NZ623102B2

NZ623102B2 - Restricted immunoglobulin heavy chain mice

Info

Publication number: NZ623102B2
Application number: NZ623102A
Authority: NZ
Inventors: Cagan Gurer; Lynn Macdonald; John Mcwhirter; Karolina A Meagher; Andrew J Murphy
Original assignee: Regeneron Pharmaceuticals Inc
Priority date: 2011-10-17
Filing date: 2012-10-17
Publication date: 2017-01-05

Abstract

Disclosed is a rat or mouse having in its germline genome a restricted endogenous immunoglobulin heavy chain locus characterized by the presence of a single unrearranged human VH gene segment, one or more unrearranged human DH gene segments, and one or more unrearranged human JH gene segments operably lined to a non-human constant region gene sequence comprising a non-human IgM gene, wherein the rat or mouse further comprises a diverse repertoire of rearranged human immunoglobulin heavy chain variable region genes, each of which is linked to the non-human constant region gene sequence comprising a non-human IgM gene and is derived from the restricted immunoglobulin heavy chain locus. ly lined to a non-human constant region gene sequence comprising a non-human IgM gene, wherein the rat or mouse further comprises a diverse repertoire of rearranged human immunoglobulin heavy chain variable region genes, each of which is linked to the non-human constant region gene sequence comprising a non-human IgM gene and is derived from the restricted immunoglobulin heavy chain locus.

Description

RESTRICTED IMMUNOGLOBULIN HEAVY CHAIN MICE FIELD Non-human animals that are genetically engineered at an immunoglobulin heavy chain le (V) region locus (or in a transgene) to make dies from a restricted number of immunoglobulin heavy chain variable (VH) segments (or a single VH segment) and/or variants thereof. man s that have a human heavy chain variable domain derived from a single globulin heavy chain variable gene segment, e.g., human immunoglobulin VH1- 69 gene segment or human VH1-2 gene t. s for making antibody sequences in non-human animals that are useful for binding pathogens, including human pathogens.

BACKGROUND Non-human animals, e.g., mice, have been genetically engineered to be useful tools in methods for making antibody sequences for use in antibody-based human therapeutics.

Mice with humanized variable region loci (e.g., VH, DH, and JH genes, and V._ and JL genes) are used to generate cognate heavy and light chain variable domains for use in antibody therapeutics. Other mice are available that generate fully human antibodies with cognate heavy and light chains.

Human antibody therapeutics are engineered based on desired characteristics with respect to certain pre-selected antigens. zed mice are immunized with the pre-selected antigens, and the zed mice are used to generate antibody populations from which to identify high-afﬁnity cognate heavy and light variable domains with desired binding characteristics. Some humanized mice, such as those having a humanization ofjust variable regions at endogenous mouse loci, generate populations of B cells that are r in character and number to wild-type mouse B cell populations. As a , an extremely large and diverse population of B cells is available in these mice from which to screen antibodies, reflecting a large number of different immunoglobulin rearrangements, to identify heavy and light variable domains with the most desirable characteristics.

But not all antigens provoke an immune response that exhibits a very large number of rearrangements from a wide selection of variable (V) segments. That is, the human humoral immune response to certain antigens is ntly cted. The restriction is reﬂected in clonal selection of B cells that express only certain V segments that bind that particular antigen with sufﬁciently high afﬁnity and speciﬁcity. Some such antigens are clinically significant, i.e., a number are well-known human ens. A presumption arises that the V segment expressed in the human immune response is a V segment that, in combination with a human D and a human J segment, is more likely to te a useful high affinity antibody than a randomly selected V segment that has not been observed in a human antibody response to that antigen.

It is hypothesized that l selection, over millennia, has selected the most efﬁcient foundation or base from which to design a most effective weapon for neutralizing human pathogens—a clonally selected V segment. There is a need in the art for more and superior antibodies that bind and/or neutralize antigens such as the ens discussed above. There is a need to more rapidly generate useful sequences from selected V segments, including polymorphic and/or somatically d selected V segments and to more rapidly generate useful populations of B cells having rearrangements of the V segments with various D and J segments, including somatically mutated versions f, and in particular rearrangements with unique and useful CDR3s. There is a need for biological systems, e.g., non-human animals (such as, e.g., mice, rats, rabbits, etc.) that can generate eutically useful antibody variable region sequences from pre-selected V segments in increased number and diversity than, e.g., can be achieved in existing modiﬁed animals. There is a need for biological systems engineered to have a committed humoral immune system for clonally selecting antibody variable ces derived from restricted, pre-selected V segments, including but not limited to e human heavy and light chain variable domains, useful in the manufacture of human antibody-based therapeutics against selected ns, including certain human pathogens.

