Non-human animals expressing exogenous terminal deoxynucleotidyl transferase

Abstract

To provide non-human animals expressing exogenous terminal deoxynucleotidyltransferase.SOLUTION: Provided herein are methods and compositions relating to non-human animals that express exogenous terminal deoxynucleotidyltransferase (TdT). In certain aspects, provided herein are genetically modified non-human animals that include in their genome an exogenous nucleic acid encoding terminal deoxynucleotidyltransferase (TdT), as well as production and use methods of such non-human animals. In some embodiments, the exogenous TdT is human TdT. In some embodiments, the exogenous TdT is from an endogenous species (eg, in mice, exogenous TdT has a mouse sequence).SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Non-human animals, especially mice and rats, have proven to be a valuable source of therapeutic antibodies and can potentially serve as a source of other antigen-binding molecules.The high level of antigen receptor diversity in such non-human animals increases the likelihood that antigen-binding molecules with desired therapeutic properties will be generated after immunization.Therefore, there is a need for genetically engineered non-human animals with increased antigen receptor diversity to improve the production of therapeutic antigen-binding molecules. Summary of the Invention [Means for solving the problem]

[0003] In certain aspects, provided herein are genetically modified non-human animals comprising in their genomes an exogenous nucleic acid encoding terminal deoxynucleotidyl transferase (TdT), as well as methods for making and using such non-human animals. In some embodiments, the exogenous TdT is human TdT. In some embodiments, the exogenous TdT is derived from an endogenous species (e.g., in a mouse, 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, e.g., 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, e.g., in double-negative (DN) thymocytes and / or double-positive (DP) thymocytes. In some embodiments, the genetically modified non-human animals comprise multiple copies (e.g., at least 2, 3, 4, 5, 6, 7, or 8 copies) of an 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 a rat).

[0004] In some embodiments, the genetically modified non-human animal comprises in its genome an immunoglobulin variable region comprising an unrearranged human immunoglobulin variable region gene segment (e.g., a heavy chain gene segment, a kappa chain gene segment, a lambda chain gene segment) operably linked to an immunoglobulin constant region gene (e.g., a heavy chain constant region gene, a kappa chain constant region gene, a lambda 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 from an endogenous species. In some embodiments, the variable region and constant region genes are located at an endogenous immunoglobulin locus (e.g., a heavy chain locus, a kappa locus, a lambda 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 the immunoglobulin constant region gene. In some embodiments, provided herein are methods of using such genetically modified non-human animals to generate antibodies, B cells, hybridomas, or nucleic acids encoding human immunoglobulin variable domains.

[0005] In certain embodiments, the genetically modified non-human animal comprises in its genome a T cell receptor (TCR) variable region comprising an unrearranged human TCR variable region gene segment (e.g., a TCR alpha gene segment, a TCR beta gene segment, a TCR gamma gene segment, a TCR delta gene segment) operably linked to a TCR constant region gene (e.g., a TCR alpha constant region gene, a TCR beta constant region gene, a TCR gamma constant region gene, a TCR delta 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 derived from an endogenous species. In some embodiments, the variable region and constant region genes are located at an endogenous TCR locus (e.g., a TCR alpha locus, a TCR beta locus, a TCR gamma locus, a TCR delta locus). In some embodiments, the genetically modified non-human organism expresses a TCR comprising a human TCR variable domain derived from a TCR variable region and a TCR constant domain encoded by a TCR constant region gene. In some embodiments, provided herein are methods of using such genetically modified non-human animals to generate TCRs, T cells, T cell hybridomas, or nucleic acids encoding human TCR variable domains.

[0006] In some embodiments, the genetically modified non-human animal comprises in its genome an immunoglobulin variable region comprising an unrearranged human immunoglobulin variable region gene segment (e.g., a heavy chain gene segment, a kappa chain gene segment, a lambda chain gene segment) operably linked to a TCR constant region gene (e.g., a TCR alpha constant region gene, a TCR beta constant region gene, a TCR gamma constant region gene, a TCR delta 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 derived from an endogenous species. In some embodiments, the variable region and constant region genes are located at an endogenous TCR locus (e.g., a TCR alpha locus, a TCR beta locus, a TCR gamma locus, a TCR delta locus). In some embodiments, the genetically modified non-human organism expresses a chimeric antigen receptor (CAR) comprising a human immunoglobulin variable domain derived from the immunoglobulin variable region and a TCR constant domain encoded by the TCR constant region gene. In some embodiments, provided herein are methods of using such genetically modified non-human animals to generate CARs, T cells, T cell hybridomas, or nucleic acids encoding human immunoglobulin variable domains.

[0007] In some embodiments, provided herein are methods of making a non-human animal disclosed herein, comprising engineering the non-human animal to contain in its germline a genetic modification described herein. In some embodiments, provided herein are non-human ES cells comprising a genetic modification described herein. The present invention provides, for example, the following items. (Item 1) 1. A genetically modified non-human animal comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operably linked to a transcriptional control element; and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to an immunoglobulin constant region gene. (Item 2) 2. The genetically modified non-human animal of item 1, wherein the transcriptional control element drives expression of a nucleic acid sequence encoding exogenous TdT in pro-B cells and / or pre-B cells. (Item 3) 3. The genetically modified non-human animal of item 2, wherein the transcriptional control element is selected from the group consisting of a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin kappa light chain transcriptional control element, and / or an immunoglobulin lambda light chain transcriptional control element. (Item 4) 4. The 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 an immunoglobulin κ light chain locus, an immunoglobulin λ light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus. (Item 5) 5. The genetically modified non-human animal of any one of items 1 to 4, wherein at least 10% of the V-J immunoglobulin light chain linkages in the animal comprise non-templated additions. (Item 6) 6. The 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) 7. The genetically modified non-human animal according to Item 6, wherein the constant region gene is a heavy chain constant region gene. (Item 8) 8. The genetically modified non-human animal of item 7, wherein the heavy chain constant region gene is derived from an endogenous species. (Item 9) 9. The genetically modified non-human animal according to any one of items 6 to 8, wherein the immunoglobulin variable region and the immunoglobulin constant region genes are located in an endogenous immunoglobulin heavy chain locus. (Item 10) 10. The genetically modified non-human animal of any one of Items 6 to 9, further comprising an immunoglobulin variable region in its genome comprising an unrearranged human light chain variable region gene segment operably linked to a second immunoglobulin constant region gene. (Item 11) 11. The genetically modified non-human animal of claim 10, wherein the immunoglobulin variable region operably linked to the second immunoglobulin constant region gene is located at an endogenous immunoglobulin light chain locus. (Item 12) 12. The genetically modified non-human animal of claim 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) 12. The genetically modified non-human animal of claim 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) 14. The genetically modified non-human animal according to any one of items 11 to 13, wherein the second constant region gene is a κ constant region gene. (Item 15) 14. The genetically modified non-human animal according to any one of items 11 to 13, wherein the second constant region gene is a λ constant region gene. (Item 16) 15. The genetically modified non-human animal of item 14, wherein the kappa constant region gene is derived from an endogenous species. (Item 17) 16. The genetically modified non-human animal of item 15, wherein the lambda constant region gene is derived from an endogenous species. (Item 18) 17. The genetically modified non-human animal of item 16, wherein the endogenous immunoglobulin light chain locus is an immunoglobulin κ locus. (Item 19) 18. The genetically modified non-human animal of item 17, wherein the endogenous immunoglobulin light chain locus is an immunoglobulin λ locus. (Item 20) 20. The genetically modified non-human animal of any one of items 1 to 19, wherein the unrearranged human immunoglobulin variable region gene segments are rearranged during B cell development to generate rearranged variable region genes in the B cells of the non-human animal. (Item 21) 21. The genetically modified non-human animal of item 20, wherein at least 10% of the rearranged variable region genes comprise non-templated additions. (Item 22) 1. A genetically modified non-human animal comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operably linked to a transcriptional control element; and a T cell receptor (TCR) variable region comprising unrearranged human TCR variable region gene segments operably linked to a TCR constant region gene. (Item 23) 23. The genetically modified non-human animal of claim 22, wherein the human TCR variable region gene segment is a human TCR alpha variable region gene segment. (Item 24) 23. The genetically modified non-human animal of claim 22, wherein the human TCR variable region gene segment is a human TCR β variable region gene segment. (Item 25) 25. The genetically modified non-human animal of any one of Aspects 22 to 24, wherein the unrearranged human TCR variable region gene segments are rearranged during T cell development to generate rearranged TCR variable region genes in the T cells of the non-human animal. (Item 26) 26. The genetically modified non-human animal of item 25, wherein at least 10% of the rearranged variable region genes comprise non-templated additions. (Item 27) 1. A genetically modified non-human animal comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT) operably linked to a transcriptional control element; and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to a TCR constant region gene. (Item 28) 28. The genetically modified non-human animal of item 27, wherein the human immunoglobulin variable region gene segment is a heavy chain gene segment. (Item 29) 28. The genetically modified non-human animal of item 27, wherein the human immunoglobulin variable region gene segment is a κ chain gene segment. (Item 30) 28. The genetically modified non-human animal of item 27, wherein the human immunoglobulin variable region gene segment is a lambda chain gene segment. (Item 31) 30. The genetically modified non-human animal of any one of items 28 or 29, wherein the TCR constant region gene is a TCR alpha constant region gene. (Item 32) 29. The genetically modified non-human animal of Item 28, wherein the TCR constant region gene is a TCR β constant region gene. (Item 33) 33. The genetically modified non-human animal of any one of items 22 to 32, wherein the transcriptional control element drives 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) 34. The genetically modified non-human animal of claim 33, wherein the transcriptional control element is a RAG1 transcriptional control element, a RAG2 transcriptional control element, a TCR alpha transcriptional control element, a TCR beta transcriptional control element, a TCR gamma transcriptional control element, and / or a TCR delta transcriptional control element. (Item 35) 35. The genetically modified non-human animal of any one of Items 22 to 34, wherein the nucleic acid sequence encoding the exogenous TdT is located at the RAG1 locus, the RAG2 locus, the TCR α chain locus, the TCR β chain locus, the TCR γ chain locus, and / or the TCR δ chain locus. (Item 36) 36. The genetically modified non-human animal according to any one of items 1 to 35, wherein the non-human animal is a mammal. (Item 37) 37. The genetically modified non-human animal of claim 36, wherein the mammal is a rodent. (Item 38) Item 39. The genetically modified non-human animal according to Item 37, wherein the rodent is a rat or a mouse. 22. A method for inducing expression of an antibody comprising a human variable domain, the method comprising exposing the genetically modified non-human animal according to any one of items 1 to 21 to an antigen, thereby causing the genetically modified non-human animal to produce an antibody comprising a human variable domain specific to the antigen. (Item 40) 1. A method for generating T cells that express a T cell receptor (TCR) comprising a human variable domain specific for a peptide presented on MHC, comprising: (a) exposing the genetically modified non-human animal according to any one of items 22 to 26 to an antigen comprising a peptide or a nucleic acid encoding an antigen comprising a peptide, whereby the peptide is presented on MHC in the non-human animal; (b) obtaining T cells expressing a TCR specific for the peptide presented on the MHC from the genetically modified non-human animal of (a). (Item 41) 1. A method for generating T cells expressing a chimeric antigen receptor (CAR) comprising a human immunoglobulin variable domain and an immunoglobulin constant, specific for a peptide presented on MHC, comprising: (a) exposing the genetically modified non-human animal according to any one of items 27 to 32 to an antigen comprising a peptide or a nucleic acid encoding an antigen comprising a peptide, whereby the peptide is presented on MHC in the non-human animal; (b) obtaining from the genetically modified non-human animal of (a) a T cell expressing a CAR specific for the peptide presented on the MHC. (Item 42) 1. A genetically modified non-human animal ES cell comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to an immunoglobulin constant region gene. (Item 43) 1. A genetically modified non-human animal ES cell comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); A genetically modified non-human animal ES cell comprising: a T cell receptor (TCR) variable region comprising an unrearranged human TCR variable region gene segment operably linked to a TCR constant region gene. (Item 44) 1. A genetically modified non-human animal ES cell comprising in its genome: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and an immunoglobulin variable region comprising an unrearranged 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 the genetically modified non-human animal ES cell according to any one of Items 42 to 44. (Item 46) 1. A method of producing a non-human animal comprising a genetic modification, wherein the non-human animal comprises in its germline: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to an immunoglobulin constant region gene. (Item 47) 1. A method of producing a non-human animal comprising a genetic modification, wherein the non-human animal comprises in its germline: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and a T cell receptor (TCR) variable region comprising unrearranged human TCR variable region gene segments operably linked to a TCR constant region gene. (Item 48) 1. A method of producing a non-human animal comprising a genetic modification, wherein the non-human animal comprises in its germline: a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT); and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to a TCR constant region gene. (Item 49) 49. The genetically modified non-human animal, non-human animal ES cell, or method according to any one of items 1 to 48, wherein the terminal deoxynucleotidyl transferase (TdT) is human TdT. (Item 50) 50. The genetically modified non-human animal, non-human animal ES cell, or method according to any one of items 1 to 49, wherein the terminal deoxynucleotidyl transferase (TdT) is the short isoform of TdT (TdTS). [Brief explanation of the drawings]

[0008] [Figure 1]A diagram (not to scale) of an exemplary targeting vector is shown, in which a portion of the mouse Rag2 gene has been replaced with a DNA sequence encoding the short isoform human TdT (hTdTs). In an exemplary embodiment, the vector is randomly integrated into the genome. Unless labeling in the diagram indicates otherwise (e.g., with respect to selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, while 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. Junctions 1-4 correspond to the junctions shown in Table 1.

