 or VH1-69.

[0074] In various embodiments, the immunoglobulin locus modifications described herein do not affect the reproductive capacity of non-human animals. In some embodiments, the heavy chain locus includes the endogenous Adam6a gene, the Adam6b gene, or both, and the gene modification does not affect the expression and / or function of the endogenous Adam6a gene, the Adam6b gene, or both. In some embodiments, the genome of the genetically modified non-human animal includes the ectopically located Adam6a gene, the Adam6b gene, or both. Exemplary non-human animals expressing exogenous Adam6a and / or Adam6b are described in U.S. Patents 8,642,835 and 8,697,940, each of which is incorporated herein by reference in whole.

[0075] In some embodiments, the human immunoglobulin heavy chain variable region gene segment is rearranged during B cell development to produce the rearranged human heavy chain variable region gene in the B cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain conjugations without non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of non-template addition-free VD and / or DJ immunoglobulin heavy chain conjugations in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least one N addition, which is greater than that of the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ immunoglobulin heavy chain conjugates in the animals contain non-template additions.In some embodiments, the non-human animal has a frequency of unique immunoglobulin heavy chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.

[0076] In certain embodiments, the immunoglobulin variable region includes an unreorganized human Igκ variable region gene segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes multiple human V κ Segments and one or more human J κ The segment includes. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes all of the human Jκ segment. In some embodiments, the immunoglobulin variable region gene segment includes four functional V κ Segment and all human J κ The segment includes. In some embodiments, the immunoglobulin variable region gene segment contains 16 functional V κ Segment and all human J κ segments (e.g., all functional human Vκ segments and J) κ (segment) included. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes all human Vκ segments and all human J κ This includes segments. Exemplary variable regions including Igκ gene segments are described, for example, Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and Supplementary Information, which are incorporated herein by reference. In some embodiments, the non-human animals provided herein are two or fewer human V L Gene segments and multiple J LHaving a restriction immunoglobulin light chain locus characterized by a gene segment (e.g., the double light chain mouse described in U.S. Patent Application Publication No. 2013 / 0198880, incorporated herein by reference, or DLC). In some embodiments, V L The gene segment is V κ It is a gene segment. In some embodiments, V L The gene segment is V λ It is a gene segment. In some embodiments, V κ The gene segments are IGKV3-20 and IGKV1-39.

[0077] In some embodiments, the human immunoglobulin κ variable region gene segment is rearranged during B cell development to produce the rearranged human κ variable region gene in the B cells of non-human organisms. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin κ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin κ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugations, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions than corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin κ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin κ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1700 unique immunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chain CDR3 sequences.

[0078] In certain embodiments, the immunoglobulin variable region includes an unreorganized human Igλ variable region gene segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes multiple human V λ Segments and one or more human J λ Includes a segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment is all human V λ Includes a segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment is human J λ This includes all segments. Exemplary variable regions containing Igλ gene segments are provided, for example, in U.S. Patent Application Publications 2012 / 0073004 and 2002 / 0088016, each of which is incorporated herein by reference.

[0079] In some embodiments, the human immunoglobulin λ variable region gene segment is rearranged during B cell development to produce the rearranged human λ variable region gene in the B cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin λ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin λ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain junctions, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin λ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin λ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 unique immunoglobulin λ chain CDR3 sequences per 10,000 immunoglobulin λ chain CDR3 sequences.

[0080] In some embodiments, the immunoglobulin variable region, including an unreorganized human immunoglobulin variable region gene segment, also includes a human immunoglobulin variable region intergene sequence. In some embodiments, the immunoglobulin variable region includes a non-human (e.g., rodent, rat, mouse) Ig variable region intergene sequence. In some embodiments, the intergene sequence is of endogenous species origin.

[0081] In some embodiments, non-human organisms include immunoglobulin loci in their germline and / or genome that contain a rearranged heavy chain variable region (universal heavy chain variable region). In some embodiments, the rearranged Ig heavy chain variable region gene is a human rearranged Ig heavy chain variable region gene. An exemplary rearranged Ig heavy chain variable region is provided in U.S. Patent Application Publication 2014 / 0245468, which is incorporated herein by reference. In some embodiments, non-human organisms containing a universal heavy chain variable region are used to produce bispecific antibodies.

[0082] In some embodiments, non-human organisms include immunoglobulin loci in their germline and / or genome that contain a rearranged light chain variable region (universal light chain variable region). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. Exemplary rearranged Ig light chain variable regions are provided, for example, in U.S. Patent Applications Publications 2011 / 0195454, 2012 / 0021409, 2012 / 0192300, 2013 / 0045492, 2013 / 0185821, 2013 / 0302836, 2015 / 0313193, 2015 / 0059009, and 2013 / 0198879, which are incorporated herein by reference. In some embodiments, non-human organisms containing a universal light chain variable region ("universal light chain" organisms) are used to produce bispecific antibodies.

[0083] In some embodiments, non-human organisms include a light chain immunoglobulin locus in their germline and / or genome that contains a limited repertoire of light chain variable gene segments (e.g., a double light chain variable region containing two light chain variable gene segments). In some embodiments, the light chain variable gene segment in the limited repertoire of light chain gene segments is a human light chain gene segment. An exemplary double light chain variable region is provided in U.S. Patent Application Publication 2013 / 0198880, which is incorporated herein by reference. In some embodiments, non-human organisms containing a double light chain variable region are used to produce bispecific antibodies.

[0084] In yet another embodiment, a non-human organism may have light and / or heavy immunoglobulin loci in its germline and / or genome that include insertions and / or substitutions of histidine codons designed to introduce pH-dependent binding properties to antibodies produced in such non-human organisms. In some such embodiments, the histidine codon is inserted and / or substituted in a nucleic acid sequence encoding CDR3. Various such light and / or heavy immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, U.S. Patent Application Publications 2013 / 0247236 and 2014 / 0013456, which are incorporated herein by reference.

[0085] In some embodiments, the immunoglobulin constant region includes a heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a human heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is of endogenous species origin. In some embodiments, the heavy chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the constant region gene is a mixture of human and non-human sequences. For example, in some embodiments, the constant region gene encodes a human CH1 region and a non-human (e.g., endogenous species origin, mouse, rat) CH2 and / or CH3 region. In some embodiments, the heavy chain constant region gene is a Cμ, Cδ, Cγ (Cγ1, Cγ2, Cγ3, Cγ4), Cα, or Cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region gene encodes a mutated CH1 region so that non-human animals express antibodies against the heavy chain only (see, for example, U.S. Patent No. 8,754,287 and U.S. Patent Application Publication 2015 / 0289489, which are incorporated herein by reference). In some embodiments, for example, when the goal is to generate a heavy chain that produces a bispecific antibody (e.g., in a universal or double light chain organism), the Fc domain of the heavy chain includes modifications to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patents No. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication 2013 / 0195849, each of which is incorporated herein by reference.

[0086] In some embodiments, the immunoglobulin constant region includes a light chain constant region gene. In some embodiments, the light chain constant region gene is a κ constant region gene. In some embodiments, the light chain constant region gene is a λ constant region gene. In some embodiments, the light chain constant region gene is of endogenous species origin. In some embodiments, the light chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the light chain constant region gene is a mixture of human and non-human sequences.

[0087] In some embodiments, the immunoglobulin variable region containing the human variable region gene segment and the immunoglobulin constant region gene to which the variable region gene segment is operably ligated are located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is the endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is the endogenous κ locus. In some embodiments, the endogenous immunoglobulin locus is the endogenous λ locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably ligated is the endogenous constant region gene.

[0088] In some embodiments, one or more endogenous immunoglobulin loci or portions of one or more endogenous loci (e.g., variable regions and / or constant regions) in the genome of a non-human animal provided herein are inactivated. Endogenous immunoglobulin variable region loci and portions thereof can be inactivated by any method known in the art, including, but not limited to, deletion of the loci or portion thereof from the genome of an organism, substitution of the loci or portion thereof with a different nucleic acid sequence, inversion of a portion of the loci to another location in the genome of a non-human organism and / or transposition of a portion of the loci. In some embodiments, the inactivation of the loci is only partial. In some embodiments, the variable region of the loci is inactivated, but the constant region remains functional (e.g., because it is operably linked to a non-endogenous variable region gene segment).

[0089] In some embodiments, the genetically modified non-human animal contains an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous variable region of the endogenous heavy chain locus. In some embodiments, at least a portion of the variable region of the endogenous heavy chain locus to be deleted, substituted, transposed, and / or inverted includes the J segment of the variable region. In some embodiments, the endogenous immunoglobulin heavy chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous constant region of the endogenous heavy chain locus. In some embodiments, at least a portion of the constant region of the endogenous heavy chain locus to be deleted, substituted, transposed, and / or inverted includes the Cμ gene of the endogenous constant region.

[0090] In some embodiments, the genetically modified non-human animal contains an inactivated endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous immunoglobulin κ chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous variable region of the endogenous κ chain locus. In some embodiments, at least a portion of the variable region of the endogenous κ chain locus that is deleted, substituted, transposed, and / or inverted includes the J segment of the variable region. In some embodiments, the endogenous immunoglobulin κ chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous constant region of the endogenous κ chain locus. In some embodiments, at least a portion of the constant region of the endogenous κ chain locus that is deleted, substituted, transposed, and / or inverted includes the Cκ gene of the endogenous constant region.

[0091] In some embodiments, the genetically modified non-human animal contains an inactivated endogenous immunoglobulin λ-chain locus. In some embodiments, the endogenous immunoglobulin λ-chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous variable region of the endogenous λ-chain locus. In some embodiments, at least a portion of at least one VJC gene cluster in the endogenous λ-chain locus is deleted, substituted, transposed, and / or inverted. In some embodiments, the endogenous immunoglobulin λ-chain locus or a portion thereof is inactivated by deletion, substitution, transposition, and / or inversion of at least a portion of the endogenous constant region of the endogenous λ-chain locus. In some embodiments, at least a portion of the constant region of the endogenous λ-chain locus that is deleted, substituted, transposed, and / or inverted contains the Cλ gene in the endogenous constant region.

[0092] In some embodiments, the genetically modified non-human animals provided herein express antibodies having a human variable domain (e.g., a human variable domain derived from an unreorganized human variable region gene segment described herein). In some embodiments, the human variable domain is a human heavy chain variable domain. In some embodiments, the antibody is a heavy chain-only antibody. In some embodiments, the human variable domain is a human light chain variable domain. In some embodiments, the antibody produced by the non-human animal has both a human heavy chain variable domain and a human light chain variable domain. In some embodiments, the antibody has a human heavy chain constant domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the heavy chain and / or light chain constant domains are of non-human origin. For example, in some embodiments, the heavy chain constant domain is of endogenous species origin. In some embodiments, the heavy chain constant domain is of mouse or rat origin. In some embodiments, the light chain constant domain is of endogenous species origin. In some embodiments, the light chain constant domain is of rat or mouse origin.

[0093] Non-human animals expressing human variable domain T cell receptor and exogenous TdT In certain embodiments, genetically modified non-human animals and non-human animal ES cells containing exogenous TdT as described herein also include TCR loci (exogenous or endogenous) in their germline and / or genome, which include a TCR variable region comprising an unreorganized human TCR variable region gene segment and a TCR constant region comprising a TCR constant region gene, the unreorganized human TCR variable region gene segment being operably linked to the TCR constant region gene. In some embodiments, various genetically modified non-human animals, such as genetically modified mice, include genetically modified T cell receptor loci (genetically modified TCRα, β, γ, and / or δ loci) in their germline and / or genome, such that the mouse expresses human, humanized, partially human, or reverse chimeric (human variable region and non-human constant region) T cell receptors. In one embodiment, exemplary non-human animals are provided in U.S. Patent No. 9,113,616 and International Patent Application Publication WO2016 / 164492, which are incorporated herein by reference.

[0094] In some embodiments, the TCR constant region gene is a non-human TCR constant region gene. In some embodiments, the TCR constant region gene is a rodent constant region gene such as a rat constant region gene or a mouse constant region gene. In some embodiments, the constant region gene is of endogenous species origin. In some embodiments, the TCR constant region gene is a human constant region gene.

[0095] In some embodiments, non-human animals and non-human ES cells include multiple such TCR loci in their germline and / or genome. For example, in some embodiments, genetically modified non-human animals and non-human animal ES cells include at least one TCR locus containing an unreorganized TCRα variable region gene segment and at least one TCR locus containing an unreorganized TCRβ variable region gene segment in their germline and / or genome. In some embodiments, genetically modified non-human animals and non-human animal ES cells include at least one TCR locus containing an unreorganized human TCRγ variable region gene segment and at least one TCR locus containing an unreorganized human TCRδ variable region gene segment in their germline and / or genome.

[0096] In some embodiments, the human unreorganized TCR variable region gene segment is the TCRα gene segment, and the TCR constant region gene is the TCRα constant region gene. In some embodiments, the human unreorganized TCR variable region gene segment is the TCRβ chain gene segment, and the TCR constant region gene is the TCRβ constant region gene. In some embodiments, the human unreorganized TCR variable region gene segment is the TCRγ chain gene segment, and the TCR constant region gene is the TCRγ constant region gene. In some embodiments, the human unreorganized TCR variable region gene segment is the TCRδ chain gene segment, and the TCR constant region gene is the TCRδ constant region gene. Exemplary variable regions including human TCR gene segments are provided, for example, in U.S. Patent No. 9,113,616 and Li et al., Nature Medicine 16:1029-1035 (2010), each of which is incorporated herein by reference.