There is a need in the art for therapeutic antibodies that are capable of neutralizing viral antigens, e.g., HIV and HCV, ing antigen-speciﬁc antibodies containing heavy chains d from a single human variable segment, and for a system that produces a diverse source of antibodies from which to select therapeutic antibody sequences. There is also a need for further methods and non-human animals for making useful antibodies, including antibodies that comprise a oire of heavy chains derived from a single human VH segment and having a e set of CDR sequences, and including such heavy chains that s with cognate human light chain variable domains. Methods are needed for selecting CDRs for immunoglobulin-based binding proteins that provide an enhanced diversity of binding ns from which to choose, and enhanced diversity of immunoglobulin variable domains, including compositions and methods for generating somatically mutated and ly selected immunoglobulin variable s for use, e.g., in making human therapeutics.

Genetically modiﬁed immunoglobulin loci are provided that comprise a restricted number of different heavy chain variable region gene segments (i.e., V genes, VH genes, VH gene segments, or V gene segments), e.g., no more than one, two, or three different V genes; or no more than one V gene segment family member present, e.g., in a single copy or in multiple copies and/or sing one or more polymorphisms.

Loci are provided that are capable of rearranging and forming a gene encoding a heavy chain variable domain that is derived from a VH gene repertoire that is restricted, e.g., that is a single VH gene t or selected from a ity of polymorphic variants of the single VH gene segment. Modiﬁed immunoglobulin loci include loci that comprise human globulin sequences are provided, e.g., a human V segment ly linked to a human or (or human/non-human chimeric) non~human immunoglobulin constant sequence (and in operable linkage with, e.g., a D and/or a J segment). Modified loci that comprise multiple copies of a single VH gene segment, including wherein one or more of the copies comprises a polymorphic variant, are provided. Modiﬁed loci that comprise multiple copies of a single VH segment, operably linked with one or more D segments and one or more J segments, operably linked to a non-human immunoglobulin constant sequence, e.g., a mouse or rat sequence, are provided. man animals comprising such humanized loci are also provided.

Non-human animals are provided that have a reduced immunoglobulin heavy chain variable gene segment complexity (Le, a limited number of heavy chain variable gene segments, or a limited heavy chain variable gene oire), wherein the reduced immunoglobulin heavy chain variable gene segment complexity is characterized by the presence of no more than one or no more than two heavy chain variable gene segments, and wherein the heavy chain variable genes present are operably linked to a human or non-human constant region sequence.

Non-human animals are provided that have a d immunoglobulin heavy chain variable gene segment complexity (e.g., a single VH gene segment, or a d number of VH gene segments that are polymorphic variants of a single VH gene segment), wherein the reduced immunoglobulin heavy chain le gene t complexity is characterized by the presence of a single VH gene segment or a plurality of VH gene segments that are polymorphic forms of a single VH gene segment (e.g., VH gene segments associated with high copy number and/or polymorphism in humans), and wherein the heavy chain variable genes present are operably linked to a human or non-human constant region sequence. In various embodiments, the heavy chain le genes present are operably linked to one or more D and/or one or more J gene segments in the germline of the non-human animal.

Non-human animals are provided that comprise an immunoglobulin heavy chain variable locus (e.g., on a transgene or as an insertion or replacement at an nous non- human animal heavy chain variable locus) that comprises a single VH t operably linked to a D and/or J gene segment. In various ments, the single VH gene segment is operably linked to one or more D and/or one or more J gene segments at the endogenous immunoglobulin heavy chain variable gene locus of the non-human animal.

] Non-human animals are provided that are modiﬁed at their immunoglobulin heavy chain le region loci to delete all or substantially all (e.g., all functional segments, or nearly all functional segments) endogenous immunoglobulin VH segments and that se a human VH1-69 segment (or a human VH1-2 segment) ly linked to a D and J segment or a J segment at the endogenous immunoglobulin heavy chain variable region locus of the non- human animal.

Non-human animals are also provided that are modiﬁed at their immunoglobulin heavy chain variable region loci to render the endogenous variable region loci incapable of rearranging to form a functional heavy chain comprising endogenous le region gene segments; wherein the non-human animals comprise a single human variable gene segment (a human VH1-2 or a human VH1-69 gene segment) operably linked to a D and a J segment or a J segment at the endogenous immunoglobulin heavy chain variable region locus of the non- human animal.

Non-human s are provided that comprise a restricted number (e.g., no more than one, or no more than two) of heavy chain gene segments operably linked to a human or non-human constant region sequence. In one embodiment, the no more than one or no more than two heavy chain gene segments linked to the constant region sequence are on a transgene, e.g., are at a position other than an endogenous heavy chain locus.