[0009] [Figure 2] A diagram (not to scale) of an exemplary targeting vector is shown in which a portion of the mouse Rag2 gene has been replaced with a DNA sequence encoding the short isoform human TdT (hTdTs). In the illustrated embodiment, the vector is used to insert hTdT driven by the mouse RAG2 promoter into the Igκ locus. Unless labeling in the diagram indicates otherwise (e.g., with respect to selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, while 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. Junctions 1-7 correspond to the junctions in Table 2.

[0010] [Figure 3]A diagram (not to scale) of an exemplary targeting vector used to insert a DNA sequence encoding human TdT (hTdTs) driven by the VH1-72 promoter and Eμ enhancer into the immunoglobulin κ locus is shown. Unless labeling in the figure indicates otherwise (e.g., with respect to selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, while 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. Junctions 1 through 4 correspond to the junctions in Table 3.

[0011] [Figure 4] Figure 1 shows hTdT mRNA expression in lymphocytes from VELOCIMMUNE® TdT mice compared to VELOCIMMUNE® control mice. VELOCIMMUNE® mice herein are mice that contain a diverse repertoire of unrearranged human heavy chain and kappa light chain variable (V(D)J) gene segments. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0012] [Figure 5] 1 shows a graph depicting 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] Graph showing the distribution of hIgκ non-templated additions 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] It has two panels. Panel (A) shows a graph depicting the distribution of hIgκ CDR3 lengths in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "AA" represents amino acid. Panel (B) shows a graph depicting the frequency of exonuclease deletion lengths 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] It has two panels. Panel (A) shows a graph depicting the distribution of hIgκ CDR3 lengths in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. "AA" represents amino acid. Panel (B) shows a graph depicting the frequency of exonuclease deletion lengths 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] There are two panels. Panel (A) shows a graph depicting Vκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Panel (B) shows a graph depicting Jκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice. [Figure 8-2]There are two panels. Panel (A) shows a graph depicting Vκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Panel (B) shows a graph depicting Jκ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice, and HO indicates homozygous mice.

[0016] [Figure 9] 1 shows a graph depicting 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] Graph showing the distribution of mIgλ non-templated additions 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] 1 shows a graph depicting the distribution of mIgλ CDR3 lengths 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] 1 shows a graph depicting Vλ usage in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice. Het indicates heterozygous mice and HO indicates homozygous mice.

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

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

[0022] [Figure 15] 1 shows a graph depicting the distribution of hIgκ CDR3 lengths in DLC mice expressing hTdT compared to DLC control mice not expressing hTdT. Het indicates mice heterozygous for hTdT, and HO indicates mice homozygous for hTdT.

[0023] [Figure 16] Figure 1 shows a graph depicting Vκ and Jκ usage in DLC mice expressing hTdT compared to DLC control mice that do not express hTdT. Het indicates mice heterozygous for hTdT, and HO indicates mice homozygous for hTdT. Only two different Rag TdT tg(HO) DLC mice are shown separately. DETAILED DESCRIPTION OF THE INVENTION

[0024] General Provided herein are methods and compositions related to non-human animals that contain an exogenous nucleic acid encoding TdT (e.g., human, mouse, or rat TdT) in their genome. In some embodiments, the genetically modified non-human animal is a mammal, such as a rodent (e.g., a mouse or a rat). In certain embodiments, the genome of the non-human animal contains further modifications such that it expresses an antigen-binding molecule (e.g., an antibody, TCR, and / or CAR) with a human variable domain.

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

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

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

[0028] As used herein, the term "antibody" can refer to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as V H Each light chain comprises a light chain variable region (abbreviated herein as V) and a heavy chain constant region. L and the light chain constant region. H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "antibody" also includes single-domain antibodies, antibodies with only a heavy chain, and antibodies with a light-chain variable gene segment on the heavy chain.

[0029] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antigen-binding molecule (e.g., an antibody, T cell receptor (TCR), chimeric antigen receptor (CAR)) refer to one or more fragments of the antigen-binding molecule that retain the ability to bind to an antigen. An antigen-binding fragment can include any antibody, TCR, or CAR fragment that retains at least a portion of the variable region of the 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-linked 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 reference to a non-human animal is used to describe the characteristics of 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, whereas the corresponding non-human animal does not.

[0031] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen-binding protein comprising 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 comprises the membrane-proximal TCR constant domain and may also comprise the TCR transmembrane domain and / or the TCR cytoplasmic tail. For example, in some embodiments, a CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR β constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to a TCR α constant domain. In some embodiments, a CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR α constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a κ or λ variable domain) linked to a TCR β constant domain.

[0032] When used in reference to a rearranged variable region gene or variable domain that is "derived from" an unrearranged variable region and / or unrearranged variable region gene segment, the phrase "derived 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 differential splicing and somatic mutations, if applicable). For example, a rearranged variable region gene that has undergone somatic mutation does not change the fact that it is derived 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 related genetic elements (e.g., genes, gene segments, regulatory elements). For example, an unrearranged immunoglobulin locus may contain immunoglobulin variable region gene segments that induce V(D)J recombination and immunoglobulin expression, one or more immunoglobulin constant region genes, and associated regulatory elements (e.g., promoters, enhancers, switch elements, etc.), while an unrearranged TCR locus may contain TCR variable region gene segments that induce V(D)J recombination and TCR expression, TCR constant region genes, and associated regulatory elements (e.g., promoters, enhancers, etc.). Similarly, an unrearranged CAR locus may contain immunoglobulin variable region gene segments that induce V(D)J recombination and CAR expression, TCR constant region genes, and associated regulatory elements (e.g., promoters, enhancers, etc.). A locus may be endogenous or non-endogenous. The term "endogenous locus" refers to the location on a chromosome where a particular genetic element is naturally found.

[0034] An unrearranged variable region gene segment is "operably linked" to an adjacent constant region gene if the unrearranged variable region gene segments are capable of rearranging to form a rearranged variable region gene that is expressed in conjunction with the 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 polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function. Coding or non-coding regions of a gene or gene fragment, locus(s) 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. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Polynucleotides may be further modified, for example, by conjugation with 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 predetermined target, e.g., a predetermined antigen. Typically, an antigen-binding molecule binds to a target of about 10 -7 K below M D specifically binds to its given target with an affinity corresponding to its K for a nonspecific, unrelated target (e.g., BSA, casein) D at least 10-fold lower, at least 100-fold lower, or at least 1000-fold lower than D In some embodiments, the antigen-binding molecule binds to a predetermined target with an affinity corresponding to about 10 -8 M or less, 10 -9 M or less, or 10 -10 K below M D The target specifically binds to its predetermined target with an affinity corresponding to

[0037] As used herein, "T cell receptor" or "TCR" refers to an antigen-binding protein that comprises 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 comprises the membrane-proximal TCR constant domain and may also comprise the TCR transmembrane domain and / or the TCR cytoplasmic tail. In certain embodiments, the TCR is a soluble TCR and does not comprise the TCR transmembrane domain or the TCR cytoplasmic tail. For example, in some embodiments, the TCR is a dimer comprising a first polypeptide comprising a TCR β variable domain linked to a TCR β constant domain (or fragment thereof) and a second polypeptide comprising a TCR α linked to a TCR α constant domain (or fragment thereof).

[0038] The term "unrearranged" includes the state of immunoglobulin, TCR or CAR variable region loci or variable region gene segments in which the V and J gene segments (and for heavy or TCR beta variable regions, the D gene segments as well) are maintained separately but can be joined to form rearranged V(D)J genes ("variable region genes") comprising a single V, (D), J of the V(D)J repertoire.

[0039] Genetically modified non-human animals and ES cells In certain aspects, provided herein are non-human animals and ES cells that comprise an exogenous nucleic acid encoding TdT (e.g., human, mouse, or rat TdT) in their genomes. In certain embodiments, the genomes of the non-human animals and ES cells comprise additional modifications, including, for example, modifications that result in the expression of antigen-binding molecules (e.g., antibodies, TCRs, and / or CARs) with human variable domains.

[0040] The genetically modified non-human animals and ES cells provided herein can be produced by any suitable method known in the art.For example, the non-human animal ES cells that contain targeted genetic modification can be produced by using VELOCIGENE® technology, as described in U.S. Patent Nos. 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, and U.S. Patent Application Publication No. 2014 / 0310828, each of which is incorporated herein by reference. Targeted modification can also be performed using the CRISPR / Cas system described, for example, in U.S. Patent No. 9,228,208, and U.S. Patent Application Publication Nos. 2015 / 0159174A1, 2016 / 0060657A1, 2015 / 0376650A1, 2015 / 0376651A1, 2016 / 0046960A1, 2015 / 0376628A1, and 2016 / 0115486A1, each of which is incorporated by reference in its entirety. Targeted modification can also be performed using the meganuclease described, for example, in U.S. Patent Nos. 8,703,485, 8,530,214, and 8,624,000, each of which is incorporated by reference in its entirety. Non-targeted genetic modifications can be performed using standard methods described, for example, in U.S. Patent Nos. 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 its entirety.

[0041] The ES cells described herein can be used to generate non-human animals using methods known in the art.For example, the mouse non-human animal ES cells described herein can be used to generate 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 of which is incorporated herein by reference.Rat ES cells can be used to generate modified rats, for example, using the method described in U.S. Patent Application Publication No. 2014 / 0310828, which is incorporated herein by reference.The obtained mice or rats can be bred to homozygotes.Multiple different modifications can be combined in a single genetically modified organism, either by breeding separately modified animals or by introducing additional modifications into already modified ES cells (for example, using the method 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 animal described herein can be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods can be used to generate non-human animals containing the genetic modifications described herein. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell), using nuclear transfer to transfer the modified genome into a suitable cell, such as an oocyte, and gestation of the modified cell (e.g., the modified oocyte) in the non-human animal under conditions suitable for forming an embryo.

[0043] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal, for example, of the superfamily Dipodoidea or Muroidea. In some embodiments, the non-human animal is a rodent. In particular 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 from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (e.g., true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (e.g., climbing mice, rock mice, white-tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny mice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In some embodiments, the rodent is selected from true mice and rats (family Muridae), gerbils, spiny mice, and crested rats. In some embodiments, the mouse is from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[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, the 129 strain is selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. In some embodiments, the genetically modified mouse is a mixture of 129 strains and C57BL strains. In some embodiments, the mouse is a mixture of 129 strains and / or a mixture of C57BL / 6 strains. In some embodiments, the 129 strain in the mixture is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mice are 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 animals provided herein can be mice derived from any combination of the aforementioned strains.

[0045] In some embodiments, the non-human animal provided herein is a rat. In some embodiments, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is selected from Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti. 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 expressing exogenous TdT In certain embodiments, provided herein are genetically modified non-human animals and ES cells that contain a nucleic acid sequence encoding exogenous terminal deoxynucleotidyl transferase (TdT) in their germline and / or genome. Deoxynucleotidyl transferase (TdT) is a DNA polymerase that catalyzes non-templated nucleotide addition (NP addition) during junction formation in V(D)J recombination, resulting in increased antigen-receptor diversity in B and T lymphocytes. Non-templated addition, non-templated 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 sequence of exogenous TdT in the genome of the genetically modified non-human animal can be from any animal encoding TdT or a TdT ortholog. In some embodiments, the TdT is vertebrate TdT. In some embodiments, the TdT is mammalian TdT. In some embodiments, the TdT is 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, the TdT is from an endogenous species (i.e., the TdT sequence is from the same species as the genetically modified non-human animal). In some embodiments, the 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 (ie, the 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 for hTdT can be found at positions 96304328-96338564 in NCBI accession number NC_000010.11, which is incorporated herein by reference. Exemplary mRNA sequences for hTdT isoforms are provided by NCBI accession numbers NM_001017520.1 and NM_004088.3, ​​each of which is incorporated herein by reference. Protein sequences encoded by these isoforms are provided by NCBI accession numbers NP_001017520.1 and NP_004079.3, respectively, each of which is incorporated herein by reference. Among TdT isoforms, there is 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 is 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 comprises an exogenous nucleic acid sequence encoding multiple isoforms (e.g., both hTdTS and hTdTL2). In certain embodiments, the non-human organism comprises 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 for mTdT can be found at positions 41029275-41059525 in NCBI accession number NC_000085.6, which is incorporated herein by reference. Exemplary mRNA sequences for 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, respectively, 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 positions 260289626-260321174 in NCBI accession number NC_005100.4, 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 the genetically modified non-human animal comprises multiple copies of a nucleic acid sequence encoding exogenous TdT. In some embodiments, the genetically modified non-human animal comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies of a nucleic acid sequence encoding exogenous TdT. In some embodiments, the genetically modified non-human animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies of a nucleic acid sequence encoding exogenous TdT.