[0097] In some embodiments, the TCR variable region includes an unreorganized human TCRβ variable region gene segment. In some embodiments, the human TCRβ variable region gene segment is rearranged during T cell development to produce the rearranged human TCRβ variable region gene in the T cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCRβ conjugation with non-template addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRβ conjugation with non-template addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRβ conjugation in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a smaller proportion of non-template addition-free VD TCRβ conjugates than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD and / or DJ TCRβ conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD and / or DJ TCRβ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VD and / or DJ TCRβ conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRβ junctions containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRβ junctions in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ TCRβ conjugates containing at least two N additions greater than that of corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRβ conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than that of the VD and / or DJ TCRβ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ TCRβ conjugates containing at least three N additions greater than that of corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRβ conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRβ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ TCRβ conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRβ conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRβ conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ TCRβ conjugates in the animals contain non-template additions.In some embodiments, the non-human animal has a frequency of unique TCRβ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.

[0098] In some embodiments, the TCR variable region includes an unreorganized human TCRα variable region gene segment. In some embodiments, the human TCRα variable region gene segment is rearranged during T cell development to produce the rearranged human TCRα variable region gene in the T cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCRα conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα conjugates without non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ TCRα conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ TCRα conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ TCRα conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRα junctions, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ TCRα conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ TCRα conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα conjugates in the corresponding non-human animals. In some embodiments, non-human animals provided herein have a higher proportion of VJ TCRα conjugates containing at least four N additions than corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRα conjugates containing at least four N additions in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRα conjugates in corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of VJ TCRα conjugates in animals contain non-template additions. In some embodiments, non-human animals have a frequency of unique TCRα CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animals.

[0099] In some embodiments, the TCR variable region includes an unreorganized human TCRδ variable region gene segment. In some embodiments, the human TCRδ variable region gene segment is rearranged during T cell development to produce the rearranged human TCRδ variable region gene in the T cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCRδ conjugates including non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRδ conjugates including non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRδ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of non-template addition-free VD TCRδ conjugates than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD and / or DJ TCRδ conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% lower than the proportion of VD and / or DJ TCRδ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCRδ conjugates with at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. The proportion of TCRδ conjugates is present. In some embodiments, the proportion of VD and / or DJ TCRδ conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRδ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ TCRδ conjugates containing at least two N additions greater than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRδ conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRδ conjugates in the corresponding non-human animals. The proportion of TCRδ conjugation is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRδ conjugation in the corresponding non-human animal. In some embodiments, the non-human animal provided herein has a proportion of VD and / or DJ TCRδ conjugation containing at least three N additions greater than that of the corresponding non-human animal that does not have nucleic acids encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ conjugation containing at least three N additions in the genetically modified non-human animal provided herein is greater than that of VD and / or DJ in the corresponding non-human animal. The proportion of VD and / or DJ TCRδ junctions is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of TCRδ junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ TCRδ junctions containing at least four N additions greater than that of corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ TCRδ junctions in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of VD and / or DJ TCRδ conjugates in animals include non-template additions. In some embodiments, non-human animals have a frequency of unique TCRδ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of corresponding non-human animals.

[0100] In some embodiments, the TCR variable region includes an unreorganized human TCRγ variable region gene segment. In some embodiments, the human TCRγ variable region gene segment is rearranged during T cell development to produce the rearranged human TCRγ variable region gene in the T cells of a non-human organism. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCRγ conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ conjugates without non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ TCRγ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ TCRγ conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ TCRγ conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ TCRγ junctions, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ TCRγ junctions containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ TCRγ junctions containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ junctions containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ junctions in the corresponding non-human animals. In some embodiments, non-human animals provided herein have a higher proportion of VJ TCRγ conjugates containing at least four N additions than corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ TCRγ conjugates containing at least four N additions in genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ TCRγ conjugates in corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of VJ TCRγ conjugates in animals contain non-template additions. In some embodiments, non-human animals have a frequency of unique TCRγ CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animals.

[0101] In some embodiments, the TCR variable region, including an unreorganized human TCR variable region gene segment, also includes human TCR variable region intergene sequences. In some embodiments, the TCR variable region includes non-human (e.g., rodent, rat, mouse) TCR variable region intergene sequences. In some embodiments, the intergene sequences are of endogenous species origin.

[0102] In some embodiments, the TCR variable region containing the human variable region gene segment and the TCR constant region gene to which the variable region gene segment is operably ligated are located at an endogenous TCR locus. In some embodiments, the endogenous TCR locus is the endogenous TCRα locus. In some embodiments, the endogenous TCR locus is the endogenous TCRβ locus. In some embodiments, the endogenous TCR locus is the endogenous TCRγ locus. In some embodiments, the endogenous TCR locus is the endogenous TCRδ locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably ligated is an endogenous constant region gene, for example, the corresponding endogenous constant region.

[0103] In some embodiments, one or more endogenous TCR loci or portions of one or more endogenous loci (e.g., variable regions and / or constant regions) in the genome of a non-human animal provided herein are inactivated. Endogenous TCR variable region loci and portions thereof can be inactivated by any method known in the art, including, but not limited to, deletion of the loci or portion thereof from the genome of an organism, substitution of the loci or portion thereof with a different nucleic acid sequence, inversion of a portion of the loci to another location in the genome of a non-human organism and / or transposition of a portion of the loci. In some embodiments, the inactivation of the loci is only partial. In some embodiments, the variable region of the loci is inactivated, but the constant region remains functional (e.g., because it is operably linked to a non-endogenous variable region gene segment). Examples of inactivated TCR loci are described, for example, in Mombaerts et al., Proc. Natl. Acad. Sci. USA 88:3084-3087 (1991) and Mombaerts et al., Nature 390:225-231 (1992), each of which is incorporated herein by reference.

[0104] In some embodiments, the genetically modified non-human animals provided herein express a TCR having a human variable domain (e.g., a human variable domain derived from an unreorganized human variable region gene segment as described herein). In some embodiments, the human variable domain is a human TCRα variable domain. In some embodiments, the human variable domain is a human TCRβ variable domain. In some embodiments, the human variable domain is a human TCRγ variable domain. In some embodiments, the human variable domain is a human TCRδ variable domain. In some embodiments, the TCR produced by a non-human animal has both a human TCRα variable domain and a human TCRβ variable domain. In some embodiments, the TCR produced by a non-human animal has both a human TCRγ variable domain and a human TCRδ variable domain. In some embodiments, the TCR produced by a non-human animal has both a human TCRα variable domain and a human TCRβ variable domain, as well as both a human TCRγ variable domain and a human TCRδ variable domain. In some embodiments, the TCR has a human constant domain. In some embodiments, the constant domain is of non-human origin. For example, in some embodiments, the steady domain is derived from an endogenous species. In some embodiments, the steady domain is derived from a mouse or rat.

[0105] Non-human animals expressing chimeric antigen receptors (CARs) and exogenous TdT Genetically modified non-human animals and non-human animal ES cells containing the exogenous TdT described herein, including, in certain embodiments, a chimeric antigen receptor (CAR) locus, are provided herein. Such CAR loci generally include a variable region and a constant region. The variable region includes an unreorganized human Ig variable region gene segment, and the constant region locus includes a TCR constant region gene, the Ig variable region gene segment being operably ligated to the constant region gene. In some embodiments, the TCR constant region gene is a non-human TCR constant region gene. In some embodiments, the TCR constant region gene is a rodent constant region gene, such as a rat constant region gene or a mouse constant region gene. In some embodiments, the constant region gene is of endogenous species origin. In some embodiments, the TCR constant region gene is a human constant region gene.

[0106] In some embodiments, the CAR loci described herein are located at endogenous TCR loci. For example, in some embodiments, a CAR locus containing a TCRα constant region gene is located at an endogenous TCRα constant region locus. In some embodiments, such a locus is created by replacing part or all of the unreorganized variable region of TCRα with an unreorganized Ig variable region. In some embodiments, a CAR locus containing a TCRβ constant region gene is located at an endogenous TCRβ constant region locus. In some embodiments, such a locus is created by replacing part or all of the unreorganized variable region of TCRβ with an unreorganized Ig variable region.

[0107] In certain embodiments, the CAR variable region locus includes an unreorganized human Ig variable region gene segment. Exemplary variable region loci including a human variable region gene segment are described in the art. For example, such loci are U.S. Patents No. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,502,018, 8,697,940, and 8,703,4 These are described in U.S. Patent Publications No. 85, 8,754,287, 8,791,323, 8,907,157, 9,035,128, 9,145,588, and 9,206,263, each of which is incorporated herein by reference in its entirety, and U.S. Patent Publications No. 2008 / 0098490, 2010 / 0146647, 2011 / 0195454, and 20 These are described in publications 12 / 0167237, 2013 / 0145484, 2013 / 0167256, 2013 / 0219535, 2013 / 0326647, 2014 / 013275, 2014 / 093908, 2015 / 0113668, and 2016 / 0081314, each of which is incorporated herein by reference in its entirety and is a PCT This is described in Patent Application Publications WO2007 / 117410, WO2008 / 151081, WO2009 / 157771, WO2010 / 039900, WO2011 / 004192, WO2011 / 123708, WO2014 / 093908, and WO2016 / 044745, each of which is incorporated herein by reference in its entirety.

[0108] In certain embodiments, the CAR variable region locus includes an unreorganized human Ig heavy chain variable region gene segment. In some embodiments, the unreorganized human Ig variable region gene segment includes multiple human V H Segment, one or more human D HSegments, and one or more human J H The segment includes. In some embodiments, the unreorganized human Ig variable region gene segment contains at least three V H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H Gene segment, or at least 80 V H Includes a gene segment. In some embodiments, the unreorganized human Ig gene segment is human D H It includes all gene segments. In some embodiments, the CAR variable region further includes TCRβ variable region gene segments (e.g., V, D, and / or J gene segments). In one embodiment, the CAR variable region further includes a distal TCR Vβ gene segment, e.g., a TCR Vβ31 gene segment. In another embodiment, the distal TCR Vβ gene segment, e.g., a TCR Vβ31 gene segment, is functionally inactivated or deleted. In some embodiments, the unreorganized human Ig gene segment is human J H This includes all gene segments. Exemplary variable regions, including the Ig heavy chain gene segment, are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and Supplementary Information, which are incorporated herein by reference.

[0109] In some embodiments, the human immunoglobulin heavy chain variable region gene segment is rearranged during T cell development to produce the rearranged human heavy chain variable region gene in T cells of non-human organisms. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain conjugations without non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VD and / or DJ immunoglobulin heavy chain conjugations in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least one N addition, which is greater than that of the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals.In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least two N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugations in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain conjugations containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD and / or DJ immunoglobulin heavy chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ immunoglobulin heavy chain conjugates in the animals contain non-template additions.In some embodiments, the non-human animal has a frequency of unique immunoglobulin heavy chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.

[0110] In some embodiments, a CAR variable locus containing an unreorganized human Ig heavy chain variable region gene segment also contains a human Ig heavy chain variable region intergene sequence. In some embodiments, a CAR variable locus contains a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergene sequence. In some embodiments, a CAR variable locus contains a human or non-human (e.g., rodent, rat, mouse) TCRβ variable region intergene sequence. For example, in some embodiments, the unreorganized variable region of a CAR locus contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) trypsinogen (TRY) genes (e.g., TRY genes and / or pseudogenes normally present at a TCRβ variable region locus). In some embodiments, the TRY gene is non-human, e.g., a mouse TRY gene. In some embodiments, the mouse TRY gene is selected from the group consisting of Try1, Try2, Try3, Try4, Try5, Try6, Try7, Try8, Try9, Try10, Try11, Try12, Try13, Try14, Try15, Try16, Try17, Try18, Try19, and Try20. In some embodiments, one or more TRY genes have an unreorganized variable region V H It is located upstream of the segment. In some embodiments, one or more TRY genes are V H D of the downstream and unreorganized variable region of the segment H It is located upstream of the segment. In some embodiments, Try1~7 is the unreorganized variable region V H Located upstream of the segment, Try8~20 is V H D of the downstream and unreorganized variable region of the segment HIt is located upstream of the segment. Additional information regarding the TRY gene located at the human and / or mouse TCRβ locus is provided in Glusman et al., Immunity 15:337-349 (2001) and Skok et al., Nature Immunology 8:378-387 (2007), each of which is incorporated by reference. In some embodiments, the CAR locus includes a non-human regulatory element (e.g., a non-human promoter and / or enhancer). In some embodiments, the non-human regulatory element is a rodent regulatory element (e.g., a rat or mouse promoter or enhancer). In some embodiments, the CAR locus includes an IgM enhancer (Eμ). In some embodiments, the IgM enhancer is a non-human Eμ (e.g., a rodent Eμ, such as a mouse or rat Eμ).

[0111] In certain embodiments, the CAR variable region locus includes an unreorganized human Igκ variable region gene segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes multiple human V κ Segments and one or more human J κ It includes a segment. In some embodiments, the immunoglobulin variable region gene segment has four functional V κ Segment and all human J κ The segment includes. In some embodiments, the immunoglobulin variable region gene segment contains 16 functional V κ Segment and all human J κ Includes segments. In some embodiments, the unreorganized human immunoglobulin variable region gene segment is all of the human Vκ segment and all of the human J κ segments (e.g., all functional human Vκ segments and J) κThe variable region includes the Igκ gene segment. Exemplary variable regions including the Igκ gene segment are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and Supplementary Information, which are incorporated herein by reference. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes all of the human Jκ segment. In some embodiments, the CAR variable region further includes the TCRα variable region gene segment (e.g., the V and / or J gene segment).

[0112] In some embodiments, the human immunoglobulin κ variable region gene segment is rearranged during T cell development to produce the rearranged human κ variable region gene in T cells of non-human organisms. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin κ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have exogenous TdT-encoding nucleic acids in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin κ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugations, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, non-human animals provided herein have a higher proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions than corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin κ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin κ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin κ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin κ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1700 unique immunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chain CDR3 sequences.

[0113] In certain embodiments, the CAR variable region locus includes an unreorganized human Igλ variable region gene segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment includes multiple human V λ Segments and one or more human J λ Includes a segment. In some embodiments, the unreorganized human immunoglobulin variable region gene segment is all human V λ Segments (e.g., all functional human V) λ Includes segments). In some embodiments, the unreorganized human immunoglobulin variable region gene segment is included in all human J λ The variable region includes a segment. In some embodiments, the CAR variable region further includes a TCRα variable region gene segment (e.g., V and / or J gene segment). Exemplary variable regions including an Igλ gene segment are provided, for example, in U.S. Patent Application Publications 2012 / 0073004 and 2002 / 0088016, each of which is incorporated herein by reference.