[0052] In some embodiments, the nucleic acid sequence encoding exogenous TdT is operably linked to one or more transcriptional control elements (e.g., promoters and / or enhancers). In some embodiments, the transcriptional control element is a constitutive (i.e., ubiquitous) promoter. Examples of constitutive promoters include, but are not limited to, 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 linked to a constitutive promoter.

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

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

[0055] In some embodiments, the nucleic acid encoding TdT is located in the genome of the non-human animal at or proximal to (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) the genomic locus 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 or proximal to an immunoglobulin kappa light chain locus, an immunoglobulin lambda light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus.

[0056] In some embodiments, the nucleic acid encoding TdT is located in the genome of the non-human animal at or proximal to (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) the genomic locus of a gene expressed during T cell development in DN and / or DP thymocytes. In some embodiments, the nucleic acid sequence encoding TdT is located at or proximal to the TCR alpha chain locus, TCR beta chain locus, TCR gamma chain locus, TCR delta chain locus, RAG1 locus, 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 a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its 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 development than the corresponding non-human animal.

[0058] In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ chain junctions that contain non-templated 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 V-J immunoglobulin κ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin κ chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that do not contain non-templated 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% less than the proportion of V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ-chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that contain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin κ-chain junctions in the animal contain non-templated 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% greater than 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 V-J immunoglobulin λ chain joints that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain non-templated 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 V-J immunoglobulin λ chain joints in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin λ chain joints that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that do not contain non-templated 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% less than the proportion of V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin λ chain joints that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain 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 V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ immunoglobulin lambda chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin λ chain junctions in the animal contain non-templated 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% greater than 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 greater proportion of VD immunoglobulin heavy chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD immunoglobulin heavy chain junctions that contain non-templated 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD immunoglobulin heavy chain junctions that do not contain non-templated 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% less than the proportion of VD immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD immunoglobulin heavy chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD immunoglobulin heavy chain junctions that contain 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 immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of VD immunoglobulin heavy chain junctions that contain at least two N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of VD immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of VD immunoglobulin heavy chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD immunoglobulin heavy chain junctions containing at least four N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD immunoglobulin heavy chain 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 immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD immunoglobulin heavy chain junctions in the animal contain non-templated 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% greater than the corresponding non-human animal.

[0061] In some embodiments, the non-human animals provided herein have a higher proportion of D-J immunoglobulin heavy chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J immunoglobulin heavy chain junctions that contain non-templated 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 D-J immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of D-J immunoglobulin heavy chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J immunoglobulin heavy chain junctions that do not contain non-templated 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% less than the proportion of D-J immunoglobulin heavy chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of D-J immunoglobulin heavy chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J immunoglobulin heavy chain junctions that contain 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 D-J immunoglobulin heavy chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of D-J immunoglobulin heavy chain junctions that contain at least two N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of D-J immunoglobulin heavy chain junctions comprising 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 D-J immunoglobulin heavy chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of D-J immunoglobulin heavy chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J immunoglobulin heavy chain junctions comprising 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 D-J immunoglobulin heavy chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a higher proportion of D-J immunoglobulin heavy chain junctions containing at least four N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J immunoglobulin heavy chain 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 D-J immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the D-J immunoglobulin heavy chain junctions in the animal contain non-templated additions.

[0062] In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR α-chain junctions that contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of V-J TCR α-chain junctions that contain non-templated 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 V-J TCR α-chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J TCR α-chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of V-J TCR α-chain junctions that do not contain non-templated 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% less than the proportion of V-J TCR α-chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR α-chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR α-chain junctions that contain 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 V-J TCR α-chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J TCR α-chain junctions comprising at least two N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome, In some embodiments, the proportion of V-J TCR α-chain junctions comprising 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 V-J TCR α-chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a proportion of V-J TCR α-chain junctions that contain at least three N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of V-J TCR α-chain junctions that contain 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 V-J TCR α-chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J TCR α-chain junctions that contain at least four N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VJ TCR α-chain junctions comprising 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 TCR α-chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ TCR α-chain junctions in the animal comprise non-templated additions. In some embodiments, the non-human animal has 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% greater than the corresponding non-human animal.

[0063] In some embodiments, the non-human animals provided herein have a greater proportion of VD TCR β chain junctions that contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR β chain junctions that contain non-templated 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 TCR β chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a smaller proportion of VD TCR β chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR β chain junctions that do not contain non-templated 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% less than the proportion of VD TCR β chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCR β chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD TCR β chain junctions that contain 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 TCR β chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCR β chain junctions comprising at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR β chain junctions comprising 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 TCR β chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of VD TCR β chain junctions containing at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR β chain 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 VD TCR β chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCR β chain junctions containing at least four N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR β chain 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 TCR β chain junctions in the corresponding non-human animal. the proportion of VD TCR β chain junctions is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of VD TCR β chain junctions in the animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD TCR β chain junctions in the animal comprise non-templated additions. In some embodiments, the non-human animal has a frequency of unique TCR β 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 the proportion of VD TCR β chain junctions in the animal.

[0064] In some embodiments, the non-human animals provided herein have a greater proportion of D-J TCR β chain junctions that contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR β chain junctions that contain non-templated 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 D-J TCR β chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a smaller proportion of D-J TCR β chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR β chain junctions that do not contain non-templated 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% less than the proportion of D-J TCR β chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of D-J TCR β chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J TCR β chain junctions that contain 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 D-J TCR β chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of D-J TCR β chain junctions comprising at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome, In some embodiments, the proportion of D-J TCR β chain junctions comprising 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 D-J TCR β chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of D-J TCR β chain junctions comprising at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR β chain junctions comprising 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 D-J TCR β chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of D-J TCR β chain junctions comprising at least four N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR β chain junctions comprising 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 D-J TCR β chain junctions in the corresponding non-human animal. at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, or 40% greater than the proportion of TCR β chain junctions. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the DJ TCR β chain junctions in the animal comprise non-templated additions.

[0065] In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR gamma chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR gamma chain junctions that contain non-templated 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 V-J TCR gamma chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J TCR gamma chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR gamma chain junctions that do not contain non-templated 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% less than the proportion of V-J TCR gamma chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR gamma chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR gamma chain junctions that contain 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 V-J TCR gamma chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR gamma chain junctions comprising at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome, hi some embodiments, the proportion of V-J TCR gamma chain junctions comprising 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 V-J TCR gamma chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a proportion of V-J TCR gamma chain junctions that contain at least three N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of V-J TCR gamma chain junctions that contain 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 V-J TCR gamma chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J TCR gamma chain junctions that contain at least four N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VJ TCR gamma chain junctions comprising 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 TCR gamma chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ TCR gamma chain junctions in the animal comprise non-templated additions. In some embodiments, the non-human animal has 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% greater than the corresponding non-human animal.

[0066] In some embodiments, the non-human animals provided herein have a higher proportion of VD TCR δ chain junctions that contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR δ chain junctions that contain non-templated 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 TCR δ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCR δ chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR δ chain junctions that do not contain non-templated 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% less than the proportion of VD TCR δ chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCR δ chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD TCR δ chain junctions that contain 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 TCR δ chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VD TCR δ chain junctions comprising at least two N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD TCR δ chain junctions comprising 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 TCR δ chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of VD TCR δ chain junctions containing at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR δ chain 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 VD TCR δ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD TCR δ chain junctions containing at least four N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD TCR delta chain junctions comprising 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 TCR delta chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD TCR delta chain junctions in the animal comprise non-templated additions. In some embodiments, the non-human animal has 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% greater than the corresponding non-human animal.

[0067] In some embodiments, the non-human animals provided herein have a higher proportion of D-J TCR delta chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J TCR delta chain junctions that contain non-templated 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 D-J TCR delta chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of D-J TCR delta chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J TCR delta chain junctions that do not contain non-templated 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% less than the proportion of D-J TCR delta chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of D-J TCR delta chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of D-J TCR delta chain junctions that contain 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 D-J TCR delta chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of D-J TCR delta chain junctions comprising at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR delta chain junctions comprising 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 D-J TCR delta chain junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of D-J TCR δ chain junctions comprising at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR δ chain junctions comprising 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 D-J TCR δ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of D-J TCR δ chain junctions comprising at least four N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of D-J TCR delta chain junctions comprising 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 D-J TCR delta chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the D-J TCR delta chain junctions in the animal comprise non-templated additions.

[0068] In some embodiments, the endogenous TdT locus in the non-human organism 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 comprising the exogenous TdT described herein also comprise in their germline and / or genome an immunoglobulin locus (exogenous or endogenous) containing an immunoglobulin variable region comprising an unrearranged human immunoglobulin variable region gene segment and an immunoglobulin constant region comprising an immunoglobulin constant region gene, wherein the unrearranged human immunoglobulin variable region gene segment is operably linked to the immunoglobulin constant region gene. In some embodiments, the non-human animals and non-human ES cells comprise 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 comprise in their germline and / or genome at least one immunoglobulin locus comprising an unrearranged human heavy chain variable region gene segment and at least one immunoglobulin locus comprising an unrearranged human light chain variable region gene segment (e.g., a κ chain gene segment and / or a λ chain gene segment). In some embodiments, genetically modified non-human animals and non-human animal ES cells comprise in their germline and / or genome at least one immunoglobulin locus comprising an unrearranged human heavy chain variable region gene segment, at least one immunoglobulin locus comprising an unrearranged human κ chain variable region gene segment, and at least one immunoglobulin locus comprising an unrearranged human λ chain variable region gene segment. In some embodiments, genetically modified non-human animals, e.g., genetically modified mice or rats, comprise in their germline and / or genome genetically modified immunoglobulin loci (genetically modified rearranged or unrearranged immunoglobulin loci) such that the mice produce human, humanized, partially human, or reverse chimeric (human variable region and non-human constant region) antibodies.

[0070] Immunoglobulin loci comprising human variable region gene segments are known in the art and are described, for example, in U.S. Patent Nos. 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, 8,5 Nos. 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 by reference herein in its entirety, and U.S. Patent Application Publication Nos. 2008 / 0098490, 2010 / 0146 Nos. 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 incorporated herein by reference in its entirety. are incorporated by reference herein and can be found in PCT Patent Application Publication Nos. WO2007 / 117410, WO2008 / 151081, WO2009 / 157771, WO2010 / 039900, WO2011 / 004192, WO2011 / 123708, and WO2014 / 093908, each of which is incorporated by reference herein in its entirety.

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

[0072] In some embodiments, the human unrearranged 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 unrearranged 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 unrearranged 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 unrearranged 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 comprises an unrearranged human Ig heavy chain variable region gene segment. In some embodiments, the unrearranged human Ig variable region gene segment comprises a plurality of human V H Segment, one or more human D H segment, and one or more human J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three V segments. 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 segments, or at least 80 V H In some embodiments, the unrearranged human Ig gene segments include human D H In some embodiments, the unrearranged human Ig gene segments include all of the human J gene segments. HExemplary variable region containing Ig heavy chain gene segments include those 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 non-human animals provided herein contain a single polymorphic human V (e.g., as described in U.S. Patent Application Publication No. 2013 / 0096287, which is incorporated herein by reference). H Gene segments, multiple D H Gene segments, and multiple J H In some embodiments, the V gene is a restricted immunoglobulin heavy chain locus characterized by a V gene segment. H The gene segment is VH1-2 or VH1-69.

[0074] In various embodiments, the immunoglobulin locus modifications described herein do not affect the fertility of the non-human animal. In some embodiments, the heavy chain locus comprises an endogenous Adam6a gene, an Adam6b gene, or both, and the genetic 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 comprises an ectopically located Adam6a gene, an Adam6b gene, or both. Exemplary non-human animals expressing exogenous Adam6a and / or Adam6b are described in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference in its entirety.

[0075] In some embodiments, human immunoglobulin heavy chain variable region gene segments rearrange during B cell development to generate rearranged human heavy chain variable region genes in B cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain non-templated 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions that do not contain non-templated 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% less than the proportion of VD and / or DJ immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain junctions containing at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain 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 VD and / or DJ immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain junctions containing at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising at least four N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ immunoglobulin heavy chain junctions in the animal comprise non-templated 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% greater than the corresponding non-human animal.

[0076] In certain embodiments, the immunoglobulin variable region comprises an unrearranged human Igκ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V κ segment and one or more human J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all of the human Jκ segments. In some embodiments, the immunoglobulin variable region gene segments comprise four functional Vκ segments. κ Segment and all human J κ In some embodiments, the immunoglobulin variable region gene segment comprises 16 functional V segments. κ Segment and all human J κ segments (e.g., all functional human Vκ segments and J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all human Vκ segments and all human J segments. κ Exemplary variable regions comprising Igκ gene segments are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementary information, which is incorporated herein by reference. In some embodiments, the non-human animals provided herein comprise no more than two human V L Gene segments and multiple J LIn some embodiments, the mouse has a restricted immunoglobulin light chain locus characterized by a V gene segment (e.g., a dual light chain mouse, or DLC, as described in U.S. Patent Application Publication No. 2013 / 0198880, which is incorporated herein by reference). L The gene segment is V κ In some embodiments, the V L The gene segment is V λ In some embodiments, the V κ The gene segments are IGKV3-20 and IGKV1-39.

[0077] In some embodiments, human immunoglobulin κ variable region gene segments rearrange during B cell development to generate rearranged human κ variable region genes in B cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ chain junctions that contain non-templated 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 V-J immunoglobulin κ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin κ chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that do not contain non-templated 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% less than the proportion of V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ-chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that contain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin κ-chain junctions in the animal contain non-templated 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% greater than 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 comprises an unrearranged human Ig λ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V λ segment and one or more human J λ In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human V λ In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise human J segments. λ Exemplary variable regions comprising Igλ gene segments are provided, for example, in U.S. Patent Application Publication Nos. 2012 / 0073004 and 2002 / 0088016, each of which is incorporated herein by reference.

[0079] In some embodiments, human immunoglobulin λ variable region gene segments rearrange during B cell development to generate rearranged human λ variable region genes in B cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin λ chain joints that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain non-templated 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 V-J immunoglobulin λ chain joints in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin λ chain joints that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that do not contain non-templated 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% less than the proportion of V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin λ chain joints that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain 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 V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ immunoglobulin lambda chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin λ chain junctions in the animal contain non-templated 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% greater than 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 comprising unrearranged human immunoglobulin variable region gene segments also comprises a human immunoglobulin variable region intergenic sequence. In some embodiments, the immunoglobulin variable region comprises a non-human (e.g., rodent, rat, mouse) Ig variable region intergenic sequence. In some embodiments, the intergenic sequence is derived from an endogenous species.

[0081] In some embodiments, the non-human organism comprises an immunoglobulin locus in its germline and / or genome that comprises 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. Exemplary rearranged Ig heavy chain variable regions are provided in U.S. Patent Application Publication No. 2014 / 0245468, which is incorporated herein by reference. In some embodiments, the non-human organism that comprises the universal heavy chain variable region is used to produce bispecific antibodies.

[0082] In some embodiments, the non-human organism comprises an immunoglobulin locus in its germline and / or genome that comprises 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 in, for example, U.S. Patent Application Publication Nos. 2011 / 0195454, 2012 / 0021409, 2012 / 0192300, 2013 / 0045492, 2013 / 0185821, 2013 / 0302836, 2015 / 0313193, 2015 / 0059009, and 2013 / 0198879, which are incorporated by reference herein. In some embodiments, non-human organisms that contain universal light chain variable regions ("universal light chain" organisms) are used to generate bispecific antibodies.

[0083] In some embodiments, the non-human organism comprises in its germline and / or genome a light chain immunoglobulin locus comprising a limited repertoire of light chain variable gene segments (e.g., a dual light chain variable region comprising two light chain variable gene segments). In some embodiments, the light chain variable gene segments in the limited repertoire of light chain gene segments are human light chain gene segments. Exemplary dual light chain variable regions are provided in U.S. Patent Application Publication No. 2013 / 0198880, which is incorporated herein by reference. In some embodiments, the non-human organism comprising the dual light chain variable region is used to produce bispecific antibodies.

[0084] In still other embodiments, the non-human organism may contain in its germline and / or genome a light and / or heavy chain immunoglobulin locus that contains an insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies produced in such non-human organisms. In some such embodiments, the histidine codon is inserted and / or substituted in the nucleic acid sequence encoding CDR3. Examples of such light and / or heavy immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and U.S. Patent Application Publication Nos. 2013 / 0247236 and 2014 / 0013456, which are incorporated herein by reference.

[0085] In some embodiments, the immunoglobulin constant region comprises 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 derived from an endogenous species. 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-derived, 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 the non-human animal expresses a heavy chain-only antibody (see, e.g., U.S. Pat. No. 8,754,287, U.S. Patent Application Publication No. 2015 / 0289489, which are incorporated by reference herein). In some embodiments, for example, when the goal is to generate a heavy chain that creates a bispecific antibody (e.g., in a universal or dual-light chain organism), the Fc domain of the heavy chain includes a modification to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Pat. Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication No. 2013 / 0195849, each of which is incorporated by reference herein.

[0086] In some embodiments, the immunoglobulin constant region comprises a light chain constant region gene. In some embodiments, the light chain constant region gene is a kappa constant region gene. In some embodiments, the light chain constant region gene is a lambda constant region gene. In some embodiments, the light chain constant region gene is from an endogenous species. 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 comprising a human variable region gene segment and the immunoglobulin constant region gene to which the variable region gene segment is operably linked are located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous κ locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous λ locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably linked is an 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 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 using any method known in the art, including, but not limited to, deleting the locus or portions thereof from the genome of the organism, replacing the locus or portions thereof with a different nucleic acid sequence, inverting a portion of the locus and / or translocating a portion of the locus to another location in the genome of the non-human organism. In some embodiments, the inactivation of the locus is only partial. In some embodiments, the variable regions of the locus are inactivated, but the constant regions remain functional (e.g., because they are operably linked to non-endogenous variable region gene segments).

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

[0090] In some embodiments, the genetically modified non-human animal comprises 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, rearrangement, and / or inversion of at least a portion of an 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, rearranged, and / or inverted comprises a variable region J segment. In some embodiments, the endogenous immunoglobulin κ chain locus, or a portion thereof, is inactivated by deletion, substitution, rearrangement, and / or inversion of at least a portion of an 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, rearranged, and / or inverted comprises an endogenous constant region CK gene.

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

[0092] In some embodiments, the genetically modified non-human animals provided herein express antibodies having human variable domains (e.g., human variable domains derived from unrearranged human variable region gene segments 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 and / or light chain constant domains are of non-human origin. For example, in some embodiments, the heavy chain constant domain is from an endogenous species. In some embodiments, the heavy chain constant domain is from a mouse or rat. In some embodiments, the light chain constant domain is from an endogenous species. In some embodiments, the light chain constant domain is from a rat or mouse.

[0093] Non-human animals expressing human variable domain T cell receptors and exogenous TdT In certain embodiments, the genetically modified non-human animals and non-human animal ES cells comprising the exogenous TdT described herein also comprise in their germline and / or genome a TCR locus (exogenous or endogenous) containing a TCR variable region comprising unrearranged human TCR variable region gene segments and a TCR constant region comprising a TCR constant region gene, wherein the unrearranged human TCR variable region gene segments are operably linked to the TCR constant region gene. In some embodiments, various genetically modified non-human animals, e.g., genetically modified mice, comprise genetically modified T cell receptor loci (genetically modified TCR α, β, γ, and / or δ loci) in their germline and / or genome such that the mice express 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. Pat. No. 9,113,616 and International Patent Application Publication No. WO 2016 / 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 derived from an endogenous species. 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 comprise 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 comprise, in their germline and / or genome, at least one TCR locus comprising an unrearranged TCR alpha variable region gene segment and at least one TCR locus comprising an unrearranged TCR beta variable region gene segment. In some embodiments, genetically modified non-human animals and non-human animal ES cells comprise, in their germline and / or genome, at least one TCR locus comprising an unrearranged human TCR gamma variable region gene segment and at least one TCR locus comprising an unrearranged human TCR delta variable region gene segment.

[0096] In some embodiments, the human unrearranged TCR variable region gene segment is a TCR alpha gene segment, and the TCR constant region gene is a TCR alpha constant region gene. In some embodiments, the human unrearranged TCR variable region gene segment is a TCR beta chain gene segment, and the TCR constant region gene is a TCR beta constant region gene. In some embodiments, the human unrearranged TCR variable region gene segment is a TCR gamma chain gene segment, and the TCR constant region gene is a TCR gamma constant region gene. In some embodiments, the human unrearranged TCR variable region gene segment is a TCR delta chain gene segment, and the TCR constant region gene is a TCR delta constant region gene. Exemplary variable region comprising 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 comprises unrearranged human TCR β variable region gene segments. In some embodiments, the human TCR β variable region gene segments rearrange during T cell development to generate rearranged human TCR β variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ TCR β junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ TCR β junctions that contain non-templated 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 TCR β junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a smaller proportion of VD TCR β junctions that do not contain non-templated additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCR β junctions that do not contain non-templated 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 VD and / or DJ TCR β junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a larger proportion of VD and / or DJ TCR β junctions that contain at least one N addition than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRβ junctions comprising 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 animal.In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCR β junctions containing at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCR β 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 VD and / or DJ TCR β junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCR β junctions containing at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCR β junctions comprising 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 TCR β junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ TCR β junctions comprising at least four N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ TCR β junctions comprising 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 TCR β junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ TCR β junctions in the animal comprise non-templated 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% greater than the corresponding non-human animal.

[0098] In some embodiments, the TCR variable region comprises unrearranged human TCR alpha variable region gene segments. In some embodiments, human TCR alpha variable region gene segments rearrange during T cell development to generate rearranged human TCR alpha variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR alpha junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR alpha junctions that contain non-templated 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 V-J TCR alpha junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-D TCR alpha junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VJ TCRα junctions that do not contain non-templated 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% less than the proportion of VJ TCRα junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of VJ TCRα junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VJ TCRα junctions that contain 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 VJ TCRα junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ TCRα junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of VJ TCRα junctions comprising 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 junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ TCRα junctions comprising at least three N additions greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VJ TCRα junctions comprising 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 junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ TCRα junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VJ TCRα 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 VJ TCRα junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VJ TCRα junctions in the animal contain non-templated additions. In some embodiments, the non-human animal has 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% greater than the corresponding non-human animal.

[0099] In some embodiments, the TCR variable region comprises unrearranged human TCRδ variable region gene segments. In some embodiments, the human TCRδ variable region gene segments rearrange during T cell development to generate rearranged human TCRδ variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ TCRδ junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions that contain non-templated 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 animal. In some embodiments, the non-human animals provided herein have a lower proportion of VD TCRδ junctions that do not contain a non-templated addition than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions that do not contain a non-templated 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% less than the proportion of VD and / or DJ TCRδ junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ TCRδ junctions that contain at least one N addition than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions comprising 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 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 comprising at least two N additions greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions comprising 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 a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. The proportion of 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 VD and / or DJ TCRδ junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ TCRδ junctions comprising at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions comprising 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 TCRδ junctions 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 VD and / or DJ TCRδ junctions in the corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ TCRδ junctions in the corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome 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 animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ TCRδ junctions in the animal comprise non-templated additions. In some embodiments, the non-human animal has 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% greater than a corresponding non-human animal.

[0100] In some embodiments, the TCR variable regions comprise unrearranged human TCR gamma variable region gene segments. In some embodiments, the human TCR gamma variable region gene segments are rearranged during T cell development to generate rearranged human TCR gamma variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCR gamma junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCR gamma junctions that contain non-templated 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 V-J TCR gamma junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-D TCR gamma junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCRγ junctions that do not contain non-templated 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% less than the proportion of V-J TCRγ junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J TCRγ junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J TCRγ junctions that contain 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 V-J TCRγ junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ TCRγ junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of VJ TCRγ junctions comprising 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 proportion of VJ TCRγ junctions comprising at least three N additions greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VJ TCRγ junctions comprising 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, the non-human animals provided herein have a proportion of V-J TCRγ junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of V-J TCRγ 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 V-J TCRγ junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J TCRγ junctions in the animal contain non-templated additions. In some embodiments, the non-human animal has 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% greater than the corresponding non-human animal.

[0101] In some embodiments, the TCR variable region comprising unrearranged human TCR variable region gene segments also comprises a human TCR variable region intergenic sequence. In some embodiments, the TCR variable region comprises a non-human (e.g., rodent, rat, mouse) TCR variable region intergenic sequence. In some embodiments, the intergenic sequence is from an endogenous species.

[0102] In some embodiments, the TCR variable region comprising a human variable region gene segment and the TCR constant region gene to which the variable region gene segment is operably linked are located at an endogenous TCR locus. In some embodiments, the endogenous TCR locus is an endogenous TCR alpha locus. In some embodiments, the endogenous TCR locus is an endogenous TCR beta locus. In some embodiments, the endogenous TCR locus is an endogenous TCR gamma locus. In some embodiments, the endogenous TCR locus is an endogenous TCR delta locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably linked is an endogenous constant region gene, e.g., a 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 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 using any method known in the art, including, but not limited to, deleting the locus or portions thereof from the genome of the organism, replacing the locus or portions thereof with a different nucleic acid sequence, inverting a portion of the locus and / or translocating a portion of the locus to another location in the genome of the non-human organism. In some embodiments, the inactivation of the locus is only partial inactivation. In some embodiments, the variable regions of the locus are inactivated, but the constant regions remain functional (e.g., due to being operably linked to non-endogenous variable region gene segments). 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 unrearranged human variable region gene segment described herein). In some embodiments, the human variable domain is a human TCR alpha variable domain. In some embodiments, the human variable domain is a human TCR beta variable domain. In some embodiments, the human variable domain is a human TCR gamma variable domain. In some embodiments, the human variable domain is a human TCR delta variable domain. In some embodiments, the TCR produced by the non-human animal has both a human TCR alpha variable domain and a human TCR beta variable domain. In some embodiments, the TCR produced by the non-human animal has both a human TCR gamma variable domain and a human TCR delta variable domain. In some embodiments, the TCR produced by the non-human animal has both a human TCR alpha variable domain and a human TCR beta variable domain, and both a human TCR gamma variable domain and a human TCR delta 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 constant domain is from an endogenous species, ie, from a mouse or rat.