[0114] In some embodiments, the human immunoglobulin λ variable region gene segment is rearranged during T cell development to produce the rearranged human λ variable region gene in the T cells of non-human organisms. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing non-template additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a lower proportion of VJ immunoglobulin λ chain conjugates that do not contain non-template additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of non-template addition-free VJ immunoglobulin λ chain conjugates in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% smaller than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a larger proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least one N addition in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% larger than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain junctions, including at least two N additions, than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome.In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least two N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least three N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions than the corresponding non-human animals that do not have nucleic acids encoding exogenous TdT in their genome. In some embodiments, the proportion of VJ immunoglobulin λ chain conjugates containing at least four N additions in the genetically modified non-human animals provided herein is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VJ immunoglobulin λ chain conjugates in the corresponding non-human animals. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ immunoglobulin λ chain conjugates in the animals contain non-template additions. In some embodiments, the non-human animal has a frequency of unique immunoglobulin λ chain CDR3 sequences that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the corresponding non-human animal.In some embodiments, the non-human animals provided herein have at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 unique immunoglobulin λ chain CDR3 sequences per 10,000 immunoglobulin λ chain CDR3 sequences.

[0115] In some embodiments, a CAR variable locus containing an unreorganized human Ig light chain variable region gene segment also contains human Ig light chain variable region intergene sequences (e.g., κ variable region intergene sequences and / or λ variable region intergene sequences). In some embodiments, a CAR variable locus contains non-human (e.g., rodent, rat, mouse) Ig light chain variable region intergene sequences (e.g., κ variable region intergene sequences and / or λ variable region intergene sequences). In some embodiments, a CAR variable locus contains human or non-human (e.g., rodent, rat, mouse) TCRα variable region intergene sequences. In some embodiments, a CAR locus contains non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0116] In some embodiments, the CAR variable region locus is a rearranged variable region locus containing an Ig heavy chain variable region gene (universal heavy chain variable region). In some embodiments, the rearranged Ig heavy chain variable region gene is a human rearranged Ig heavy chain variable region gene. The use of the universal heavy chain variable region facilitates the production of bispecific antibodies in which at least one antigen-binding domain has specificity for a peptide / MHC complex. An exemplary rearranged Ig heavy chain variable region is provided in U.S. Patent Application Publication 2014 / 0245468, which is incorporated herein by reference.

[0117] In some embodiments, the CAR variable region locus is a rearranged variable region locus containing an Ig light chain variable region gene (universal light chain variable region). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. The use of the universal light chain variable region facilitates the production of bispecific antibodies in which at least one antigen-binding domain has binding specificity for peptide / MHC complexes. An exemplary rearranged Ig heavy chain variable region is provided in U.S. Patent Application Publication 2013 / 0185821, which is incorporated herein by reference.

[0118] Other genetic modifications In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci that encode humanized MHC class Iα chain polypeptides (e.g., humanized HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K, and / or HLA-L). In some embodiments, the humanized MHC class Iα chain polypeptides are entirely human. In some embodiments, the humanized MHC class Iα chain polypeptides include human extracellular domains (e.g., human α1, α2, and α3 domains) and endogenous species-derived cytoplasmic domains. Humanized MHC class Iα chain polypeptides, loci encoding humanized MHC class Iα chain polypeptides, and non-human animals expressing humanized MHC class Iα chain polypeptides are described in U.S. Patent Applications Publications 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467, each incorporated herein by reference.

[0119] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci that encode humanized β-2 microglobulin polypeptide. Humanized β-2 microglobulin polypeptide, loci encoding humanized β-2 microglobulin polypeptide, and non-human animals expressing humanized β-2 microglobulin polypeptide are described in U.S. Patent Application Publications 2013 / 0111617 and 2013 / 0185819, each of which is incorporated herein by reference.

[0120] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci encoding humanized MHC class IIα chain polypeptides (e.g., humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA). In some embodiments, the humanized MHC class IIα chain polypeptides are entirely human. In some embodiments, the humanized MHC class IIα chain polypeptides include a human extracellular domain and an endogenous species-derived cytoplasmic domain. Humanized MHC class IIα chain polypeptides, loci encoding humanized MHC class IIα chain polypeptides, and non-human animals expressing humanized MHC class IIα chain polypeptides are described in U.S. Patent Nos. 8,847,005 and 9,043,996, and U.S. Patent Application Publication 2014 / 0245467, each of which is incorporated herein by reference.

[0121] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci that encode humanized MHC class IIβ chain polypeptides (e.g., humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB). In some embodiments, the humanized MHC class IIβ chain polypeptides are entirely human. In some embodiments, the humanized MHC class IIβ chain polypeptides include a human extracellular domain and an endogenous species-derived cytoplasmic domain. Humanized MHC class IIβ chain polypeptides, loci that encode humanized MHC class IIβ chain polypeptides, and non-human animals that express humanized MHC class IIβ chain polypeptides are described in U.S. Patent Nos. 8,847,005 and 9,043,996, and U.S. Patent Application Publication 2014 / 0245467, each of which is incorporated herein by reference.

[0122] Genetically modified non-human animals containing exogenous TdT, humanized TCR loci, and humanized MHC I and / or MHC II (MHCIIα / IIβ) loci can be produced by breeding using conventional methods. Alternatively, they can be produced by homologous recombination in ES cells that already contain one or more genetically modified loci (e.g., humanized TCR loci), and non-human animals can be produced from these ES cells.

[0123] Genetically modified non-human animals containing exogenous TdT, humanized CAR loci, and humanized MHC I and / or MHC II (MHCIIα / IIβ) loci can be produced by breeding using conventional methods. Alternatively, they can be produced by homologous recombination in ES cells that already contain one or more genetically modified loci (e.g., humanized CAR loci), and non-human animals can be produced from these ES cells.

[0124] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci that encode humanized CD8α chain polypeptide. In some embodiments, the humanized CD8α chain polypeptide is entirely human. In some embodiments, the humanized CD8α chain polypeptide comprises a human extracellular immunoglobulin domain and an endogenous species-derived cytoplasmic domain. The humanized CD8α chain polypeptide, the loci encoding the humanized CD8α chain polypeptide, and the non-human animals expressing the humanized CD8α chain polypeptide are described in U.S. Patent Application Publication 2014 / 0245466, which is incorporated herein by reference.

[0125] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome loci that encode humanized CD8β chain polypeptide. In some embodiments, the humanized CD8β chain polypeptide is entirely human. In some embodiments, the humanized CD8β chain polypeptide comprises a human extracellular immunoglobulin domain and an endogenous species-derived cytoplasmic domain. The humanized CD8β chain polypeptide, the loci encoding the humanized CD8β chain polypeptide, and the non-human animals expressing the humanized CD8β chain polypeptide are described in U.S. Patent Application Publication 2014 / 0245466, which is incorporated herein by reference.

[0126] In some embodiments, the genetically modified non-human animals and ES cells described herein that express exogenous TdT, humanized TCR, or CAR also express and / or include in their genome a locus encoding a humanized CD4 polypeptide. In some embodiments, the humanized CD4 polypeptide is entirely human. In some embodiments, the humanized CD4 polypeptide comprises at least one human extracellular immunoglobulin domain and an endogenous species-derived cytoplasmic domain. In some embodiments, the humanized CD4 polypeptide comprises at least one human D1 immunoglobulin domain, a human D2 immunoglobulin domain, and a human D3 immunoglobulin domain, as well as an endogenous species-derived cytoplasmic domain. In some embodiments, the humanized CD4 polypeptide comprises at least one human D1 immunoglobulin domain, a human D2 immunoglobulin domain, a human D3 immunoglobulin domain, an endogenous species-derived D4 immunoglobulin domain, and an endogenous species-derived cytoplasmic domain. A humanized CD4 polypeptide, a locus encoding the humanized CD4 polypeptide, and a non-human animal expressing the humanized CD4 polypeptide are described in U.S. Patent Application Publication 2014 / 0245466, which is incorporated herein by reference.

[0127] Genetically modified non-human animals containing exogenous TdT, humanized TCR loci, and humanized CD4 and / or CD8 (CD8α / CD8β) loci can be produced by breeding using conventional methods. Alternatively, they can be produced by homologous recombination in ES cells that already contain one or more genetically modified loci (e.g., humanized TCR loci), and non-human animals can be produced from these ES cells.

[0128] Genetically modified non-human animals containing exogenous TdT, humanized CAR loci, and humanized CD4 and / or CD8 (CD8α / CD8β) loci can be produced by breeding using conventional methods. Alternatively, they can be produced by homologous recombination in ES cells that already contain one or more genetically modified loci (e.g., humanized CAR loci), and non-human animals can be produced from these ES cells.

[0129] Methods for using genetically modified non-human animals In certain embodiments, methods are provided herein for using genetically modified non-human animals described herein to produce antigen-binding proteins (e.g., antibodies, CARs, TCRs), cells expressing such antigen-binding proteins (e.g., B cells, T cells, B cell hybridomas, T cell hybridomas), and nucleic acids encoding such antigen-binding proteins or portions thereof (e.g., variable domains). In some embodiments, methods are provided herein for producing a wider variety of antigen-binding proteins (e.g., antibodies, CARs, TCRs). In some embodiments, methods are provided herein for producing a rearranged variable region of an antigen-binding protein (e.g., antibody, CAR, TCR) having an increased number of nucleotide additions.

[0130] In certain embodiments, the method involves exposing a genetically modified non-human animal described herein, which has been modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen, so that the genetically modified non-human animal produces an antibody or an antigen-binding fragment thereof containing a human variable domain specific to the antigen.

[0131] In some embodiments, the method comprises exposing a genetically modified non-human animal described herein, modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen, and obtaining B cells from the non-human animal that express an antibody containing an antigen-specific human variable domain or an antigen-binding fragment thereof.

[0132] In some embodiments, the method comprises exposing a genetically modified non-human animal described herein, modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen; obtaining B cells from the non-human animal that express an antibody containing an antigen-specific human variable domain or an antigen-binding fragment thereof; and producing a hybridoma from the B cells.

[0133] In some embodiments, the method comprises exposing a genetically modified non-human animal described herein, modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen, and obtaining a nucleic acid encoding an antigen-specific human immunoglobulin variable domain from the non-human animal.

[0134] In certain embodiments, the method includes exposing a genetically modified non-human animal described herein, modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen; obtaining B cells from the non-human animal that express an antibody containing an antigen-specific human variable domain or an antigen-binding fragment thereof; optionally, constructing a hybridoma from the B cells; and obtaining nucleic acids encoding an antigen-specific human immunoglobulin variable domain from the B cells or hybridoma.

[0135] In some embodiments, the method includes exposing a non-human animal described herein, modified to express an antibody having an exogenous TdT and a human variable domain or an antigen-binding fragment thereof, to an antigen; obtaining B cells from the non-human animal that express an antibody containing an antigen-specific human variable domain or an antigen-binding fragment thereof; optionally constructing a hybridoma from the B cells; obtaining a nucleic acid encoding an antigen-specific human immunoglobulin variable domain from the B cells or hybridoma; operably linking the nucleic acid encoding the immunoglobulin variable domain and the nucleic acid encoding the human immunoglobulin constant domain in a host cell; and culturing the host cell under conditions such that the host cell expresses a human antibody containing an immunoglobulin variable domain and an immunoglobulin constant domain.

[0136] In some embodiments, the method comprises exposing genetically modified non-human animals described herein, which have been modified to express an exogenous TdT and a TCR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animals, and obtaining T cells from the genetically modified non-human animals that express a TCR specific to the peptide presented on the MHC.

[0137] In some embodiments, the method comprises: exposing a genetically modified non-human animal described herein, modified to express an exogenous TdT and a TCR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a TCR specific to the peptide presented on the MHC from the genetically modified non-human animal; and constructing T cell hybridomas from the T cells.

[0138] In some embodiments, the method comprises: exposing a non-human animal described herein, modified to express an exogenous TdT and a TCR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a TCR specific to the peptide presented on the MHC from the genetically modified non-human animal; and isolating the nucleic acid encoding the human TCR variable domain of the TCR from the T cells.

[0139] In some embodiments, the method includes: exposing a non-human animal described herein, modified to express a TCR having an exogenous TdT and a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a TCR specific to the peptide presented on the MHC from the genetically modified non-human animal; isolating the nucleic acid encoding the TCR variable domain of the TCR from the T cells; and operably linking the nucleic acid encoding the TCR variable domain and the TCR constant domain in the cell so that the cell expresses a TCR comprising the TCR variable domain and the TCR constant domain.

[0140] In some embodiments, the method comprises exposing a genetically modified non-human animal described herein, modified to express an exogenous TdT and a CAR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal, and obtaining T cells from the genetically modified non-human animal that express a CAR specific to the peptide presented on the MHC.

[0141] In some embodiments, the method comprises: exposing a genetically modified non-human animal described herein, modified to express an exogenous TdT and a CAR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a CAR specific to the peptide presented on the MHC from the genetically modified non-human animal; and constructing T cell hybridomas from the T cells.

[0142] In some embodiments, the method comprises: exposing a non-human animal described herein, modified to express an exogenous TdT and a CAR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a chimeric antigen receptor (CAR) specific to the peptide presented on the MHC from the genetically modified non-human animal; and isolating the nucleic acid encoding the human TCR variable domain of the CAR from the T cells.