[0105] Non-human animals expressing chimeric antigen receptors (CARs) and exogenous TdT In certain aspects, provided herein are genetically modified non-human animals and non-human animal ES cells comprising the exogenous TdT described herein, which also comprise a chimeric antigen receptor (CAR) locus. Such CAR loci generally comprise a variable region and a constant region. The variable region comprises unrearranged human Ig variable region gene segments, and the constant region locus comprises a TCR constant region gene, wherein the Ig variable region gene segment is operably linked 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 derived from an endogenous species. In some embodiments, the TCR constant region gene is a human constant region gene.

[0106] In some embodiments, the CAR locus described herein is located at an endogenous TCR locus. For example, in some embodiments, a CAR locus comprising a TCR alpha constant region gene is located at an endogenous TCR alpha constant region locus. In some embodiments, such a locus is created by replacing part or all of the TCR alpha unrearranged variable region with an unrearranged Ig variable region. In some embodiments, a CAR locus comprising a TCR beta constant region gene is located at an endogenous TCR beta constant region locus. In some embodiments, such a locus is created by replacing part or all of the TCR beta unrearranged variable region with an unrearranged Ig variable region.

[0107] In certain embodiments, the CAR variable region locus comprises unrearranged human Ig variable region gene segments.Exemplary variable region loci comprising human variable region gene segments are described in the art.For example, such loci are described in U.S. Patent Nos. 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, 8,703,4 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 by reference herein in its entirety, and in U.S. Patent Application Publication Nos. 2008 / 0098490, 2010 / 0146647, 2011 / 0195454, 2012 / 0195456, 2013 / 0195458, 2014 / 0195459, 2015 / 0195461, 2016 / 0195462, 2017 / 0195463, 2018 / 0195464, 2019 / 0195465, 2019 / 0195466, 2019 / 0195467, 2019 / 0195468, 2019 / 0195469 ... Nos. 12 / 0167237, 2013 / 0145484, 2013 / 0167256, 2013 / 0219535, 2013 / 0326647, 2014 / 013275, 2014 / 093908, 2015 / 0113668, and 2016 / 0081314, each of which is incorporated by reference herein in its entirety, and are described in PCT and in published patent applications WO2007 / 117410, WO2008 / 151081, WO2009 / 157771, WO2010 / 039900, WO2011 / 004192, WO2011 / 123708, WO2014 / 093908, and WO2016 / 044745, each of which is incorporated by reference herein in its entirety.

[0108] In certain embodiments, the CAR variable region locus comprises an unrearranged human Ig heavy chain variable region gene segment. In some embodiments, the unrearranged human Ig variable region gene segment comprises a plurality of human V H Segment, one or more human D Hsegment, and one or more human J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three V segments. 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 segments, or at least 80 V H In some embodiments, the unrearranged human Ig gene segments include human D H In some embodiments, the CAR variable region further comprises a TCR β variable region gene segment (e.g., a V, D, and / or J gene segment). In one embodiment, the CAR variable region further comprises 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 unrearranged human Ig gene segment comprises a human J H Exemplary variable region, including Ig heavy chain gene segments, are described, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and in the supplementary information, which is incorporated herein by reference.

[0109] In some embodiments, human immunoglobulin heavy chain variable region gene segments rearrange during T cell development to generate rearranged human heavy chain variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain non-templated 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of VD immunoglobulin heavy chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions that do not contain non-templated 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% less than the proportion of VD and / or DJ immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal.In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain junctions containing at least two N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain 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 VD and / or DJ immunoglobulin heavy chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a higher proportion of VD and / or DJ immunoglobulin heavy chain junctions containing at least three N additions than a corresponding non-human animal that does not have a nucleic acid encoding exogenous TdT in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a greater proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising at least four N additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of VD and / or DJ immunoglobulin heavy chain junctions comprising 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 junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the VD and / or DJ immunoglobulin heavy chain junctions in the animal comprise non-templated 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% greater than the corresponding non-human animal.

[0110] In some embodiments, the CAR variable locus comprising an unrearranged human Ig heavy chain variable region gene segment also comprises a human Ig heavy chain variable region intergenic sequence. In some embodiments, the CAR variable locus comprises a non-human (e.g., rodent, rat, mouse) Ig heavy chain variable region intergenic sequence. In some embodiments, the CAR variable locus comprises a human or non-human (e.g., rodent, rat, mouse) TCR β variable region intergenic sequence. For example, in some embodiments, the unrearranged variable region of the CAR locus comprises 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 in TCR β variable region loci). In some embodiments, the TRY genes are non-human, e.g., mouse TRY genes. In some embodiments, the mouse TRY genes are selected from the group consisting of Try, 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 are selected from the group consisting of the V of the unrearranged variable region. H In some embodiments, one or more TRY genes are located upstream of the V H D of the downstream segment and unrearranged variable region H In some embodiments, Try1 to Try7 are located upstream of the V segment of the unrearranged variable region. H Located upstream of the segment, Try 8 to 20 are V H D of the downstream segment and unrearranged variable region HIt is located upstream of the segment. Additional information regarding the TRY gene located in the human and / or mouse TCR β 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 comprises 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 comprises 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 comprises an unrearranged human Igκ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V κ segment and one or more human J κ In some embodiments, the immunoglobulin variable region gene segment comprises four functional V κ Segment and all human J κ In some embodiments, the immunoglobulin variable region gene segment comprises 16 functional V segments. κ Segment and all human J κ In some embodiments, the unrearranged human immunoglobulin variable region gene segments include all of the human Vκ segments and all of the human J segments. κ segments (e.g., all functional human Vκ segments and J κsegments). Exemplary variable regions comprising Igκ gene segments 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 unrearranged human immunoglobulin variable region gene segments comprise all of the human Jκ segments. In some embodiments, the CAR variable region further comprises a TCR α variable region gene segment (e.g., a V and / or J gene segment).

[0112] In some embodiments, human immunoglobulin κ variable region gene segments rearrange during T cell development to generate rearranged human κ variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ chain junctions that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ chain junctions that contain non-templated 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 V-J immunoglobulin κ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin κ chain junctions that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that do not contain non-templated 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% less than the proportion of V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin κ-chain junctions that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions that contain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain junctions comprising 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin κ-chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin κ-chain 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 V-J immunoglobulin κ-chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin κ-chain junctions in the animal contain non-templated 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% greater than 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 comprises an unrearranged human Igλ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human V λ segment and one or more human J λ In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human V λ segments (e.g., all functional human V λ In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human J segments. λ In some embodiments, the CAR variable region further comprises a TCR alpha variable region gene segment (e.g., a V and / or a J gene segment). Exemplary variable regions comprising Ig lambda gene segments are provided, for example, in U.S. Patent Application Publication Nos. 2012 / 0073004 and 2002 / 0088016, each of which is incorporated herein by reference.

[0114] In some embodiments, human immunoglobulin λ variable region gene segments rearrange during T cell development to generate rearranged human λ variable region genes in T cells of the non-human organism. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin λ chain joints that contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain non-templated 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 V-J immunoglobulin λ chain joints in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a lower proportion of V-J immunoglobulin λ chain joints that do not contain non-templated additions than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that do not contain non-templated 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% less than the proportion of V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a greater proportion of V-J immunoglobulin λ chain joints that contain at least one N addition than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain joints that contain 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 V-J immunoglobulin λ chain joints in the corresponding non-human animals. In some embodiments, the non-human animals provided herein have a proportion of VJ immunoglobulin lambda chain junctions that contain at least two N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome.In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions comprising at least three N additions greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain junctions comprising 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, the non-human animals provided herein have a proportion of V-J immunoglobulin λ chain junctions containing at least four N additions that is greater than a corresponding non-human animal that does not have an exogenous TdT-encoding nucleic acid in its genome. In some embodiments, the proportion of V-J immunoglobulin λ chain 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 V-J immunoglobulin λ chain junctions in the corresponding non-human animal. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the V-J immunoglobulin λ chain junctions in the animal contain non-templated 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% greater than 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, the CAR variable locus comprising an unrearranged human Ig light chain variable region gene segment also comprises a human Ig light chain variable region intergenic sequence (e.g., a κ variable region intergenic sequence and / or a λ variable region intergenic sequence). In some embodiments, the CAR variable locus comprises a non-human (e.g., rodent, rat, mouse) Ig light chain variable region intergenic sequence (e.g., a κ variable region intergenic sequence and / or a λ variable region intergenic sequence). In some embodiments, the CAR variable locus comprises a human or non-human (e.g., rodent, rat, mouse) TCR α variable region intergenic sequence. In some embodiments, the CAR locus comprises 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).

[0116] In some embodiments, the CAR variable region locus is a rearranged variable region locus comprising 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 a universal heavy chain variable region facilitates the generation of bispecific antibodies in which at least one antigen-binding domain has specificity for a peptide / MHC complex. Exemplary rearranged Ig heavy chain variable regions are provided in U.S. Patent Application Publication No. 2014 / 0245468, which is incorporated herein by reference.

[0117] In some embodiments, the CAR variable region locus is a rearranged variable region locus comprising 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 a universal light chain variable region facilitates the generation of bispecific antibodies in which at least one antigen-binding domain has binding specificity for a peptide / MHC complex. Exemplary rearranged Ig heavy chain variable regions are provided in U.S. Patent Application Publication No. 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 genomes loci encoding humanized MHC class I α 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 I α chain polypeptides are fully human. In some embodiments, the humanized MHC class I α chain polypeptides include a human extracellular domain (e.g., human α1, α2, and α3 domains) and a cytoplasmic domain derived from an endogenous species. Humanized MHC class I α chain polypeptides, loci encoding humanized MHC class I α chain polypeptides, and non-human animals expressing humanized MHC class I α chain polypeptides are described in U.S. Patent Application Publication Nos. 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467, each of which is 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 a locus encoding a humanized beta-2 microglobulin polypeptide. Humanized beta-2 microglobulin polypeptides, loci encoding humanized beta-2 microglobulin polypeptides, and non-human animals that express humanized beta-2 microglobulin polypeptides are described in U.S. Patent Application Publication Nos. 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 genomes loci encoding humanized MHC class II α chain polypeptides (e.g., humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA). In some embodiments, the humanized MHC class II α chain polypeptides are fully human. In some embodiments, the humanized MHC class II α chain polypeptides comprise a human extracellular domain and a cytoplasmic domain derived from an endogenous species. Humanized MHC class II α chain polypeptides, loci encoding humanized MHC class II α chain polypeptides, and non-human animals expressing humanized MHC class II α chain polypeptides are described in U.S. Patent Nos. 8,847,005 and 9,043,996, and U.S. Patent Application Publication No. 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 genomes loci encoding humanized MHC class II β chain polypeptides (e.g., humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB). In some embodiments, the humanized MHC class II β chain polypeptides are fully human. In some embodiments, the humanized MHC class II β chain polypeptides comprise a human extracellular domain and a cytoplasmic domain derived from an endogenous species. Humanized MHC class II β chain polypeptides, loci encoding humanized MHC class II β chain polypeptides, and non-human animals expressing humanized MHC class II β chain polypeptides are described in U.S. Patent Nos. 8,847,005 and 9,043,996, and U.S. Patent Application Publication No. 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 engineered loci (e.g., humanized TCR loci), and non-human animals can be produced from the ES cells.

[0123] Genetically modified non-human animals containing exogenous TdT, a humanized CAR locus, and a humanized MHC I and / or MHC II (MHCIIα / IIβ) locus 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 engineered loci (e.g., a humanized CAR locus), and non-human animals can be produced from the 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 a locus encoding a humanized CD8 α-chain polypeptide. In some embodiments, the humanized CD8 α-chain polypeptide is fully human. In some embodiments, the humanized CD8 α-chain polypeptide comprises a human extracellular immunoglobulin domain and a cytoplasmic domain derived from an endogenous species. Humanized CD8 α-chain polypeptides, loci encoding humanized CD8 α-chain polypeptides, and non-human animals that express humanized CD8 α-chain polypeptides are described in U.S. Patent Application Publication No. 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 a locus encoding a humanized CD8 β chain polypeptide. In some embodiments, the humanized CD8 β chain polypeptide is fully human. In some embodiments, the humanized CD8 β chain polypeptide comprises a human extracellular immunoglobulin domain and a cytoplasmic domain derived from an endogenous species. Humanized CD8 β chain polypeptides, loci encoding humanized CD8 β chain polypeptides, and non-human animals that express humanized CD8 β chain polypeptides are described in U.S. Patent Application Publication No. 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 contain a locus in their genome encoding a humanized CD4 polypeptide. In some embodiments, the humanized CD4 polypeptide is fully human. In some embodiments, the humanized CD4 polypeptide comprises at least one human extracellular immunoglobulin domain and a cytoplasmic domain derived from an endogenous species. 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, and a cytoplasmic domain derived from an endogenous species. 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, a D4 immunoglobulin domain derived from an endogenous species, and a cytoplasmic domain derived from an endogenous species. Humanized CD4 polypeptides, loci encoding humanized CD4 polypeptides, and non-human animals expressing humanized CD4 polypeptides are described in U.S. Patent Application Publication No. 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 engineered loci (e.g., humanized TCR loci), and non-human animals can be produced from the ES cells.

[0128] Genetically modified non-human animals containing exogenous TdT, a humanized CAR locus, and a humanized CD4 and / or CD8 (CD8α / CD8β) locus 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 engineered loci (e.g., a humanized CAR locus), and non-human animals can be produced from the ES cells.

[0129] Methods of using genetically modified non-human animals In certain aspects, provided herein are methods of using the genetically modified non-human animals described herein to generate 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, provided herein are methods of generating more diverse antigen binding proteins (e.g., antibodies, CARs, TCRs). In some embodiments, provided herein are methods of generating rearranged variable regions of antigen binding proteins (e.g., antibodies, CARs, TCRs) with an increased number of nucleotide additions.

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

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

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

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

[0134] In certain embodiments, the method comprises exposing a genetically modified non-human animal described herein that has been modified to express exogenous TdT and an antibody or antigen-binding fragment thereof having a human variable domain to an antigen; obtaining B cells from the non-human animal that express the antibody or antigen-binding fragment thereof comprising a human variable domain specific for the antigen; optionally producing hybridomas from the B cells; and obtaining nucleic acid encoding a human immunoglobulin variable domain specific for the antigen from the B cells or hybridomas.

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

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

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

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

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

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

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

[0143] In some embodiments, the method comprises exposing a non-human animal described herein that has been modified to express exogenous TdT and a CAR with a human variable domain to an antigen comprising a peptide or a nucleic acid encoding an antigen comprising the peptide, such that the peptide is presented on MHC in the non-human animal; obtaining a T cell from the genetically modified non-human animal that expresses a chimeric antigen receptor (CAR) specific for the peptide presented on the MHC; isolating a nucleic acid encoding a human immunoglobulin variable domain of the CAR from the T cell; and operably linking the nucleic acid encoding the human immunoglobulin variable domain and the human immunoglobulin constant domain in the cell such 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 exposing a non-human animal described herein to an antigen (immunization) to induce an immune response (e.g., a B cell immune response and / or a T cell immune response). In some embodiments, the genetically modified non-human animal is immunized with a whole protein antigen or a fragment thereof. Rodents can be immunized by any method known in the art (e.g., see 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, the genetically modified non-human animal is exposed to the antigen by administering to the non-human animal a virus (e.g., a retrovirus, adenovirus, vaccinia virus, or lentivirus) containing a nucleic acid sequence encoding the antigen. Viral vaccination methods are described, for example, in U.S. Patent Nos. 6,001,349, 8,663,622, 8,691,502, 8,377,688, and Precopio et al. al., JEM 204:1405-1416 (2007), each of which is incorporated by reference in its entirety. In some embodiments, the non-human animal is directly administered the virus. 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 the non-human animal. In some embodiments, the virus encodes a peptide / MHC complex (e.g., a single-chain 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, the genetically modified non-human animal is exposed to an antigen by administering a nucleic acid encoding the antigen to the animal. In some embodiments, the non-human animal is administered a nucleic acid encoding a single-chain 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. 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 gene transfer and liposome-mediated gene transfer. The polynucleotide of interest is associated with liposome to form gene delivery vehicle, as described in, for example, United States Patent No. 6,770,291, United States Patent No. 7,001,614, United States Patent No. 6,749,863, United States Patent No. 5,512,295 and United States Patent No. 7,112,338, each of which is incorporated herein by reference.In some embodiments, nucleic acid is mRNA vector.The exemplary method for producing and administering mRNA vector is described in, for example, United States Patent No. 8,278,036 and United States Patent Application Publication No. 2013 / 151736 and United States Patent Application Publication No. 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 include, but are not limited to, adipophilin, 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, malic enzyme, mammaglobin A, MART2, MATN, MC1R,MCSP, mdm-2, ME1, Melan-A / MART-1, Meroe, 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, polymorphic 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, cecernin1, SIRT2, SLAMF7, SLC39A6, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, survivin, SYT-SSX1 or -SSX2 fusion proteins, TAG-1, TAG-2, telomerase, TERT, TGF-βRII, Thompson-nouvelle antigen, TMPRSS2, TNFRSF17, TPBG, TRAG-3, triosephosphate 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 neoantigen.

[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, herpesvirus (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 arboviral encephalitis virus antigens. In some embodiments, the parasite is malaria. In some embodiments, the pathogen is 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 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 obtaining T cells and / or B cells from a genetically modified non-human animal. 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 the 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 generating B cell hybridomas from the B cells. Methods useful for generating B cell hybridomas are known in the art, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory, Malik and Lillehoj (1994) Antibody Techniques, Academic Press, CA, incorporated herein by reference.

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

[0152] In some embodiments, the methods provided herein include isolating nucleic acids encoding Ig or TCR variable regions. In some embodiments of the methods described herein, any method can be used to isolate nucleic acids encoding Ig or TCR variable regions.

[0153] In some embodiments, isolating the nucleic acid comprises generating a B cell or T cell hybridoma from the B cell or T cell, respectively, and isolating the nucleic acid from the hybridoma. In some embodiments, the nucleic acid is isolated using a nucleic acid amplification process. For example, in some aspects, the nucleic acid amplification process is polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), self-sustained 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, the nucleic acid is isolated by sequencing rearranged Ig or TCR variable region genes in B cells, T cells, B cell hybridomas, or T cell hybridomas and synthesizing a nucleic acid sequence comprising the rearranged Ig or TCR variable region genes. Exemplary nucleic acid sequencing processes include, but are not limited to, chain termination sequencing, sequencing by ligation, sequencing by synthesis, pyrosequencing, ion semiconductor sequencing, single molecule real-time sequencing, 454 sequencing, and / or Dilute-'N'-Go sequencing.

[0155] When 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, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding the variable region is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0156] Isolated DNA encoding a heavy chain variable region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the variable region to another DNA molecule encoding heavy chain constant regions (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, USDapartment 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 heavy chain constant domain can be, for example, an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant domain. For a Fab fragment heavy chain gene, the V H The encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0157] Thus, in some embodiments, the methods described herein comprise operably linking a nucleic acid sequence encoding a heavy chain Ig variable domain to a nucleic acid sequence encoding a heavy chain Ig constant domain in a host cell, such that the host cell expresses an Ig heavy chain polypeptide comprising an Ig heavy chain variable domain and an Ig heavy chain constant domain. In some embodiments, the methods comprise operably linking a nucleic acid sequence encoding a light chain Ig variable domain to a nucleic acid sequence encoding a light chain Ig constant domain in a host cell, such that the host cell expresses an Ig light chain polypeptide comprising an Ig light chain variable domain and an Ig heavy chain constant domain. In some embodiments, the methods comprise operably linking a nucleic acid sequence encoding a heavy chain Ig variable domain to a nucleic acid sequence encoding a heavy chain Ig constant domain in a host cell, and operably linking a nucleic acid sequence encoding a light chain Ig variable domain to a nucleic acid sequence encoding a light chain Ig constant domain in a host cell, such 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. The Ig variable region can be linked to an 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, a HEK-293 cell, a BHK cell, an NS0 cell, a SP2 / 0 cell, or a Vero cell, or a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

[0158] In some embodiments, the nucleic acid encoding the heavy chain constant domain encodes a constant domain comprising a modified Fc domain (e.g., a mutation that alters the interaction between Fc and an Fc receptor). For example, in some embodiments, the constant domain comprises a modification 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, the constant domain comprises 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 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, H268D / / K326A / A327H.Additional Fc modifications and combinations of Fc modifications are described 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, and 8,218,805. Nos. 8,388,955, and 8,937,158, and U.S. Patent Publication 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 proteins In certain aspects, provided herein are antigen binding proteins (e.g., antibodies, TCRs, CARs, and antigen-binding fragments thereof) obtainable and / or obtainable by the methods described herein (e.g., using the non-human animals described herein).

[0160] In certain embodiments, the antigen-binding molecules provided herein are -6 , 10 -7 , 10 -8 , or 10 -9 In some embodiments, the binding affinity of an antigen-binding protein for an antigen (K D (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 an unrelated antigen. In some embodiments, the antigen binding protein is -6 , 10 -7 , 10 -8 , or 10 -9In some embodiments, the binding affinity (K D The affinity of the antigen-binding protein for the same MHC protein presenting an irrelevant peptide (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 irrelevant peptide. Standard assays for assessing the binding ability of antigen-binding proteins include, for example, ELISA, Western blot, and RIA, and are known in the art. The binding kinetics (e.g., binding affinity) of antigen-binding proteins can also be assessed by standard assays known in the art, such as Biacore analysis.

[0161] In some embodiments, the antigen comprises an epitope of and / or is a cancer-associated antigen. Examples of cancer-associated antigens include, but are not limited to, adipophilin, 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, KMHN1, LAGE-1, LDLR-fucosyltransferase, also known as CCDC110, KMHN1, KM-HN- ... M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, meroe, 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, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, S Examples of antigens include AGE, cecernin 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, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a. In some embodiments, the antigen is a neoantigen.

[0162] In some embodiments, the antigen comprises an epitope of an antigen expressed by an infectious pathogen and / or is 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), human immunodeficiency viruses (HIV-1 and HIV-2), herpes viruses 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 pathogen is 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. 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 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 an epitope of and / or is a protein 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, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture's syndrome, infertility, 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, celiac disease, and Cushing's syndrome. , dermatomyositis, discoid erythema, lupus, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis nodosa, polyglandular autoimmune syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, morphea (or CREST syndrome), sympathetic ophthalmia, systemic cachectic peritonitis, ankylosing cystitis, polyglandular autoimmune syndrome, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type B insulin resistance, 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 antibodies provided herein comprise a human heavy chain variable domain. In some embodiments, the antibodies comprise a human heavy chain constant domain. In some embodiments, the antibodies provided herein comprise 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, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001). In some embodiments, the antibodies provided herein lack a heavy chain constant domain or a portion thereof.

[0165] In some embodiments, the antibodies provided herein comprise a modified Fc domain (e.g., a mutation that alters the interaction between Fc and an Fc receptor). For example, in some embodiments, the antibodies provided herein comprise modifications to their 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, the antibodies comprise multiple modifications to their 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 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, H268D / / K326A / A327H.Additional Fc modifications and combinations of Fc modifications are described 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, 8,220,805, and 8,222,805. ,388,955, and 8,937,158, as well as U.S. Patent Application Publication 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 by reference herein.

[0166] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the two antigen-binding domains of a bispecific antibody have separate heavy chain variable domains but identical light chain variable domains. In some embodiments, the Fc domain of the heavy chain contains modifications to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication No. 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. Sequences of human light chain constant domains are known in the art (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health). and Human Services, NIH Publication No. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press 2001).

[0168] In some embodiments, the antibodies described herein are intact antibodies. In some embodiments, the antibodies described herein are antibody fragments that retain antigen binding. In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked 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 comprising an Ig heavy chain variable domain and a TCR β constant domain and a second CAR polypeptide comprising an Ig light chain variable domain (e.g., an Ig κ variable domain or an Ig λ variable domain) and a TCR α constant domain. In some embodiments, the Ig heavy chain variable domain and / or the Ig light chain variable domain are human Ig variable domains. In some embodiments, the TCR β constant domain and / or the TCR α constant domain are non-human constant domains (e.g., rat or mouse constant domains). In some embodiments, the TCR β constant domain and / or the TCR α constant domain are human constant domains.

[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 comprising a TCR β variable domain and a TCR β constant domain and a second TCR polypeptide comprising a TCR α variable domain and a TCR α constant domain. In some embodiments, the TCR α variable domain and / or the TCR β variable domain are human TCR variable domains. In some embodiments, the TCR β constant domain and / or the TCR α constant domain are non-human constant domains (e.g., rat or mouse constant domains). In some embodiments, the TCR β constant domain and / or the TCR α constant domain are human constant domains.