[0143] In some embodiments, the method includes: exposing a non-human animal described herein, modified to express an exogenous TdT and a CAR having a human variable domain, to an antigen comprising a peptide or a nucleic acid encoding a peptide-containing antigen, such that the peptide is presented on the MHC in the non-human animal; obtaining T cells expressing a chimeric antigen receptor (CAR) specific to the peptide presented on the MHC from the genetically modified non-human animal; isolating the nucleic acid encoding the human immunoglobulin variable domain of the CAR from the T cells; and operably linking the nucleic acid encoding the human immunoglobulin variable domain and the human immunoglobulin constant domain in the cell so that the cell expresses an antibody comprising the human immunoglobulin variable domain and the human immunoglobulin constant domain.

[0144] In certain embodiments, the methods described herein include the step of exposing a non-human animal described herein to an antigen (immunization) in order to induce an immune response (e.g., a B-cell immune response and / or a T-cell immune response). In some embodiments, genetically modified non-human animals are immunized with a whole protein antigen or a fragment thereof. Rodents can be immunized by any method known in the art (e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory, Malik). and Lillehoj (1994) Antibody Techniques, Academic Press, CA).

[0145] In some embodiments, genetically modified non-human animals are exposed to the antigen by administering a virus (e.g., a retrovirus, adenovirus, vaccinia virus, or lentivirus) containing a nucleic acid sequence encoding the antigen to the non-human animal. Methods for viral vaccination are described, for example, in U.S. Patents 6,001,349, 8,663,622, 8,691,502, 8,377,688, and Precopio et al. These are provided in al., JEM 204:1405-1416 (2007), each of which is incorporated herein by reference in whole. In some embodiments, non-human animals are administered the virus directly. In some embodiments, cells (e.g., antigen-presenting cells such as dendritic cells) are infected with the virus in vitro or ex vivo and then administered to non-human animals. In some embodiments, the virus encodes a peptide / MHC complex (e.g., a single-stranded peptide / MHC complex). Examples of single-chain peptide / MHC-based vaccines are provided in Truscott et al., J.Immunol. 178:6280-6289 (2007), EP1773383, Kim et al., Vaccine 30:2178-2186 (2012), and Kim et al., J.Immunol. 184:4423-4430 (2010), each of which is incorporated herein by reference.

[0146] In some embodiments, genetically modified non-human animals are exposed to an antigen by administering a nucleic acid encoding the antigen to the animal. In some embodiments, non-human animals are administered a nucleic acid encoding a single-stranded peptide / MHC complex. Examples of single-stranded peptide / MHC-based vaccines are provided in Truscott et al., J.Immunol. 178:6280-6289 (2007), EP1773383, Kim et al., Vaccine 30:2178-2186 (2012), and Kim et al., J.Immunol. 184:4423-4430 (2010), each of which is incorporated herein by reference. In certain embodiments, the nucleic acid is a DNA vector. Delivery of the nucleic acid can be by any technique known in the art, including virus-mediated and liposome-mediated gene delivery. The target polynucleotides associate with liposomes to form a gene delivery vehicle, as described, for example, in U.S. Patents 6,770,291, 7,001,614, 6,749,863, 5,512,295, and 7,112,338, each of which is incorporated herein by reference. In some embodiments, the nucleic acid is an mRNA vector. Exemplary methods for producing and administering mRNA vectors are described, for example, in U.S. Patent 8,278,036 and U.S. Patent Application Publications 2013 / 151736 and 2012 / 135805, each of which is incorporated herein by reference.

[0147] In some embodiments, the antigen is a cancer-associated antigen. Examples of cancer-associated antigens, but not limited to these, include adipophyllin, AIM-2, ALDH1A1, α-actinin-4, α-fetoprotein ("AFP"), ALK, ANKRD30A, ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, β-catenin, BING-4, BIRC7, CA-125, CA9, CALCA, carcinoembryonic antigen ("CEA"), CALR, CASP-5, CASP-8, CCR5, CD19, CD20, CD22, CD27, CD274, CD30, and CD3 3, CD38, CD40, CD44, CD45, CD52, CD56, CD79, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLEC12A, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, Cyclin D1, Cyclin-A1, Dek-Can fusion protein, DKK1, EFTUD2, EGFR, EGFR variant III, Elongation factor 2, ENAH(hMena), Ep-CAM, EpCAM, EphA2, EphA3, Epithelial tumor antigen ("ETA"), ERBB3, ERBB4, ETV6- AML1 fusion protein, EZH2, FCRL3, FGF5, FLT3-ITD, FN1, FOLR1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, Glypican-3, GnTV, gp100 / Pmel17, GPNMB, GM3, GPR112, IL3RA, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Rα2, Intestinal carboxylesterase, K-ras Kallikrein 4, KIF20A, KIT, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), KRAS, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, LGR5, LMP2, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malate enzyme, mammoglobin A, MART2, MATN, MC1R,MCSP, mdm-2, ME1, Melane-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC2, MUC3, MUC4, MUC5, MUC5AC, MUC16, Mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin Class I, N-raw, NA88-A, Neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LA GE-2, OA1, OGT, OS-9, OX40, P polypeptide, p53, PAP, PAX3, PAX5, PBF, PLAC1, PMEL, pml-RARα fusion protein, pleomorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PRLR, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RET, Examples include RGS5, RhoC, RNF43, ROR1, RU2AS, SAGE, SART1, SART3, Cesernin 1, SIRT2, SLAMF7, SLC39A6, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, Survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TERT, TGF-βRII, Thompson-nouvelle antigen, TMPRSS2, TNFRSF17, TPBG, TRAG-3, Triose phosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, Tyrosinase, Tyrosinase ("TYR"), UPK3A, VEGF, VTCN1, WT1, and XAGE-1b / GAGED2a. In some embodiments, the antigen is a nascent antigen.

[0148] In some embodiments, the antigen is an antigen expressed by an infectious pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, helminth, or protozoan. Non-limiting examples of viruses include HIV, hepatitis A, hepatitis B, hepatitis C, herpesviruses (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, Ebola virus, and arbovirus encephalitis virus antigens. In some embodiments, the parasite is malaria. In some embodiments, the pathogens are Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Klebsiella, Legionella, Leishmania, Meningococci, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibriocholerae. Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridium Examples include botulinum, or fungi such as Paracoccidioides brasiliensis, or other pathogens such as Plasmodium falciparum.

[0149] In some embodiments of the methods described herein, the method includes the step of obtaining T cells and / or B cells from genetically modified non-human animals. In certain embodiments, such cells can be obtained using any method known in the art. For example, such T cells and / or B cells can be obtained from the spleen, lymph nodes, and / or peripheral blood of an animal. Such T cells and / or B cells can be screened for binding specificity using methods available in the art.

[0150] In some embodiments, the methods described herein include the step of producing B cell hybridomas from B cells. Useful methods for producing B cell hybridomas are known in the art, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring. This is described in Harbor Laboratory, Malik and Lillehoj (1994), Antibody Techniques, Academic Press, CA, and is incorporated herein by reference.

[0151] In some embodiments, the methods described herein include the step of producing T cell hybridomas from T cells. Useful methods for producing T cell hybridomas are known in the art, for example, Hedrick et al., Cell 30:141-152 (1982) and Kruisbeek Curr. Protoc. Immunol. Chapter 3 (2001) and White et al., Methods This is described in Molecular Biology 134:185-193 (2000), each of which is incorporated herein by reference.

[0152] In some embodiments, the methods provided herein include the step of isolating a nucleic acid encoding an Ig or TCR variable region. In some embodiments of the methods described herein, the nucleic acid encoding an Ig or TCR variable region can be isolated using any method.

[0153] In some embodiments, the step of isolating nucleic acids includes the step of constructing B cell or T cell hybridomas from B cells or T cells, respectively, and isolating nucleic acids from the hybridomas. In some embodiments, nucleic acids are isolated using a nucleic acid amplification process. For example, in some embodiments, the nucleic acid amplification process is polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), autologous persistent sequence replication (3SR), Qβ replicase-based amplification, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), boomerang DNA amplification (BDA), or rolling circle amplification (RCA).

[0154] In some embodiments, nucleic acids are isolated by sequencing a rearranged Ig or TCR variable region gene in a B cell, T cell, B cell hybridoma, or T cell hybridoma, and by synthesizing a nucleic acid sequence containing the rearranged Ig or TCR variable region gene. Exemplary nucleic acid sequencing processes, but not limited to these, include linkage arrest sequencing, ligation sequencing, synthesis sequencing, pyrosequencing, ion semiconductor sequencing, single-molecule real-time sequencing, 454 sequencing, and / or Dilute-'N'-Go sequencing.

[0155] If DNA fragments encoding heavy and / or light chain Ig variable regions are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region gene to a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding the variable region is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. As used in this context, the term "operably ligated" is intended to mean that the two DNA fragments are ligated in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0156] Isolated DNA encoding the heavy chain variable region can be converted into a full-length heavy chain gene by operably ligating the variable region-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant domains may be, for example, IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant domains. For Fab fragment heavy chain genes, V H The coding DNA can be operably ligated to another DNA molecule that codes only for the heavy chain CH1 constant region.

[0157] Accordingly, in some embodiments, the method described herein includes the step of operably linking a nucleic acid sequence encoding a heavy chain Ig variable domain in a host cell with a nucleic acid sequence encoding a heavy chain Ig constant domain so that the host cell expresses an Ig heavy chain polypeptide comprising the Ig heavy chain variable domain and the Ig heavy chain constant domain. In some embodiments, the method includes the step of operably linking a nucleic acid sequence encoding a light chain Ig variable domain in a host cell with a nucleic acid sequence encoding a light chain Ig constant domain so that the host cell expresses an Ig light chain polypeptide comprising the Ig light chain variable domain and the Ig heavy chain constant domain. In some embodiments, the method includes the step of operably linking a nucleic acid sequence encoding a heavy chain Ig variable domain in a host cell with a nucleic acid sequence encoding a heavy chain Ig constant domain so that the host cell expresses an antibody having a heavy chain comprising a heavy chain Ig variable domain and a heavy chain Ig constant domain, and a light chain comprising a light chain Ig variable domain and a light chain Ig constant domain, and operably linking a nucleic acid sequence encoding a light chain Ig variable domain in a host cell with a nucleic acid sequence encoding a light chain Ig constant domain. The Ig variable region can be linked to the Ig constant region using standard molecular biology techniques well known in the art. In some embodiments, any host cell capable of expressing an immunoglobulin polypeptide can be used. In some embodiments, the cell is a CHO cell, HEK-293 cell, BHK cell, NS0 cell, SP2 / 0 cell, or Vero cell, or a retinal cell expressing a viral nucleic acid sequence (e.g., PERC.6® cell).

[0158] In some embodiments, the nucleic acid encoding the heavy chain constant domain encodes a constant domain that includes a modified Fc domain (e.g., a mutation that alters the interaction between Fc and the Fc receptor). For example, in some embodiments, the constant domain includes modifications to its Fc domain at positions 235, 236, 237, 239, 265, 267, 268, 269, 270, 298, 326, 327, 330, 332, 350, 351, 366, 392, 394, 405, and / or 407 (using the EU numbering system). In some embodiments, the modifications are selected from the group consisting of L235A, G236E, G237F, S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268E, H268D, E269L, D270N, D270E, S298A, K326A, K326D, A327H, A327V, A327L, A330I, A330S, I332E, T350V, L351Y, T366L, K392M, K392L, T394W, F405A, and / or Y407V (using the EU numbering system). In some embodiments, a steady domain includes multiple modifications to its Fc domain. In some embodiments, the multiple modifications are D270N / K326D, S239E / S298A / K326A / A327H, L235A / S239E / D265E / A327H, G236E / G237F / S239E, G237F / S239E / D265E, G327F / S239E / H268D, G236E / D270N / A327V / I332E, G237F / S239E / A327H, G237F / A327L / A330I, S23 The group is selected from the following: 9D / D265S / S298A / I332E, S239E / D265S / H268D / I332E, S239E / D265S / I332E, S239E / S267E / H268D, S239E / A327L / A330I, D265E / S267D / A330S, S267G / H268E / D270E, H268D / E269L / S298A / K326A / A327H, and H268D / / K326A / A327H.Additional Fc modifications and combinations of Fc modifications are provided in U.S. Patent Nos. 5,624,821, 5,648,260, 6,528,624, 6,737,056, 7,122,637, 7,183,387, 7,297,775, 7,317,091, 7,332,581, 7,632,497, 7,662,925, 7,695,936, 8,093,359, 8,216,805, 8,218,805, 8,388,955, and 8,937,158, and U.S. Patent Publications Nos. 2005 / 0054832, 2006 / 0222653, 2006 / 0275282, 2006 / 0275283, 2007 / 0190063, 2008 / 0154025, 2009 / 0042291, 2013 / 0108623, and 2013 / 0089541, each of which is incorporated herein by reference.

[0159] antigen-binding protein In certain embodiments, antigen-binding proteins (e.g., antibodies, TCRs, CARs, and antigen-binding fragments thereof) that can be obtained by and / or are obtained by the methods described herein (e.g., using the non-human animals described herein) are provided herein.

[0160] In certain embodiments, the antigen-binding molecules provided herein have a dissociation constant of 10 -6 , 10 -7 , 10 -8 , or 10 -9 M or less and can specifically bind to a target antigen. In some embodiments, the binding affinity (K D represented thereby) of the antigen-binding protein for the antigen is at least 10-fold, at least 100-fold, or at least 1000-fold lower than the affinity of the antigen-binding protein for an irrelevant antigen. In some embodiments, the antigen-binding protein has a dissociation constant of 10 -6 , 10 -7 , 10 -8 , or 10 -9It binds to the peptide / MHC complex with a dissociation constant of M or less. In some embodiments, the binding affinity (K) of the antigen-binding protein to the peptide / MHC complex is determined to be less than or equal to M. D The affinity of the antigen-binding protein (represented by ) is at least 10-fold, at least 100-fold, or at least 1000-fold lower than the affinity of the antigen-binding protein for the same MHC protein presenting an unrelated peptide. Standard assays for evaluating the binding ability of antigen-binding proteins include, for example, ELISA, Western blotting, and RIA, which are known in the art. The binding kinetics (e.g., binding affinity) of antigen-binding proteins can also be evaluated by standard assays known in the art, such as Biacore analysis.