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

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

[0173] Pharmaceutical compositions provided herein suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

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

[0175] In certain embodiments, the composition comprises an antibody, TCR, and / or CAR described herein at a concentration that provides a weight / volume suitable for the desired dose. The antibody, TCR, and / or CAR may be present in the composition at a concentration of 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, at least 80 mg / mL, at least 90 mg / mL, at least 100 mg / mL, at least 150 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, at least 80 mg / mL, at least 90 mg / mL, at least 10 ... 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 compositions contain more than one active compound necessary for the particular indication being treated, typically with complementary activities that do not adversely affect each other, and such additional active compounds are suitably present in combination in amounts effective for the intended purpose.

[0177] In some embodiments, compositions comprise an antibody, TCR, and / or CAR described herein in any physiologically acceptable carrier, excipient, or stabilizer, including, but not limited to, buffers, sugars, salts, detergents, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelating agents, preservatives, and the like (Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, L. Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)) to prepare a lyophilized composition or aqueous solution of the desired final concentration. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as histidine, phosphate, citrate, glycine, acetate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, e.g., serum albumin, and the like. The composition may comprise a hydrophilic polymer such as PEG, gelatin, or immunoglobulin, a hydrophilic polymer such as polyvinylpyrrolidone, an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine, a monosaccharide, a disaccharide, and other carbohydrates including trehalose, glucose, mannose, or dextrin, a chelating agent such as EDTA, a sugar such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion such as sodium, a metal complex (e.g., Zn-protein complex), and / or a non-ionic surfactant such as TWEEN, polysorbate 80, PLURONICS®, or polyethylene glycol (PEG).

[0178] In some embodiments, the buffering agent 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 compositions include a buffer or pH adjusting agent to provide improved pH control. Such compositions may have a pH 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 certain embodiments, the composition has a pH of about 6.0. Those skilled in the art will understand that the pH of a composition generally should not be equal to the isoelectric point of the specific antibody, TCR, or CAR used in the composition. Typically, the buffer is a salt prepared from an organic or inorganic acid or base. Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers. Additionally, amino acid components can also function in a buffering capacity. Representative amino acid components that can be utilized as buffering agents in the 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 specific embodiment, the buffering agent is citrate. In yet another specific embodiment, the buffering agent is glycine. The purity of the buffering agent should 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 the histidine or glycine as understood in the art, e.g., as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

[0180] In certain embodiments, the composition comprises histidine as a buffering agent, hi certain embodiments, the histidine is present in the composition at a concentration 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 histidine. In another embodiment, the composition comprises about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 100 mM, about 1 mM to about 75 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 75 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM histidine. In further embodiments, the composition comprises 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 histidine. In certain embodiments, the composition may comprise about 10 mM, about 25 mM, or no histidine.

[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 an amount of from about 1% to about 99% by weight or volume, e.g., from about 0.1% to about 20%, from about 0.1% to about 15%, from about 0.1% to about 5%, from about 1% to about 20%, from about 5% to about 15%, from about 8% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from 0.1% to 20%, from 5% to 15%, from 8% to 10%, from 10% to 15%, from 15% to 20%, from about 0.1% to about 5%, from about 5% to about 10%, or from about 15% to about 20%. In still other specific embodiments, the carbohydrate excipient is present at 1%, or 1.5%, or 2%, or 2.5%, or 3%, or 4%, or 5%, or 10%, or 15%, or 20%.

[0182] In some embodiments, the composition comprises a carbohydrate excipient. Carbohydrate excipients suitable 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, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, and xylitol sorbitol (glucitol). In certain embodiments, the carbohydrate excipient for use in the compositions provided herein is selected from sucrose, trehalose, lactose, mannitol, and raffinose. In certain embodiments, the carbohydrate excipient is trehalose. In another specific embodiment, the carbohydrate excipient is mannitol. In yet another specific embodiment, the carbohydrate excipient is sucrose. In yet another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.

[0183] In some embodiments, the composition comprises trehalose. In specific embodiments, the composition comprises 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 comprises between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 2% and about 40%, between about 2% and about 30%, between about 2% and about 20%, between about 4% and about 40%, between about 4% and about 30%, or between about 4% and about 20% trehalose. In further embodiments, the composition comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% trehalose. In specific embodiments, the composition comprises about 4%, about 6%, or about 15% trehalose.

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

[0185] In some embodiments, the composition comprises an amino acid, such as lysine, arginine, glycine, histidine, or an amino acid salt. The composition may comprise 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 the amino acid. In another embodiment, the composition may contain 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 of amino acids. In further embodiments, the composition comprises 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 acid.

[0186] In some embodiments, the composition includes a surfactant. As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure. That is, they consist of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of the surface-active moiety, surfactants can be classified as anionic, cationic, and nonionic surfactants. 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); poloxamers (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 methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUA® series (Mona Pharmaceutically acceptable surfactants, such as polysorbate 20, polysorbate 40, polysorbate 60, and copolymers of ethylene and propylene glycol (e.g., Polysorbate Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS® PF68, etc.), can optionally be added to the composition to reduce aggregation. In certain embodiments, the composition comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Surfactants are particularly useful when administering the composition using a pump or plastic container. The presence of a pharmaceutically acceptable surfactant reduces the tendency of proteins to aggregate. The composition may comprise a polysorbate at a concentration ranging from about 0.001% to about 1%, or from about 0.001% to about 0.1%, or from about 0.01% to about 0.1%.In other specific embodiments, the composition comprises polysorbate at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

[0187] In some embodiments, the composition further comprises other excipients and / or additives, including, but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, etc. 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., but not limited to, EDTA, DTPA, or EGTA), can 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, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (for example, but not limited to, hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, can be optionally added to the composition at any suitable concentration ranging from about 0.001% to about 5%, or any range or value therein. The concentration of the preservative used in the composition is sufficient to achieve microbial effectiveness. Such a concentration depends on the preservative selected and can be easily determined by one skilled in the art.

[0189] In some embodiments, the composition is isotonic with human blood, meaning that the composition 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 freeze osmometer. The tonicity of the composition is adjusted by using a tonicity adjuster. A "tonicity adjuster" is a pharmaceutically acceptable inert substance that can be added to a composition to provide isotonicity to the composition. Suitable tonicity adjusters for the compositions provided herein include, but are not limited to, saccharides, salts, and amino acids.

[0190] In certain embodiments, the composition is pyrogen-free and substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins trapped inside microorganisms and are released only when the microorganisms decompose or die. Pyrogens 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 potential adverse effects, even small amounts of endotoxin must be removed from intravenously administered pharmaceutical solutions. The U.S. Food and Drug Administration (FDA) has set a limit of 5 endotoxin units (EU) per kilogram of body weight per administration within one hour 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), even traces of harmful and dangerous endotoxins must be removed. In some embodiments, the endotoxin and pyrogen levels 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, radiation, etc. In certain embodiments, the compositions are sterile filtered through a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as that described in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins, (2005). Compositions containing a protein of interest (e.g., an antibody, TCR, or CAR) as disclosed herein are typically stored in lyophilized form or in solution. It is contemplated that a sterile composition containing a protein of interest (e.g., an antibody, TCR, or CAR) may be placed in a container with a sterile access port, such as an intravenous solution bag or vial with an adapter that allows for withdrawal of the composition, such as a stopper that can be pierced with a hypodermic injection needle. In certain embodiments, the composition is provided as a pre-filled syringe.

[0192] In certain embodiments, the composition is a lyophilized formulation. The terms "lyophilization" or "freeze-dried" include a state of matter that has been subjected to a drying procedure, such as lyophilization, in which at least 50% of the water has been removed.

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

[0194] treatment method In certain aspects, provided herein are methods of treating a disease or disorder comprising administering to a subject an antigen binding protein (e.g., an antibody, TCR and / or CAR 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 obtainable using the methods described herein (e.g., using a non-human animal described herein).

[0195] In certain embodiments, provided herein are methods for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising an antibody described herein, such as a fully human antibody, TCR, or CAR described herein, obtained from a non-human animal described herein). 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, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. Non-limiting examples of various histological types of cancer include neoplasms (malignant); carcinoma; carcinoma (undifferentiated); giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma (malignant); cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, Familial polyposis coli;Solid tumors;Carcinoid tumors (malignant);Bronchioloalveolar adenocarcinoma;Papillary adenocarcinoma;Chromophobe carcinoma;Osinophilic carcinoma;Osinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Cutaneous adenocarcinoma;Apocrine adenocarcinoma;Sebaceous adenocarcinoma;Ear wax adenocarcinoma;Mucous epidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous adenocarcinoma Liquid cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease (breast); Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma (malignant); Ovarian stromal tumor (malignant); Theca cell tumor (malignant); Granulosa cell tumor (malignant); and Robustoma (malignant); Sertoli cell carcinoma; Leydig cell tumor (malignant); Lipid cell tumor (malignant) ;Paragaglioma (malignant);Extramammary paraganglioma (malignant);Pheochromocytoma;Hemangioangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Blue nevus (malignant);Sarcoma;Fibrosarcoma;Fibrous histiocytoma (malignant);Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor (malignant);Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymal cell tumor (malignant);Brenner tumor (malignant);Pseudoleiomyoma (malignant);Synovial sarcoma;Mesothelioma (malignant);Dysgerminoma;Embryonal carcinoma;Teratoma (malignant);Ovarian goiter (malignant);Choriocarcinoma;Mesonephroma (malignant);Angiosarcoma;Hemangioendothelioma (malignant);Kaposi's sarcoma;Hemangiopericytoma (malignant);Lymphangiosarcoma;Bone Sarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma (malignant); Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor (malignant); Ameloblastic odontoma; Ameloblastic epithelioma (malignant); Ameloblastic fibrosarcoma; Pinealoma (malignant); Chordoma; Glioma (malignant); Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Glioastroblastoma; Glioblastoma; Rare Dendroglioma;Oligodendroglioma;Primary neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma (malignant);Neurofibrosarcoma;Neurilemoma (malignant);Granular cell tumor (malignant);Malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;Side granuloma;Malignant lymphoma (small lymphocytic);Malignant lymphoma (large cell, diffuse);Malignant lymphoma (follicular); mycosis fungoides; other certain non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0196] In certain embodiments, the antibody, TCR, or CAR in the pharmaceutical composition administered to a subject has binding specificity for an epitope of a cancer-associated antigen (e.g., an epitope expressed by the cancer being treated). Examples of cancer-associated antigens include adipophilin, 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, 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, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, meroe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myoglobin Osin 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, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, SI, cernin. 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 proteins, TAG-1, TAG-2, telomerase, TGF-βRII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a. In some embodiments, the antigen is a neoantigen.

[0197] In certain embodiments, provided herein are methods for treating a subject suffering from an infectious disease, such as a viral infection, a fungal infection, a bacterial infection, a helminth infection, or a protozoan infection, comprising administering to the subject a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising an antibody, TCR, or CAR described herein obtained from a non-human animal described herein). Non-limiting examples of viral infections include retroviruses such as HPV, HBV, hepatitis C virus (HCV), human immunodeficiency viruses (HIV-1 and HIV-2), herpes viruses 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-limiting 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 for an epitope of an antigen expressed by an infectious pathogen (e.g., an epitope expressed by the infectious pathogen being treated).

[0199] In some embodiments, provided herein are methods of treating an inflammatory disease, skin or organ transplant rejection, graft-versus-host disease (GVHD), or an autoimmune disease, comprising administering to a subject a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising an antibody, TCR, or CAR described herein obtained from a non-human animal described herein). Examples of autoimmune diseases include glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, anemia with malignant fever, Goodpasture's syndrome, aseptic disease, 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, celiac disease, and These include Cushing's syndrome, dermatomyositis, discoid lupus, lupus erythematosus, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes mellitus, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphocytopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis nodosa, autoimmune polyglandular syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, localized scleroderma (or CREST syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type B insulin resistance, ulcerative colitis, and Wegener's granulomatosis.

[0200] In certain embodiments, the antibody, TCR, or CAR in the pharmaceutical composition administered to a subject has binding specificity for a target of autoreactive T cells in the disease being treated (e.g., an epitope targeted by autoreactive T cells in an autoimmune disease). Exemplary proteins targeted by 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, including, for example, oral, nasal, rectal, vaginal, parenteral, intracisternal, and topical, buccal, and sublingual administration by powder, ointment, or drops. In certain embodiments, the pharmaceutical compositions are delivered conventionally (e.g., orally or parenterally).

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

[0203] The selected dosage level will depend upon a variety of factors, including the activity of the particular agent used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound used, the duration of treatment, as well as the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0204] In some embodiments, CAR and / or TCR described herein are used for T cell-based therapy.For example, in certain embodiments, T cells that express CAR and / or TCR described herein are administered to subjects to induce T cell-based immune response in subjects.Methods useful for T cell-based therapy are described in, for example, 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 embodiments, provided herein are methods for 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) that expresses a CAR or TCR described herein.

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

[0207] In some embodiments, provided herein are methods for inhibiting an immune response in a subject. In some embodiments, the methods involve administering regulatory T cells (e.g., CD4 T cells) expressing a described CAR or TCR. + , CD-25 + , and Foxp3 + The method includes administering to a subject a T cell-specific antibody (regulatory T cells or Treg17 T cells).