[0161] In some embodiments, the antigen includes and / or is a cancer-associated antigen epitope. Examples of cancer-associated antigens include, but are not limited to, adipophyllin, AIM-2, ALDH1A1, α-actinin-4, α-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, β-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, Cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, Epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, Glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Rα2, Intestinal Carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-Fucosyltransfer Gelase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malate enzyme, mammoglobin A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM -3, Myosin, Myosin class I, N-raw, NA88-A, Neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARα fusion protein, Pleomorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, S Examples include AGE, sesernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, Survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-βRII, TPBG, TRAG-3, triose phosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a. In some embodiments, the antigen is a nascent antigen.

[0162] In some embodiments, the antigen is an antigen expressed by an infectious pathogen, and / or an antigen expressed by an infectious pathogen. In some embodiments, the pathogen is a virus, bacterium, fungus, helminth, or protozoan. Some non-limiting examples of viruses include retroviruses such as HPV, HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 and HIV-2), herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza viruses. In some embodiments, the parasite is malaria. In some embodiments, the pathogens are Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibriocholerae. Examples of species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, and Brucella abortus.Brucella melitensis coli fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridium botulinum, and Paracoccidioides brasiliensis strains and Plasmodium falciparum strains.

[0163] In some embodiments, the antigen comprises and / or is a protein epitope that is a target of autoreactive T cells in inflammatory diseases, skin or organ transplant rejection, graft-versus-host disease (GVHD), or autoimmune diseases. Examples of autoimmune diseases include, for example, glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjögren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, polyarteritis nodosa, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, Behçet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture syndrome, infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, autoimmune achlorhydria, celiac disease, and Cushing's syndrome. This includes dermatomyositis, discoid erythema, lupus, Goodpasture syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, several cases of lymphopenia, mixed connective tissue disease, bullous pemphigoid, pemphigus vulgaris, pernicious anemia, lens-induced uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt syndrome, focal scleroderma (or Crest syndrome), sympathetic ophthalmitis, systemic cachexic peritonitis, ankylosing cystitis, polyglandular parasymptomatic syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type B, ulcerative colitis, and Wegener's granulomatosis. Exemplary proteins targeted by autoreactive T cells include, for example, p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1, and myelin.

[0164] In some embodiments, the antigen-binding protein is an antibody. In some embodiments, the antibody provided herein includes a human heavy chain variable domain. In some embodiments, the antibody includes a human heavy chain constant domain. In some embodiments, the antibody provided herein includes an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant domain. The sequences of human heavy chain constant domains are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001). In some embodiments, the antibody provided herein lacks a heavy chain constant domain or a portion thereof.

[0165] In some embodiments, the antibodies provided herein include modified Fc domains (e.g., mutations that alter the interaction between Fc and the Fc receptor). For example, in some embodiments, the antibodies provided herein include modifications to their Fc domains at positions 235, 236, 237, 239, 265, 267, 268, 269, 270, 298, 326, 327, 330, 332, 350, 351, 366, 392, 394, 405, and / or 407 (using the EU numbering system). In some embodiments, the modifications are selected from the group consisting of L235A, G236E, G237F, S239E, S239D, D265E, D265S, S267E, S267D, S267G, H268E, H268D, E269L, D270N, D270E, S298A, K326A, K326D, A327H, A327V, A327L, A330I, A330S, I332E, T350V, L351Y, T366L, K392M, K392L, T394W, F405A, and / or Y407V (using the EU numbering system). In some embodiments, the antibodies include multiple modifications to their Fc domains. In some embodiments, the multiple modifications are D270N / K326D, S239E / S298A / K326A / A327H, L235A / S239E / D265E / A327H, G236E / G237F / S239E, G237F / S239E / D265E, G327F / S239E / H268D, G236E / D270N / A327V / I332E, G237F / S239E / A327H, G237F / A327L / A330I, S23 The group is selected from the following: 9D / D265S / S298A / I332E, S239E / D265S / H268D / I332E, S239E / D265S / I332E, S239E / S267E / H268D, S239E / A327L / A330I, D265E / S267D / A330S, S267G / H268E / D270E, H268D / E269L / S298A / K326A / A327H, and H268D / / K326A / A327H.Additional Fc modifications and combinations of Fc modifications are U.S. Patent Nos. 5,624,821, 5,648,260, 6,528,624, 6,737,056, 7,122,637, 7,183,387, 7,297,775, 7,317,091, 7,332,581, 7,632,497, 7,662,925, 7,695,936, 8,093,359, 8,216,805, 8,218,805, and 8 These are provided in U.S. Patent Publications No. 388,955 and No. 8,937,158, and in U.S. Patent Application Publications No. 2005 / 0054832, No. 2006 / 0222653, No. 2006 / 0275282, No. 2006 / 0275283, No. 2007 / 0190063, No. 2008 / 0154025, No. 2009 / 0042291, No. 2013 / 0108623, and No. 2013 / 0089541, each of which is incorporated herein by reference.

[0166] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the two antigen-binding domains of the bispecific antibody have distinct heavy chain variable domains but identical light chain variable domains. In some embodiments, the Fc domain of the heavy chain includes modifications to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patents 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication 2013 / 0195849, each of which is incorporated herein by reference.

[0167] In some embodiments, the antibodies provided herein have a human light chain variable domain. In some embodiments, the light chain variable domain has a λ light chain variable domain. In some embodiments, the light chain variable domain has a κ light chain variable domain. In some embodiments, the antibody has a human light chain constant domain. In some embodiments, the light chain constant domain is a λ light chain constant domain. In some embodiments, the light chain constant domain is a κ light chain constant domain. The sequence of the human light chain constant domain is known in the art (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health (See *and Human Services*, NIH Publication No. 91-3242, or Lefranc, *The Immunoglobulin Handbook*, London: Academic Press 2001).

[0168] In some embodiments, the antibody described herein is an intact antibody. In some embodiments, the antibody described herein is an antibody fragment that retains antigen binding. In some embodiments, the antibody fragment is Fab, Fab', F(ab')2, Fv, scFv, disulfide-bonded Fv, Fd, single-chain antibody, isolated CDRH3, or another antibody fragment that retains at least a portion of the variable domain of an intact antibody.

[0169] In certain embodiments, the antigen-binding protein is a CAR. In some embodiments, the CAR is membrane-bound. In some embodiments, the CAR is a soluble CAR (e.g., lacking a transmembrane or cytoplasmic domain). In some embodiments, such a CAR comprises a first CAR polypeptide containing an Ig heavy chain variable domain and a TCRβ constant domain, and a second CAR polypeptide containing an Ig light chain variable domain (e.g., an Igκ variable domain or an Igλ variable domain) and a TCRα constant domain. In some embodiments, the Ig heavy chain variable domain and / or Ig light chain variable domain is a human Ig variable domain. In some embodiments, the TCRβ constant domain and / or TCRα constant domain is a non-human constant domain (e.g., a rat or mouse constant domain). In some embodiments, the TCRβ constant domain and / or TCRα constant domain is a human constant domain.

[0170] In certain embodiments, the antigen-binding protein is a TCR. In some embodiments, the TCR is membrane-bound. In some embodiments, the TCR is a soluble TCR (e.g., lacking a transmembrane or cytoplasmic domain). In some embodiments, such a TCR comprises a first TCR polypeptide containing a TCRβ variable domain and a TCRβ constant domain, and a second TCR polypeptide containing a TCRα variable domain and a TCRα constant domain. In some embodiments, the TCRα variable domain and / or the TCRβ variable domain is a human TCR variable domain. In some embodiments, the TCRβ constant domain and / or the TCRα constant domain is a non-human constant domain (e.g., a rat or mouse constant domain). In some embodiments, the TCRβ constant domain and / or the TCRα constant domain is a human constant domain.

[0171] Pharmaceutical composition In certain embodiments, pharmaceutical compositions are provided herein, for example, comprising a composition, formulated with a pharmaceutically acceptable carrier, at least one agent described herein (e.g., an antigen-binding molecule described herein, such as an antibody, CAR, or TCR obtained from a non-human animal described herein).

[0172] The pharmaceutical compositions provided herein can be specifically formulated for administration in solid or liquid form and include those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for oral, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, or (2) parenteral administration, e.g., sterile solutions or suspensions, or as sustained-release formulations by subcutaneous, intramuscular, intravenous, or epidural injection.

[0173] The pharmaceutical compositions provided herein for parenteral administration include one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the recipient to whom the formulation is intended, or suspending agents or thickeners, and are combined with one or more of the agents described herein.

[0174] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0175] In certain embodiments, the composition comprises the antibody, TCR, and / or CAR described herein at a concentration that yields a suitable weight / volume for the desired dose. The antibody, TCR, and / or CAR in the composition is at least 1 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 35 mg / mL, at least 40 mg / mL, at least 45 mg / mL, at least 50 mg / mL, at least 55 mg / mL, at least 60 mg / mL, at least 65 mg / mL, at least 70 mg / mL, at least 75 mg / mL, and at least 80 mg / mL. It may be present in concentrations of mg / mL, at least 85 mg / mL, at least 90 mg / mL, at least 95 mg / mL, at least 100 mg / mL, at least 105 mg / mL, at least 110 mg / mL, at least 115 mg / mL, at least 120 mg / mL, at least 125 mg / mL, at least 130 mg / mL, at least 135 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 200 mg / mL, at least 250 mg / mL, or at least 300 mg / mL.

[0176] In some embodiments, the composition includes one or more active compounds necessary for the specific indication being treated, typically having complementary activities that do not adversely affect each other. Such additional active compounds are appropriately present in combination in amounts effective for the intended purpose.

[0177] In some embodiments, the composition contains the antibodies, TCRs, and / or CARs described herein, along with any physiologically acceptable carriers, excipients, or stabilizers (Goodman and By mixing with Gilman's *The Pharmacological Basis of Therapeutics*, 12th edition, L. Brunton, et al. and Remington's *Pharmaceutical Sciences*, 16th edition, Osol, A. Ed. (1999)), it is prepared in the form of a lyophilized composition or aqueous solution of the desired final concentration. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as histidine, phosphates, citrates, glycine, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin This includes hydrophilic polymers such as gelatin, or immunoglobulins, polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including trehalose, glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

[0178] In some embodiments, the buffer is histidine, citrate, phosphate, glycine, or acetate. The sugar excipient may be trehalose, sucrose, mannitol, maltose, or raffinose. The surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl2, or CaCl2.

[0179] In some embodiments, the composition includes a buffer or pH adjuster that provides improved pH control. Such compositions may have pH values ​​of about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.5, about 5.5 to about 8.0, about 5.5 to about 7.0, or about 5.5 to about 6.5. In further embodiments, such compositions have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In particular embodiments, the composition has a pH of about 6.0. Those skilled in the art will understand that the pH of a composition should generally not be equal to the isoelectric point of a particular antibody, TCR, or CAR used in the composition. Typically, buffers are salts prepared from organic or inorganic acids or bases. Typical buffering agents include, but are not limited to, organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, tromethamine hydrochloride, or phosphate buffers. Furthermore, amino acid components can also function in buffering capacity. Typical amino acid components that can be used as buffering agents in a composition include, but are not limited to, glycine and histidine. In certain embodiments, the buffering agent is selected from histidine, citrate, phosphate, glycine, and acetate. In certain embodiments, the buffering agent is histidine. In another particular embodiment, the buffering agent is citrate. In yet another particular embodiment, the buffering agent is glycine. The purity of the buffering agent must be at least 98%, or at least 99%, or at least 99.5%. As used herein, the term “purity” in the context of histidine and glycine refers to the chemical purity of histidine or glycine as understood in the art, for example, as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

[0180] In certain embodiments, the composition contains histidine as a buffer. In certain embodiments, histidine is present in the composition at concentrations of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM. In another embodiment, the composition contains histidine in amounts of approximately 1 mM to approximately 200 mM, approximately 1 mM to approximately 150 mM, approximately 1 mM to approximately 100 mM, approximately 1 mM to approximately 75 mM, approximately 10 mM to approximately 200 mM, approximately 10 mM to approximately 150 mM, approximately 10 mM to approximately 100 mM, approximately 10 mM to approximately 75 mM, approximately 10 mM to approximately 50 mM, approximately 10 mM to approximately 40 mM, approximately 10 mM to approximately 30 mM, approximately 20 mM to approximately 75 mM, approximately 20 mM to approximately 50 mM, approximately 20 mM to approximately 40 mM, or approximately 20 mM to approximately 30 mM. In further embodiments, the composition may contain about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM of histidine. In certain embodiments, the composition may contain about 10 mM, about 25 mM of histidine, or may not contain histidine at all.

[0181] In some embodiments, the composition includes a carbohydrate excipient. The carbohydrate excipient can act, for example, as a thickener, stabilizer, bulking agent, solubilizer, etc. The carbohydrate excipient is generally present in amounts ranging from about 1% by weight or volume to about 99% by weight or volume, for example, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 5%, about 1% to about 20%, about 5% to about 15%, about 8% to about 10%, about 10% to about 15%, about 15% to about 20%, 0.1% to about 20%, 5% to about 15%, 8% to 10%, 10% to about 15%, 15% to about 20%, about 0.1% to about 5%, about 5% to about 10%, or about 15% to about 20%. In other specific embodiments, the carbohydrate excipient is present in an amount of 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, or 20%.

[0182] In some embodiments, the composition includes a carbohydrate excipient. Suitable carbohydrate excipients for use in the composition include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol). In certain embodiments, the carbohydrate excipient for use in the composition provided herein is selected from sucrose, trehalose, lactose, mannitol, and raffinose. In certain embodiments, the carbohydrate excipient is trehalose. In another particular embodiment, the carbohydrate excipient is mannitol. In yet another particular embodiment, the carbohydrate excipient is sucrose. In yet another particular embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient must be at least 98%, or at least 99%, or at least 99.5%.