[0208] In some embodiments, the subject is a subject in need thereof, for example, a subject with an autoimmune disease. In such embodiments, the T cell is a regulatory T cell (i.e., a suppressor T cell), and the peptide in the peptide / MHC complex recognized by the TCR or CAR is an autoantigen against which the subject is undergoing an autoimmune response.

[0209] nucleic acid molecule Provided herein are nucleic acid molecules encoding the antibodies, TCRs, or CARs described herein, and / or portions of the antibodies, TCRs, and CARs described herein. In some embodiments, the nucleic acid encodes the variable domain of the antibodies, TCRs, or CARs described herein. The nucleic acid molecules can be present, for example, in whole cells, cell lysates, or in a partially purified or substantially pure form.

[0210] In certain embodiments, the present specification provides nucleic acids encoding the antibody, TCR and / or CAR polypeptides described herein or portions thereof.The nucleic acids can be, for example, in whole cells, cell lysates, or in partially purified or substantially pure form.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 CAR, TCR, or antibodies expressed by hybridomas, cDNAs encoding each chain of the antibody, TCR, or CAR produced by hybridomas 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" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop that can be ligated to additional DNA segments. Another type of vector is a viral vector, in which additional DNA segments can be ligated to the viral genome. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Additionally, certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0212] In certain embodiments, provided herein are cells containing a nucleic acid described herein (e.g., a nucleic acid encoding an antibody, TCR, or CAR described herein, or a nucleic acid encoding a portion thereof). The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine, and / or human. In certain embodiments, the nucleic acid described herein is operably linked to a transcriptional control element, such as a promoter. In some embodiments, the cell transcribes the nucleic acid described herein, thereby expressing an antibody, antigen-binding fragment thereof, or polypeptide described herein. The nucleic acid molecule may be integrated into the genome of the cell, or it 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] For the antibodies and CAR nucleic acids described herein, VH and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V H The DNA fragment encoding the antibody is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0215] The isolated DNA encoding the heavy chain variable region can be converted into a full-length heavy chain gene by operably linking the heavy chain variable region DNA to another DNA molecule encoding a 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, USDapartment 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 heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgD constant region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0216] The isolated DNA encoding the 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, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment 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 comprises in its genome a nucleic acid sequence encoding human terminal deoxynucleotidyl transferase (hTdT).

[0218] In exemplary embodiment 2, provided herein is a genetically modified non-human animal according to 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 a genetically modified non-human animal according to embodiment 2, wherein the transcriptional control element drives expression of a nucleic acid sequence encoding exogenous hTdT in pro-B cells and / or pre-B cells.

[0220] In exemplary embodiment 4, provided herein is a genetically modified non-human animal according to embodiment 2, wherein the transcriptional control element is selected from the group consisting of a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin kappa light chain transcriptional control element, and / or an immunoglobulin lambda light chain transcriptional control element.

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

[0222] In exemplary embodiment 6, provided herein is a genetically modified non-human animal as described in embodiment 2, wherein the transcriptional control element drives 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, provided herein is a genetically modified non-human animal according to embodiment 2, wherein the transcriptional control element is a RAG1 transcriptional control element, a RAG2 transcriptional control element, a TCR alpha transcriptional control element, a TCR beta transcriptional control element, a TCR gamma transcriptional control element, and / or a TCR delta transcriptional control element.

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

[0225] In exemplary embodiment 9, provided herein is a genetically modified non-human animal according to any one of embodiments 1 to 8, wherein the nucleic acid sequence encoding hTdT is located at an immunoglobulin kappa light chain locus, an immunoglobulin lambda light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, a RAG2 locus, a TCR alpha chain locus, a TCR beta chain locus, a TCR gamma chain locus, and / or a TCR delta chain locus.

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

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

[0228] In exemplary embodiment 12, provided herein is a genetically modified non-human animal of any one of embodiments 1-11, wherein at least 10% of the VJ immunoglobulin light chain junctions in the animal comprise non-templated additions.

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

[0230] In exemplary embodiment 14, provided herein is a genetically modified non-human animal according to embodiment 12, wherein at least 40% of the VJ immunoglobulin light chain junctions in the animal comprise non-templated additions.

[0231] In exemplary embodiment 15, provided herein is a genetically modified non-human animal comprising in its genome a nucleic acid sequence encoding an exogenous terminal deoxynucleotidyl transferase (TdT), and an immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to an immunoglobulin constant region gene.

[0232] In exemplary embodiment 16, provided herein is a genetically modified non-human animal according to embodiment 15, wherein the exogenous TdT is human TdT.

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

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

[0235] In exemplary embodiment 19, provided herein is a genetically modified non-human animal of embodiment 17, wherein the transcriptional control element is selected from the group consisting of a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin kappa light chain transcriptional control element, and / or an immunoglobulin lambda light chain transcriptional control element.

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

[0237] In exemplary embodiment 21, provided herein is a genetically modified non-human animal according to embodiment 17, wherein the transcriptional control element drives 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, provided herein is a genetically modified non-human animal according to embodiment 17, wherein the transcriptional control element is a RAG1 transcriptional control element or a RAG2 transcriptional control element.

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

[0240] In exemplary embodiment 24, provided herein is a genetically modified non-human animal according to any one of embodiments 15 to 23, wherein the nucleic acid sequence encoding exogenous TdT is located at an immunoglobulin kappa light chain locus, an immunoglobulin lambda light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, a RAG2 locus, a TCR alpha chain locus, a TCR beta chain locus, a TCR gamma chain locus, and / or a TCR delta chain locus.

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

[0242] In an exemplary embodiment 26, provided herein is a genetically modified non-human animal according to any one of embodiments 15 to 25, wherein exogenous TdT is not constitutively expressed.

[0243] In exemplary embodiment 27, provided herein is a genetically modified non-human animal of any one of embodiments 15-26, wherein at least 10% of the VJ immunoglobulin light chain junctions in the animal comprise non-templated additions.

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

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

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

[0247] In exemplary embodiment 31, provided herein is a genetically modified non-human animal according to embodiment 30, wherein the constant region gene is a heavy chain constant region gene.

[0248] In exemplary embodiment 32, provided herein is a genetically modified non-human animal according to embodiment 31, 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, provided herein is a genetically modified non-human animal according to embodiment 31 or 32, wherein the heavy chain constant region gene is from an endogenous species.

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

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

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

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

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

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

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

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

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

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

[0260] In exemplary embodiment 44, provided herein is a genetically modified non-human animal of embodiment 43, 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, provided herein is a genetically modified non-human animal of embodiment 43, 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, provided herein is a genetically modified non-human animal according to any one of embodiments 43-45, wherein the second constant region gene is a light chain constant region gene.

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

[0264] In exemplary embodiment 48, provided herein is a genetically modified non-human animal according to embodiment 46, wherein the second constant region gene is a lambda constant region gene.

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

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

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

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

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

[0270] In exemplary embodiment 54, provided herein is a genetically modified non-human animal according to embodiment 53, 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] In exemplary embodiment 55, provided herein is a genetically modified non-human animal according to embodiment 53, wherein the second constant region gene is a lambda constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin lambda locus.

[0272] In exemplary embodiment 56, provided herein is a genetically modified non-human animal of any one of embodiments 30-42, further comprising in its genome an immunoglobulin variable region comprising 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, provided herein is a genetically modified non-human animal of embodiment 56, wherein the rearranged human light chain variable region (V / J) gene segment operably linked to the second immunoglobulin constant region gene comprises a Vκ gene segment selected from Vκ1-39 and Vκ3-20, and is rearranged to a Jκ gene segment.

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

[0275] In exemplary embodiment 59, provided herein is a genetically modified non-human animal of any one of embodiments 30-42, further comprising in its genome an immunoglobulin variable region comprising 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, provided herein is a genetically modified non-human animal according to embodiment 59, wherein the limited repertoire of human light chain variable region (V and J) gene segments operably linked to the second immunoglobulin constant region gene comprises two V gene segments and at least two, preferably five J gene segments.

[0277] In exemplary embodiment 61, provided herein is a genetically modified non-human animal according to embodiment 60, wherein the two V gene segments are Vκ1-39 and Vκ3-20 gene segments.

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

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

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

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

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

[0283] In exemplary embodiment 67, provided herein is a genetically modified non-human animal according to embodiment 65, wherein the constant region gene is a lambda constant region gene.

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

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

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

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

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

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

[0290] In exemplary embodiment 74, provided herein is a genetically modified non-human animal according to embodiment 73, 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, provided herein is a genetically modified non-human animal according to embodiment 73, wherein the constant region gene is a lambda constant region gene and the endogenous immunoglobulin light chain locus is an immunoglobulin lambda locus.

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

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

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

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

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

[0297] In exemplary embodiment 81, provided herein is a genetically modified non-human animal according to embodiment 80, wherein the inactivated endogenous immunoglobulin locus is an endogenous immunoglobulin heavy chain locus.

[0298] In exemplary embodiment 82, provided herein is a genetically modified non-human animal according to embodiment 81, wherein 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.

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

[0300] In exemplary embodiment 84, provided herein is a genetically modified non-human animal according to embodiment 81, wherein 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, provided herein is a genetically modified non-human animal according to embodiment 84, wherein the deletion of at least a portion of the constant region comprises a deletion of the Cμ gene of the constant region.

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

[0303] In exemplary embodiment 87, provided herein is a genetically modified non-human animal according to embodiment 86, wherein 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.

[0304] In exemplary embodiment 88, provided herein is a genetically modified non-human animal according to embodiment 87, wherein the deletion of at least a portion of the variable region comprises a deletion of a J gene segment of the variable region.

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

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

[0307] In exemplary embodiment 91, provided herein is a genetically modified non-human animal according to embodiment 80, wherein the inactivated endogenous immunoglobulin locus is an endogenous λ immunoglobulin chain locus.

[0308] In exemplary embodiment 92, provided herein is a genetically modified non-human animal according to embodiment 91, 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, provided herein is a genetically modified non-human animal of any one of embodiments 15-92, wherein unrearranged human immunoglobulin variable region gene segments are rearranged during B cell development to generate rearranged variable region genes in B cells of the non-human animal.

[0310] In exemplary embodiment 94, provided herein is a genetically modified non-human animal according to embodiment 93, wherein at least 10% of the rearranged variable region genes comprise non-templated additions.

[0311] In an exemplary embodiment, provided herein is a genetically modified non-human animal according to embodiment 93, wherein at least 20% of the rearranged variable region genes comprise non-templated additions.

[0312] In an exemplary embodiment, provided herein is a genetically modified non-human animal according to embodiment 93, wherein at least 40% of the rearranged variable region genes comprise non-templated additions.

[0313] In exemplary embodiment 97, provided herein is a genetically modified non-human animal of any one of embodiments 93-96, wherein 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.

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

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

[0316] In exemplary embodiment 100, provided herein is a genetically modified non-human animal according to embodiment 99, wherein the exogenous TdT is human TdT.

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

[0318] In exemplary embodiment 102, provided herein is a genetically modified non-human animal according to embodiment 101, wherein the transcriptional control element drives 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] In exemplary embodiment 103, provided herein is a genetically modified non-human animal according to embodiment 101, wherein the transcriptional control element is a RAG1 transcriptional control element, a RAG2 transcriptional control element, a TCR alpha transcriptional control element, a TCR beta transcriptional control element, a TCR gamma transcriptional control element, and / or a TCR delta transcriptional control element.

[0320] In exemplary embodiment 104, provided herein is a genetically modified non-human animal 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, provided herein is a genetically modified non-human animal according to any one of embodiments 99 to 104, wherein the nucleic acid sequence encoding exogenous TdT is located at the RAG1 locus, the RAG2 locus, the TCR alpha chain locus, the TCR beta chain locus, the TCR gamma chain locus, and / or the TCR delta chain locus.

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

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

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

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

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

Claims

1. A targeting vector comprising: (i) a 5' homology arm comprising a nucleotide sequence corresponding to a genomic target sequence comprising the immunoglobulin κ 3' enhancer and 3' immunoglobulin κ recombination sequence (RS) within an unmodified endogenous rodent κ light chain locus; (ii) a transcriptional control element capable of driving expression in rodent pre-B cells; (iii) the short isoform of the human TdT gene (TdTS); (iv) a 3' homology arm comprising a nucleotide sequence corresponding to a genomic target sequence immediately downstream of the sequence corresponding to the 5' homology arm in the genome of the rodent; wherein the rodent is a rat or a mouse.

2. 2. The targeting vector of claim 1, wherein the TdTS sequence has been modified to remove (i) the splice donor site of exon 7 and (ii) the splice acceptor site of exon 12.

3. 2. The targeting vector of claim 1, wherein the transcriptional control element is capable of driving expression in pro-B cells of a rodent.

4. The targeting vector of claim 1 , wherein the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin kappa light chain transcriptional control element, and / or an immunoglobulin lambda light chain transcriptional control element.

5. 2. The targeting vector of claim 1, wherein the transcriptional control element comprises (i) a rodent IgVH1-72 promoter and a rodent Eμ enhancer, and / or (ii) a RAG2 promoter.

6. The targeting vector according to any one of claims 1 to 5, wherein the human TdT gene is the short isoform of TdT (TdTS).

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