[0183] In some embodiments, the composition contains trehalose. In certain embodiments, the composition contains at least about 1%, at least about 2%, at least about 4%, at least about 8%, at least about 20%, at least about 30%, or at least about 40% trehalose. In other embodiments, the composition contains about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 2% to about 40%, about 2% to about 30%, about 2% to about 20%, about 4% to about 40%, about 4% to about 30%, or about 4% to about 20% trehalose. In further embodiments, the composition contains about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% trehalose. In certain embodiments, the composition contains about 4%, about 6%, or about 15% trehalose.

[0184] In certain embodiments, the composition includes an excipient. In certain embodiments, the composition includes at least one excipient selected from sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. In certain embodiments, the composition includes a salt, for example, a salt selected from NaCl, KCl, CaCl2, and MgCl2. In certain embodiments, the composition includes NaCl.

[0185] In some embodiments, the composition comprises amino acids, such as lysine, arginine, glycine, histidine, or amino acid salts. The composition may contain at least about 1 mM, at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of amino acids. In another embodiment, the composition may contain amino acids in amounts of about 1 mM to about 100 mM, about 10 mM to about 150 mM, about 25 mM to about 250 mM, about 25 mM to about 300 mM, about 25 mM to about 350 mM, about 25 mM to about 400 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 100 mM to about 250 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 150 mM to about 250 mM, about 150 mM to about 300 mM, or about 150 mM to about 400 mM. In further embodiments, the composition contains about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of amino acids.

[0186] In some embodiments, the composition includes a surfactant. As used herein, the term “surfactant” refers to an organic substance having an amphiphilic structure; that is, it consists of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified into anionic, cationic, and nonionic surfactants depending on the charge of their surfactant moiety. Surfactants are often used as wetting agents, emulsifiers, solubilizers, and dispersants for various pharmaceutical compositions and preparations of biological materials. Polysorbates (e.g., polysorbate 20 or 80); polyoxomers (e.g., poloxamer 188); triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, paImidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl taurate, or disodium methyloleyl taurate; and MONAQUA® series (Mona Aggregation can be optionally reduced by adding pharmaceutically acceptable surfactants to the composition, such as polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68). In certain embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when the composition is administered using a pump or plastic container. The presence of a pharmaceutically acceptable surfactant mitigates the tendency of proteins to aggregate. The composition may contain polysorbate in concentrations ranging from about 0.001% to about 1%, or about 0.001% to about 0.1%, or about 0.01% to about 0.1%.In other specific embodiments, the composition comprises a polysorbate at a concentration of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.015%, or 0.02%.

[0187] In some embodiments, the composition may further include, but is not limited to, other excipients and / or additives, such as diluents, binders, stabilizers, lipophilic solvents, preservatives, and adjuvants. pharmaceutically acceptable excipients and / or additives may be used in the compositions provided herein. Commonly used excipients / additives, such as pharmaceutically acceptable chelating agents (e.g., EDTA, DTPA, or EGTA), may optionally be added to the composition to reduce aggregation. These additives are particularly useful when the composition is administered using a pump or plastic container.

[0188] In some embodiments, the composition contains a preservative. Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate, but not limited to these), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal or mixtures thereof may be optionally added to the composition in a preferred concentration ranging from about 0.001% to about 5%, or any range or value within that range. The concentration of the preservative used in the composition is sufficient to obtain a microbial effect. Such a concentration depends on the selected preservative and is readily determined by those skilled in the art.

[0189] In some embodiments, the composition is isotonic with human blood and has essentially the same osmotic pressure as human blood. Such isotonic compositions generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, using a vapor pressure or a freeze-type osmometer. The tonicity of the composition is adjusted by the use of a tonicity modifier. A “tonicity modifier” is a pharmaceutically acceptable inert substance that, in addition to the composition, can provide the composition with isotonicity. Suitable tonicity modifiers for the compositions provided herein include, but are not limited to, saccharides, salts, and amino acids.

[0190] In certain embodiments, the composition is pyrogenic and substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins trapped inside microorganisms that are released only when the microorganism decomposes or dies. Pyromogenic substances also include heat-stable substances derived from the outer membranes of bacteria and other microorganisms that cause fever. Both of these substances can cause fever, hypotension, and shock when administered to humans. Due to the potential for adverse effects, even small amounts of endotoxins must be removed from medicinal solutions administered intravenously. The U.S. Food and Drug Administration ("FDA") has set a limit of 5 endotoxin units (EU) per kilogram of body weight per hour per dose for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223(2000)). When therapeutic proteins are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, such as in the case of proteins of interest (e.g., antibodies), trace amounts of harmful and dangerous endotoxins must also be removed. In some embodiments, the levels of endotoxin and pyrogens in the composition are less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg.

[0191] When used for in vivo administration, the compositions described herein must be sterile. The compositions can be sterilized by various sterilization methods, including sterile filtration and radiation. In certain embodiments, the compositions are sterilized by filtration through a pre-sterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, as described in “Remington: The Science & Practice of Pharmacy”, 21st ed., Lippincott Williams & Wilkins, (2005). Compositions containing the protein of interest (e.g., antibody, TCR, or CAR) as disclosed herein are typically stored in lyophilized form or in solution. Sterile compositions containing the protein of interest (e.g., antibody, TCR, or CAR) may be placed in an intravenous solution bag or vial having an adapter that allows for the recovery of the composition, such as a stopper that can be punctured with a subcutaneous needle, or a container with a sterile outlet. In certain embodiments, the compositions are provided as pre-filled syringes.

[0192] In certain embodiments, the composition is a lyophilized formulation. The term “lyophilized” or “freeze-dried” includes a state of a substance subjected to a drying procedure such as lyophilization, from which at least 50% of the moisture has been removed.

[0193] Regardless of the selected route of administration, the agents and / or pharmaceutical compositions provided herein, which can be used in an appropriate hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0194] treatment method In certain embodiments, methods for treating diseases or disorders are provided herein that involve administering an antigen-binding protein (e.g., an antibody, TCR, or CAR as described herein, such as a fully human antibody, TCR, or CAR). In some embodiments, the antibody, TCR, and / or CAR is an antibody, TCR, and / or CAR obtained from or using the methods described herein (e.g., using a non-human animal as described herein).

[0195] This specification provides a method for treating a target cancer, comprising, in certain embodiments, administering a pharmaceutical composition described herein (for example, a pharmaceutical composition comprising an antibody described herein, such as a fully human antibody, TCR, or CAR, obtained from a non-human animal described herein) to the target. In some embodiments, the methods described herein can be used to treat any cancerous or precancerous tumor. Cancers that can be treated by the methods and compositions described herein include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Non-limiting examples of various histological types of cancer include: neoplasms (malignant); carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilosa carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrin-producing tumors (malignant); cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, Familial adenomatous polyposis; solid tumors; carcinoid tumors (malignant); bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; acid-affinity adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous adenocarcinoma; ceruminous gland carcinoma; mucinous epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serosa Humoral cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease (breast); acinar cell carcinoma; adenosquamous cell carcinoma; squamous metaplastic adenocarcinoma; thymoma (malignant); ovarian stromal tumor (malignant); follicular cell tumor (malignant); granulosa cell tumor (malignant); and lovastoma (malignant); Sertoli cell carcinoma; Leydig cell tumor (malignant); lipid cell tumor (malignant) Paraganglioma (malignant); Extramammary paraganglioma (malignant); Pheochromocytoma; Angioglobular angiosarcoma; Malignant melanoma; Melanin-deficient melanoma; Superficial spreading melanoma; Malignant melanoma of a giant pigmented nevus; Epithelioid cell melanoma; Blue nevus (malignant); Sarcoma; Fibrosarcoma; Fibrous histiocytoma (malignant); Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma;Mixed tumor (malignant); Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal cell tumor (malignant); Brenner tumor (malignant); pseudopapillary tumor (malignant); synovial sarcoma; mesothelioma (malignant); undifferentiated embryonal cell tumor; fetal carcinoma; teratoma (malignant); ovarian teratoma (malignant); choriocarcinoma; mesonephroma (malignant); angiosarcoma; vascular endothelial tumor (malignant); Kaposi sarcoma; perivascular cell tumor (malignant); lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma (malignant); mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor (malignant); ameloblastic fibrosarcoma; ameloblastoma (malignant); ameloblastic fibroadenosarcoma; pinealoma (malignant); chordoma; glioma (malignant); epithelioma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; glioblastoma; glioblastoma; oligodendroglioma; anaplastic glioblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma (malignant); neurofibrosarcoma; neurinoma (malignant); granular cell tumor (malignant); malignant lymphoma; Hodgkin disease; Hodgkin lymphoma; granuloma; malignant lymphoma (small lymphocyte); malignant lymphoma (large cell, diffuse); malignant lymphoma (follicular); fungating polypoid tumor; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblast leukemia; myelosarcoma; and hairy cell leukemia are included.;

[0196] In certain embodiments, the antibody, TCR, or CAR in the pharmaceutical composition administered to the subject has binding specificity to an epitope of a cancer-associated antigen (e.g., an epitope expressed by the cancer being treated). Examples of cancer-associated antigens include adipophyllin, AIM-2, ALDH1A1, α-actinin-4, α-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, β-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, and CLP. P, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, Cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, Epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, Glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal Carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-Fucosyltransferase AS Fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malate enzyme, mammoglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, mi Osinclase I, N-raw, NA88-A, Neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARα fusion protein, pleomorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, Cesernin 1, SI Examples of antigens include, but are not limited to, RT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, Survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-βRII, TPBG, TRAG-3, triose phosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a. In some embodiments, the antigen is a nascent antigen.

[0197] In certain embodiments, methods for treating subjects suffering from infectious diseases such as viral, fungal, bacterial, helminthic, or protozoan infections are provided herein, comprising administering a pharmaceutical composition described herein (for example, a pharmaceutical composition comprising an antibody, TCR, or CAR obtained from a non-human animal described herein) to the subject. Non-limiting examples of viral infections include retroviruses such as HPV, HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 and HIV-2), herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, and influenza viruses. Non-limiting examples of parasitic infections include malaria. Non-exclusive examples of bacterial, fungal, and other pathogenic diseases include Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibriocholerae.Drugs include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Haemophilus ducreyi,Granuloma inguinale,Lymphopathia venereum,Treponema pallidum,Brucella abortus.Brucella melitensis,Brucella suis,Brucella canis,Campylobacter fetus,Campylobacter fetus intestinalis,Leptospira pomona,Listeria monocytogenes,Brucella ovis,Chlamydia psittaci,Trichomonas. fetus,Toxoplasma gondii,Escherichia coli,Actinobacillus equuli,Salmonella abortus ovis,Salmonella abortus equi,Pseudomonas aeruginosa,Corynebacterium equi,Corynebacterium pyogenes,Actinobacillus seminis,Mycoplasma bovigenitalium,Aspergillus fumigatus,Absidia ramosa,Trypanosoma equiperdum,Babesia caballi,Clostridium tetani,Clostridium botulinum; falciparum and other cases.

[0198] In certain embodiments, the antibody, TCR, or CAR in the pharmaceutical composition administered to the subject has binding specificity to an antigen epitope expressed by an infectious pathogen (e.g., an epitope expressed by an infectious pathogen under treatment).

[0199] In some embodiments, methods for treating inflammatory diseases, skin or organ transplant rejection, graft-versus-host disease (GVHD), or autoimmune diseases are provided herein, comprising administering a pharmaceutical composition described herein (for example, a pharmaceutical composition comprising an antibody, TCR, or CAR obtained from a non-human animal described herein) to a subject. For example, examples of autoimmune diseases include glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjögren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, polyarteritis nodosa, rheumatic fever, vitiligo, insulin-dependent diabetes mellitus, Behçet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, malignant fever, Goodpasture syndrome, aseptic diseases, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, antiphospholipid antibody syndrome, atopic allergy, autoimmune atrophic gastritis, autoimmune achlorhydria, abdominal diseases, These include Cushing's syndrome, dermatomyositis, lupus discoid, lupus erythematosus, Goodpasture syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, certain cases of lymphopenia, mixed connective tissue disease, bullous pemphigoid, pemphigus vulgaris, pernicious anemia, lens-induced uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt syndrome, focal scleroderma (or Crest syndrome), sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type B, ulcerative colitis, and Wegener's granulomatosis.

[0200] In certain embodiments, the antibody, TCR, or CAR in the pharmaceutical composition administered to the subject has binding specificity to targets of autoreactive T cells in the disease being treated (e.g., epitopes that are targets of autoreactive T cells in autoimmune diseases). Exemplary proteins that are targets of autoreactive T cells include, for example, p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1, and myelin.

[0201] The pharmaceutical compositions described herein may be delivered by any suitable route of administration, such as oral or nasal administration by spray, rectal, vaginal, parenteral, intracisional, and topical administration by powder, ointment, or intravenous infusion, oral administration, and sublingual administration. In certain embodiments, the pharmaceutical compositions are generally delivered (for example, by oral or parenteral administration).

[0202] The actual dose levels of the active ingredients in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0203] The selected dosage level depends on various factors, including the activity of the specific drug used, the route of administration, the timing of administration, the rate of excretion or metabolism of the specific compound used, the duration of treatment, and other factors such as the patient's age, sex, weight, condition, overall health, and medical history, as well as similar factors well known in the medical field.

[0204] In some embodiments, the CARs and / or TCRs described herein are used in T cell-based therapies. For example, in certain embodiments, T cells expressing the CARs and / or TCRs described herein are administered to a subject to induce a T cell-based immune response in the subject. Methods useful for T cell-based therapies are described, for example, in Schumacher Nat.Rev.Immunol.2:512-519(2002) and Bitton et al,Frontiers in Bioscience 4:d386-393(1999), each of which is incorporated herein by reference.

[0205] In some aspects, provided herein are methods of inducing an immune response (e.g., a T cell-based immune response) in a subject. In some embodiments, the method comprises administering to the subject a cell (e.g., a human T cell such as a CD4 T cell or a CD8 T cell) expressing a CAR or TCR described herein.

[0206] In some embodiments, the subject is a subject that needs it. In some embodiments, the subject is a subject having cancer or a subject infected with a pathogen. In such embodiments, the peptide in the peptide / MHC complex recognized by the CAR or TCR is a peptide of a cancer antigen or a peptide derived from an antigen expressed by an infectious pathogen.

[0207] In some aspects, provided herein are methods of inhibiting an immune response in a subject. In some embodiments, the method comprises administering to the subject a regulatory T cell (e.g., a CD4 + 、CD-25 + 、and Foxp3 + regulatory T cell or a Treg17 T cell) expressing the described CAR or TCR.

[0208] In some embodiments, the subject is one that needs it, for example, a subject with an autoimmune disease. In such embodiments, the T cells are regulatory T cells (i.e., suppressor T cells), and the peptide in the peptide / MHC complex recognized by the TCR or CAR is an autoantigen that is causing the subject to an autoimmune response.

[0209] nucleic acid molecule This specification provides nucleic acid molecules encoding antibodies, TCRs, or CARs described herein, and / or portions thereof. In some embodiments, the nucleic acid encodes a variable domain of an antibody, TCR, or CAR described herein. The nucleic acid molecules may exist, for example, in whole cells, cell lysates, or in partially purified or substantially pure forms.

[0210] In certain embodiments, nucleic acids encoding antibodies, TCRs and / or CAR polypeptides or parts thereof are provided herein. Nucleic acids may exist, for example, in whole cells, cell lysates, or in partially purified or substantially pure forms. The nucleic acids described herein can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules described herein can be cloned using standard PCR techniques or chemically synthesized. For nucleic acids encoding CARs, TCRs, or antibodies expressed by hybridomas, cDNAs encoding the respective strands of the antibody, TCR, or CAR produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.

[0211] In certain embodiments, vectors containing the nucleic acid molecules described herein are provided herein. As used herein, the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which means a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to a viral genome. Certain vectors (e.g., bacterial vectors having bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. Furthermore, certain vectors can direct gene expression. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”).

[0212] In certain embodiments, cells containing the nucleic acids described herein (e.g., antibodies, nucleic acids encoding TCRs or CARs, or nucleic acids encoding a portion thereof) are provided herein. The cells may be, for example, prokaryotes, eukaryotes, mammals, birds, mice, and / or humans. In certain embodiments, the nucleic acids described herein are operably linked to transcriptional regulatory elements such as promoters. In some embodiments, cells transcribe the nucleic acids described herein, thereby expressing antibodies, their antigen-binding fragments, or polypeptides described herein. The nucleic acid molecules may be integrated into the cell's genome, or they may be extrachromosomal.

[0213] The nucleic acid molecules provided herein can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules described herein can be cloned using standard PCR techniques or chemically synthesized.

[0214] The antibodies and CAR nucleic acids described herein are VH and V L Once DNA fragments encoding the segment are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, V L or V H A DNA fragment encoding an antibody is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. In this context, the term "operably ligated" means that the two DNA fragments are ligated in such a way that the amino acid sequences encoded by those two DNA fragments remain in frame.

[0215] Isolated DNA encoding the heavy chain variable region can be converted into a full-length heavy chain gene by operably ligating the heavy chain variable region DNA to another DNA molecule encoding the heavy chain constant region (e.g., CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgD constant region. For Fab fragment heavy chain genes, see V H The DNA encoding this can be operably ligated to another DNA molecule that encodes only the heavy chain CH1 constant region.

[0216] Isolated DNA encoding a light chain variable region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the light chain variable region to another DNA molecule encoding a light chain constant region. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0217] Additional exemplary embodiments In Exemplary Embodiment 1, provided herein is a genetically modified non-human animal that contains in its genome a nucleic acid sequence encoding human terminal deoxynucleotidyl transferase (hTdT).

[0218] In Exemplary Embodiment 2, provided herein is the genetically modified non-human animal of Embodiment 1, wherein the nucleic acid sequence encoding hTdT is operably linked to a transcriptional control element.

[0219] In Exemplary Embodiment 3, provided herein is the genetically modified non-human animal of Embodiment 2, wherein the transcriptional control element drives the expression of the nucleic acid sequence encoding exogenous hTdT in pro-B cells and / or pre-B cells.

[0220] In an exemplary embodiment 4, the gene-modified non-human animal described herein is provided, wherein the transcriptional regulatory element is selected from the group consisting of a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, an immunoglobulin heavy chain transcriptional regulatory element, an immunoglobulin κ light chain transcriptional regulatory element, and / or an immunoglobulin λ light chain transcriptional regulatory element.

[0221] In exemplary embodiment 5, a genetically modified non-human animal according to any one of embodiments 1 to 4 is provided herein, wherein the non-human animal expresses hTdT in pro-B cells and / or pre-B cells.

[0222] In an exemplary embodiment 6, the gene-modified non-human animal described herein is provided, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding hTdT in CD4 / CD8 double-negative (DN) thymocytes and / or CD4 / CD8 double-positive (DP) thymocytes.

[0223] In exemplary embodiment 7, the gene-modified non-human animal described herein is provided, wherein the transcriptional regulatory element is a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, a TCRα transcriptional regulatory element, a TCRβ transcriptional regulatory element, a TCRγ transcriptional regulatory element, and / or a TCRδ transcriptional regulatory element.

[0224] In exemplary embodiment 8, the gene-modified non-human animal described herein is provided, which expresses hTdT in DN thymocytes and / or DP thymocytes, as described in any one of embodiments 1 to 7.

[0225] In exemplary embodiment 9, the nucleic acid sequence encoding hTdT is located at the immunoglobulin κ light chain locus, immunoglobulin λ light chain locus, immunoglobulin heavy chain locus, RAG1 locus, RAG2 locus, TCRα chain locus, TCRβ chain locus, TCRγ chain locus and / or TCRδ chain locus, as described herein, a genetically modified non-human animal according to any one of embodiments 1 to 8.

[0226] In exemplary embodiment 10, a genetically modified non-human animal according to any one of embodiments 1 to 9 is provided herein, wherein the nucleic acid sequence encoding hTdT is not operably linked to a constitutive transcriptional regulatory element.

[0227] In exemplary embodiment 11, a genetically modified non-human animal according to any one of embodiments 1 to 10 is provided herein, wherein hTdT is not constitutively expressed.

[0228] In exemplary embodiment 12, a genetically modified non-human animal according to any one of embodiments 1 to 11 is provided herein, wherein at least 10% of the VJ immunoglobulin light chain conjugates in the animal involve non-template addition.

[0229] In exemplary embodiment 13, a genetically modified non-human animal according to embodiment 12 is provided herein, wherein at least 20% of the VJ immunoglobulin light chain conjugates in the animal involve non-template addition.

[0230] In exemplary embodiment 14, the genetically modified non-human animal described herein is provided, wherein at least 40% of the VJ immunoglobulin light chain conjugates within the animal are non-template additions, as described in embodiment 12.

[0231] In exemplary embodiment 15, the Specified herein provides a genetically modified non-human animal having an immunoglobulin variable region in its genome, which includes a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyltransferase (TdT) and an unreorganized human immunoglobulin variable region gene segment operably linked to an immunoglobulin constant region gene.

[0232] In exemplary embodiment 16, the gene-modified non-human animal described in embodiment 15 is provided herein, wherein the exogenous TdT is human TdT.

[0233] In exemplary embodiment 17, a genetically modified non-human animal according to embodiment 15 or 16 is provided herein, wherein a nucleic acid sequence encoding exogenous TdT is operably linked to a transcriptional regulatory element.

[0234] In an exemplary embodiment 18, the gene-modified non-human animal described herein, as in embodiment 17, is provided, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding exogenous TdT in pro-B cells and / or pre-B cells.

[0235] In exemplary embodiment 19, the gene-modified non-human animal described herein is provided, wherein the transcriptional regulatory element is selected from the group consisting of a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, an immunoglobulin heavy chain transcriptional regulatory element, an immunoglobulin κ light chain transcriptional regulatory element, and / or an immunoglobulin λ light chain transcriptional regulatory element.

[0236] In exemplary embodiment 20, the gene-modified non-human animal described herein is provided, which expresses exogenous TdT in pro-B cells and / or pre-B cells, as described in any one of embodiments 15 to 19.

[0237] In an exemplary embodiment 21, the gene-modified non-human animal described herein, as in Embodiment 17, is provided, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding exogenous TdT in CD4 / CD8 double-negative (DN) thymocytes and / or CD4 / CD8 double-positive (DP) thymocytes.

[0238] In exemplary embodiment 22, the gene-modified non-human animal described herein, as in embodiment 17, is provided, wherein the transcriptional regulatory element is either a RAG1 transcriptional regulatory element or a RAG2 transcriptional regulatory element.

[0239] In exemplary embodiment 23, the gene-modified non-human animal described herein is provided as described in any one of embodiments 15 to 22, wherein the non-human animal expresses exogenous TdT in DN thymocytes and / or DP thymocytes.

[0240] In exemplary embodiment 24, the nucleic acid sequence encoding exogenous TdT is located at the immunoglobulin κ light chain locus, immunoglobulin λ light chain locus, immunoglobulin heavy chain locus, RAG1 locus, RAG2 locus, TCRα chain locus, TCRβ chain locus, TCRγ chain locus and / or TCRδ chain locus, as described herein, a genetically modified non-human animal according to any one of embodiments 15 to 23.

[0241] In exemplary embodiment 25, a genetically modified non-human animal according to any one of embodiments 15 to 24 is provided herein, wherein the nucleic acid sequence encoding exogenous TdT is not operably linked to a constitutive transcriptional regulatory element.

[0242] In exemplary embodiment 26, the gene-modified non-human animal described herein, according to any one of embodiments 15 to 25, is provided, in which exogenous TdT is not constitutively expressed.

[0243] In exemplary embodiment 27, a genetically modified non-human animal according to any one of embodiments 15 to 26 is provided herein, wherein at least 10% of the VJ immunoglobulin light chain conjugates in the animal involve non-template addition.

[0244] In exemplary embodiment 28, a genetically modified non-human animal according to embodiment 27 is provided herein, wherein at least 20% of the VJ immunoglobulin light chain conjugates in the animal involve non-template addition.

[0245] In exemplary embodiment 29, a genetically modified non-human animal according to embodiment 27 is provided herein, wherein at least 40% of the VJ immunoglobulin light chain conjugates in the animal involve non-template addition.

[0246] In exemplary embodiment 30, a genetically modified non-human animal according to any one of embodiments 15 to 26 is provided herein, wherein the human immunoglobulin variable region gene segment is a human heavy chain variable region gene segment.

[0247] This specification provides a genetically modified non-human animal according to Embodiment 30, wherein in Exemplary Embodiment 31, the constant region gene is a heavy chain constant region gene.

[0248] In exemplary embodiment 32, the genetically modified non-human animal described herein, as in embodiment 31, is provided, wherein the heavy chain constant region gene is a Cμ gene, a Cδ gene, a Cγ gene, a Cε gene, or a Cα gene.

[0249] In exemplary embodiment 33, the genetically modified non-human animal described herein, as in embodiment 31 or 32, is provided, wherein the heavy chain constant region gene is derived from an endogenous species.

[0250] In exemplary embodiment 34, a genetically modified non-human animal according to embodiment 31 or 32 is provided herein, wherein the heavy chain constant region gene is a mouse constant region gene.

[0251] In exemplary embodiment 35, the genetically modified non-human animal according to embodiment 31 or 32 is provided herein, wherein the heavy chain constant region gene is a rat constant region gene.

[0252] In exemplary embodiment 36, the genetically modified non-human animal according to embodiment 31 or 32 is provided herein, wherein the heavy chain constant region gene is a human constant region gene.

[0253] In exemplary embodiment 37, the genetically modified non-human animal described herein, as in embodiment 31 or 32, is provided, wherein the heavy chain constant region gene has a human CH1 domain and non-human CH2 and CH3 domains.

[0254] In exemplary embodiment 38, the genetically modified non-human animal described in embodiment 37 is provided herein, wherein the non-human CH2 and CH3 domains are derived from an endogenous species.

[0255] In exemplary embodiment 39, the genetically modified non-human animal described herein, as in embodiment 37, is provided, wherein the non-human CH2 and CH3 domains are mouse CH2 and CH3 domains.

[0256] In exemplary embodiment 40, the genetically modified non-human animal described herein, as in embodiment 37, is provided, wherein the non-human CH2 and CH3 domains are rat CH2 and CH3 domains.

[0257] In exemplary embodiment 10, the genetically modified non-human animals described herein, as in embodiments 15 to 40, are provided, in which the animal lacks a functional CH1 domain in the constant region of an immunoglobulin heavy chain selected from IgG, IgA, IgE, IgD, or a combination thereof.

[0258] In exemplary embodiment 20, the immunoglobulin variable region and immunoglobulin constant region genes are located at the endogenous immunoglobulin heavy chain locus, as described herein, in any one of embodiments 31 to 41, and a genetically modified non-human animal is provided herein.

[0259] In exemplary embodiment 43, the genetically modified non-human animal according to any one of embodiments 30 to 42 is provided herein, further comprising in the genome an immunoglobulin variable region including an unreorganized human light chain variable region gene segment operably linked to a second immunoglobulin constant region gene.

[0260] In exemplary embodiment 44, the genetically modified non-human animal described herein is provided, wherein the human immunoglobulin variable region gene segment operably linked to the second immunoglobulin constant region gene is a human κ-chain variable region gene segment.

[0261] In exemplary embodiment 45, the genetically modified non-human animal described herein is provided, wherein the human immunoglobulin variable region gene segment operably linked to the second immunoglobulin constant region gene is a human λ-chain variable region gene segment.

[0262] In exemplary embodiment 46, a genetically modified non-human animal according to any one of embodiments 43 to 45 is provided herein, wherein the second constant region gene is a light chain constant region gene.

[0263] In exemplary embodiment 47, the gene-modified non-human animal according to embodiment 46 is provided herein, wherein the second constant region gene is a κ constant region gene.

[0264] In exemplary embodiment 48, the genetically modified non-human animal described herein, as in embodiment 46, is provided, wherein the second constant region gene is a λ constant region gene.

[0265] In exemplary embodiment 49, a genetically modified non-human animal according to any one of embodiments 43 to 48 is provided herein, wherein the second constant region gene is derived from an endogenous species.

[0266] In exemplary embodiment 50, a genetically modified non-human animal according to any one of embodiments 43 to 48 is provided herein, wherein the second constant region gene is a mouse constant region gene.

[0267] In exemplary embodiment 51, a genetically modified non-human animal according to any one of embodiments 43 to 48 is provided herein, wherein the second constant region gene is a rat constant region gene.

[0268] In exemplary embodiment 52, a genetically modified non-human animal according to any one of embodiments 43 to 48 is provided herein, wherein the second constant region gene is a human constant region gene.

[0269] In exemplary embodiment 53, a genetically modified non-human animal according to any one of embodiments 43 to 52 is provided herein, wherein an immunoglobulin variable region, operably linked to a second immunoglobulin constant region gene, is located at an endogenous immunoglobulin light chain locus.

[0270] This specification provides a genetically modified non-human animal according to Embodiment 53, in exemplary embodiment 54, wherein the second constant region gene is a κ constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin κ locus.

[0271] This specification provides a genetically modified non-human animal according to Embodiment 53, wherein in exemplary Embodiment 55, the second constant region gene is a λ constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin λ locus.

[0272] In exemplary embodiment 56, the genetically modified non-human animal according to any one of embodiments 30 to 42 further comprises an immunoglobulin variable region in the genome, which includes a rearranged human light chain variable region (V / J) gene segment operably linked to a second immunoglobulin constant region gene.

[0273] In exemplary embodiment 57, a rearranged human light chain variable region (V / J) gene segment operably linked to a second immunoglobulin constant region gene comprises a Vκ gene segment selected from Vκ1-39 and Vκ3-20, which is rearranged into a Jκ gene segment, as described herein, and is provided herein.

[0274] In exemplary embodiment 58, the gene-modified non-human animal described herein is provided, wherein the animal contains an immunoglobulin light chain variable region comprising a Vκ1-39 / Jκ5 or Vκ3-20 / Jκ1 sequence in its genome, as described in embodiment 57.

[0275] In exemplary embodiment 59, the genetically modified non-human animal according to any one of embodiments 30 to 42 further comprises an immunoglobulin variable region in the genome, which includes a limited repertoire of human light chain variable region (V and J) gene segments operably linked to a second immunoglobulin constant region gene.

[0276] In exemplary embodiment 60, the genetically modified non-human animal described herein, as in embodiment 59, is provided, wherein the limited repertoire of human light chain variable region (V and J) gene segments operably linked to a second immunoglobulin constant region gene comprises two V gene segments and at least two, preferably five J gene segments.

[0277] In exemplary embodiment 61, the genetically modified non-human animal described herein is provided, wherein the two V gene segments are the Vκ1-39 and Vκ3-20 gene segments.

[0278] In exemplary embodiment 62, a genetically modified non-human animal according to any one of embodiments 15 to 29 is provided herein, wherein the human immunoglobulin variable region gene segment is a human light chain variable region gene segment.

[0279] In exemplary embodiment 63, the genetically modified non-human animal described herein, as in embodiment 62, is provided, wherein the human immunoglobulin variable region gene segment is a human κ-chain variable region gene segment.

[0280] In exemplary embodiment 64, the genetically modified non-human animal described herein, as in embodiment 63, is provided, wherein the human immunoglobulin variable region gene segment is a human λ-chain variable region gene segment.

[0281] In exemplary embodiment 65, a genetically modified non-human animal according to any one of embodiments 62 to 64 is provided herein, wherein the constant region gene is a light chain constant region gene.

[0282] In exemplary embodiment 66, the gene-modified non-human animal according to embodiment 65 is provided herein, wherein the constant region gene is a κ constant region gene.

[0283] This specification provides a genetically modified non-human animal according to embodiment 65, wherein in exemplary embodiment 67, the constant region gene is a λ constant region gene.

[0284] In exemplary embodiment 68, a genetically modified non-human animal according to any one of embodiments 62 to 64 is provided herein, wherein the constant region gene is a heavy chain constant region gene.

[0285] In exemplary embodiment 69, a genetically modified non-human animal according to any one of embodiments 62 to 68 is provided herein, wherein the constant region gene is derived from an endogenous species.

[0286] In exemplary embodiment 70, a genetically modified non-human animal according to any one of embodiments 62 to 68 is provided herein, wherein the constant region gene is a mouse constant region gene.

[0287] In exemplary embodiment 71, a genetically modified non-human animal according to any one of embodiments 62 to 68 is provided herein, wherein the constant region gene is a rat constant region gene.

[0288] In exemplary embodiment 72, a genetically modified non-human animal according to any one of embodiments 62 to 68 is provided herein, wherein the constant region gene is a human constant region gene.

[0289] In exemplary embodiment 73, a genetically modified non-human animal according to any one of embodiments 62 to 72 is provided herein, wherein the immunoglobulin variable region and immunoglobulin constant region genes are located at the endogenous immunoglobulin light chain locus.

[0290] In exemplary embodiment 74, the gene-modified non-human animal described herein is provided, wherein the constant region gene is a κ constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin κ locus.

[0291] In exemplary embodiment 75, the gene-modified non-human animal described herein is provided, wherein the constant region gene is a λ constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin λ locus.

[0292] In an exemplary embodiment 76, the genetically modified non-human animal described herein, according to any one of embodiments 15 to 75, wherein the immunoglobulin variable region comprises an intergeneric sequence of a human-derived immunoglobulin variable region.

[0293] In exemplary embodiment 77, a genetically modified non-human animal according to any one of embodiments 15 to 75 is provided herein, wherein the immunoglobulin variable region includes an intergeneric sequence of an endogenous species immunoglobulin variable region.

[0294] In exemplary embodiment 78, the genetically modified non-human animal according to any one of embodiments 15 to 75 is provided herein, wherein the immunoglobulin variable region comprises an intergeneric sequence of an immunoglobulin variable region derived from a mouse.

[0295] In exemplary embodiment 79, the genetically modified non-human animal according to any one of embodiments 15 to 75 is provided herein, wherein the immunoglobulin variable region comprises an intergeneric sequence of an immunoglobulin variable region derived from a rat.

[0296] In exemplary embodiment 80, a genetically modified non-human animal according to any one of embodiments 15 to 79 is provided herein, further comprising an inactivated endogenous immunoglobulin locus in its genome.

[0297] This specification provides a genetically modified non-human animal according to Embodiment 80, wherein in exemplary Embodiment 81, the inactivated endogenous immunoglobulin locus is an endogenous immunoglobulin heavy chain locus.

[0298] In exemplary embodiment 82, the endogenous immunoglobulin heavy chain locus is inactivated by deletion of at least a portion of the variable region of the endogenous heavy chain locus, as described herein, in which a genetically modified non-human animal according to embodiment 81 is provided.

[0299] In exemplary embodiment 83, the genetically modified non-human animal described herein, as in embodiment 82, is provided, wherein the deletion of at least a portion of the variable region includes a deletion of the J gene segment of the variable region.

[0300] In exemplary embodiment 84, the gene-modified non-human animal described herein is provided, in which the endogenous immunoglobulin heavy chain locus is inactivated by deletion of at least a portion of the constant region of the endogenous heavy chain locus.

[0301] In exemplary embodiment 85, a genetically modified non-human animal according to embodiment 84 is provided herein, wherein the deletion of at least a portion of the constant region includes a deletion of the Cμ gene in the constant region.

[0302] In exemplary embodiment 86, the genetically modified non-human animal according to embodiment 80 is provided herein, wherein the inactivated endogenous immunoglobulin locus is the endogenous immunoglobulin κ chain locus.

[0303] In exemplary embodiment 87, the endogenous immunoglobulin κ chain locus is inactivated by deletion of at least a portion of the variable region of the endogenous κ chain locus, as described herein, in which a genetically modified non-human animal according to embodiment 86 is provided.

[0304] In exemplary embodiment 88, the genetically modified non-human animal described herein is provided, wherein the deletion of at least a portion of the variable region includes a deletion of the J gene segment of the variable region.

[0305] In exemplary embodiment 89, the endogenous immunoglobulin κ locus is inactivated by deletion of at least a portion of the constant region of the endogenous κ chain locus, as described herein, in which a genetically modified non-human animal according to embodiment 86 is provided.

[0306] In exemplary embodiment 90, the genetically modified non-human animal described herein, as in embodiment 89, is provided, wherein the deletion of at least a portion of the constant region includes a deletion of the Cκ gene in the constant region.

[0307] In exemplary embodiment 91, the genetically modified non-human animal described herein, as in embodiment 80, is provided, wherein the inactivated endogenous immunoglobulin locus is the endogenous immunoglobulin λ chain locus.

[0308] In exemplary embodiment 92, the gene-modified non-human animal described herein, as in embodiment 91, is provided, wherein the endogenous immunoglobulin λ-chain locus is inactivated by deletion of at least a portion of the VJC cluster of the endogenous λ-chain locus.

[0309] In exemplary embodiment 93, an unreorganized human immunoglobulin variable region gene segment is rearranged during B cell development to generate a rearranged variable region gene in the B cells of the non-human animal, as described herein, and a genetically modified non-human animal according to any one of embodiments 15 to 92 is provided herein.

[0310] In exemplary embodiment 94, the genetically modified non-human animal described herein, as in embodiment 93, is provided, wherein at least 10% of the rearranged variable region genes include non-template addition.

[0311] In exemplary embodiment 95, the genetically modified non-human animal described herein is provided, wherein at least 20% of the rearranged variable region genes include non-template addition, as described in embodiment 93.

[0312] In exemplary embodiment 96, the genetically modified non-human animal described herein, as in embodiment 93, is provided, wherein at least 40% of the rearranged variable region genes include non-template addition.

[0313] In exemplary embodiment 97, the animal expresses an antibody comprising a variable domain encoded by a rearranged variable region gene and a constant domain encoded by a constant region gene, as described herein, a genetically modified non-human animal according to any one of embodiments 93 to 96.

[0314] In exemplary embodiment 98, a genetically modified non-human animal according to any one of embodiments 15 to 97 is provided herein, further comprising a functionally ectopic mouse Adam6 gene.

[0315] In exemplary embodiment 99, a genetically modified non-human animal is provided herein, comprising a T cell receptor (TCR) variable region in its genome, which includes a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyltransferase (TdT) and a non-reorganized human TCR variable region gene segment operably linked to a TCR constant region gene.

[0316] In exemplary embodiment 100, the gene-modified non-human animal described herein, in which the exogenous TdT is human TdT, is provided.

[0317] In exemplary embodiment 101, a genetically modified non-human animal according to embodiment 99 or 100 is provided herein, wherein a nucleic acid sequence encoding exogenous TdT is operably linked to a transcriptional regulatory element.

[0318] In exemplary embodiment 102, the gene-modified non-human animal described herein is provided, wherein the transcriptional regulatory element drives the expression of a nucleic acid sequence encoding exogenous TdT in CD4 / CD8 double-negative (DN) thymocytes and / or CD4 / CD8 double-positive (DP) thymocytes.

[0319] The following gene-modified non-human animal according to Embodiment 101 is provided herein, wherein in exemplary embodiment 103, the transcriptional regulatory element is a RAG1 transcriptional regulatory element, a RAG2 transcriptional regulatory element, a TCRα transcriptional regulatory element, a TCRβ transcriptional regulatory element, a TCRγ transcriptional regulatory element, and / or a TCRδ transcriptional regulatory element.

[0320] In exemplary embodiment 104, the gene-modified non-human animal described herein is provided as described in any one of embodiments 99 to 103, wherein the non-human animal expresses exogenous TdT in DN thymocytes and / or DP thymocytes.

[0321] In exemplary embodiment 105, a genetically modified non-human animal according to any one of embodiments 99 to 104 is provided herein, wherein the nucleic acid sequence encoding exogenous TdT is located at the RAG1 locus, RAG2 locus, TCRα chain locus, TCRβ chain locus, TCRγ chain locus and / or TCRδ chain locus.

[0322] In exemplary embodiment 106, a genetically modified non-human animal according to any one of embodiments 99 to 105 is provided herein, wherein the nucleic acid sequence encoding exogenous TdT is not operably linked to a constitutive transcriptional regulatory element.

[0323] In exemplary embodiment 107, the gene-modified non-human animal according to any one of embodiments 99 to 106 is provided herein, wherein exogenous TdT is not constitutively expressed.

[0324] In exemplary embodiment 108, a genetically modified non-human animal according to any one of embodiments 99 to 107 is provided herein, wherein the human TCR variable region gene segment is a human TCRα variable region gene segment.

[0325] In exemplary embodiment 109, a genetically modified non-human animal according to any one of embodiments 99 to 107 is provided herein, wherein the human TCR variable region gene segment is a human TCRβ variable region gene segment.

[0326] In exemplary embodiment 110, a genetically modified non-human animal according to any one of embodiments 99 to 108 is provided herein, wherein the TCR constant region gene is a TCRα constant region gene.

[0327] In exemplary embodiment 111, a genetically modified non-human animal according to any one of embodiments 99 to 107 and 109 is provided herein, wherein the TCR constant region gene is a TCRβ constant region gene.

[0328] In exemplary embodiment 112, a genetically modified non-human animal according to any one of embodiments 99 to 111 is provided herein, wherein the TCR constant region gene is derived from an endogenous species.

[0329] In exemplary embodiment 113, a genetically modified non-human animal according to any one of embodiments 99 to 111 is provided herein, wherein the TCR constant region gene is a mouse constant region gene.

[0330] In exemplary embodiment 114, the gene-modified non-human animal according to any one of embodiments 99 to 111 is provided herein, wherein the TCR constant region gene is a rat constant region gene.

[0331] In exemplary embodiment 115, a genetically modified non-human animal according to any one of embodiments 99 ...

Claims

[Claim 1] The invention as shown in the drawings.

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