Methods are provided for making human immunoglobulin sequences in non-human animals. In various ments, the human immunoglobulin sequences are derived from a repertoire of immunoglobulin V sequences that consist essentially of a single human V t, e.g., VH1-69 or VH1-2, and one or more D and J segments or one or more J segments. Methods for making human immunoglobulin sequences in non-human animals. tissues, and cells are provided. wherein the human immunoglobulin sequences bind a s are provided for making mice characterized by a restricted immunoglobulin heavy chain locus, wherein the restriction is with respect to the number of immunoglobulin VH gene segments. in various aspects, the restriction is to one or no more than two, or a single VH gene family member (e.g., one or more VH alleles, variants. or polymorphic variants thereof). in various aspects, the heavy chain locus r ses one or more DH gene segments and one or more JH gene segments. In various aspects, the VH, DH and JH gene segments are human. In various aspects, the V”, DH and JH gene segments are operably linked to a non- human constant region (e.g., an lgM and/or an lgG). In various s, the constant region is a mouse or rat nt region.

In one aspect, a method for making a mouse having a restricted immunoglobulin heavy chain locus is provided, comprising introducing a nucleic acid construct as described herein into a mouse embryonic stem (ES) cell, and isolating or identifying a mouse ES cell that comprises the nucleic acid construct.

In one ment, the nucleic acid construct comprises a single human VH gene segment, one or more human DH gene ts, and one or more human JH gene segments.

In one embodiment, the nucleic acid construct comprises one or more site-speciﬁc recombination sites (e.g., a loxP or a Frt site).

In one aspect, a mouse made using a targeting vector, nucleic acid sequence, or cell as described herein is provided. In various embodiments, the targeting vector, nucleic acid sequence or cell comprises a DNA sequence that contains a single human VH gene segment (or rphic variants thereof), one or more human DH gene segments, and one or more human JH gene segments operably linked to a non-human constant gene.

In one aspect, a method for making a mouse comprising a restricted immunoglobulin heavy chain locus is provided, comprising replacing a mouse globulin heavy chain locus with a human genomic sequence comprising a single human VH gene segment (or polymorphic variants thereof), one or more human DH gene segments, and one or more human JH gene segments, wherein the human VH, DH and JH gene segments are e of rearranging to form a chimeric heavy chain that contains a human variable domain operably linked to a non-human constant region. In one embodiment, the non-human nt region is a mouse or rat constant .

In various aspects, the non-human animals are rodents. In various aspects, the rodents are mice and/or rats.

] In one aspect, a modified immunoglobulin heavy chain locus is provided that comprises a heavy chain V t repertoire that is restricted with respect to the identity of the V segment, and that comprises one or more D ts and one or more J segments, or one or more J segments. In one embodiment, the heavy chain V segment is a human segment. In one embodiment, the one or more D ts are human D ts. In one embodiment, the one or more J ts are human J segments. In one embodiment, the one or more D segments and one or more J ts are human D and human J segments.

In one embodiment, the modified locus is a non-human locus. In one embodiment, the man locus is modiﬁed with at least one human immunoglobulin sequence.

In one embodiment, the restriction is to one V segment family member. In one embodiment, the one V segment family member is present in two or more copies. In one embodiment, the one V segment family member is present as two or more variants (e.g., two or more polymorphic forms of the V segment family member). In one embodiment, the one V segment is a human V segment family member. In one embodiment, the one V segment family member is present in a number of variants as is observed in the human population with respect to that variant. In one embodiment, the V segment family member is selected from Table 1. In one embodiment, the V segment family member is present in a number of variants as shown, for each V segment, in a number of alleles from 1 allele to the number of alleles shown in the right column of Table 1.

In one embodiment, the restriction is to a human VH1-69 gene segment. In one embodiment, the human VH1-69 gene segment is present in two or more copies. In one embodiment, the human VH1-69 gene segment is present as two or more variants (e.g., two or more polymorphic forms the human VH1-69 gene). In one embodiment, the human VH1-69 gene segment is present in a number of variants as is observed in the human population with respect to the human VH1-69 gene segment. In one ment, the human VH1-69 gene segment is selected from Table 2. In one ment, the human VH1-69 gene segment is t in a number of variants as shown, for each VH1-69 gene segment, in a number of alleles from one allele to the number of alleles shown in Table 2.

In one ment, the restriction is to a human VH1-2 gene segment. In one embodiment, the human VH1-2 gene t is present in two or more copies. In one embodiment, the human VH1-2 gene segment is present as two or more variants (e.g., two or more polymorphic forms the human VH1-2 gene). In one embodiment, the human VH1-2 gene segment is present in a number of variants as is observed in the human tion with respect to the human VH1-2 gene segment. In one embodiment, the human VH1-2 gene segment is selected from Table 3. In one embodiment, the human VH1-2 gene segment is present in a number of variants as shown, for each VH1-2 gene t, in a number of alleles from one allele to the number of alleles shown in Table 3.

In one aspect, a heavy chain immunoglobulin locus is provided that comprises a single functional human V segment. In one ment, the single functional human V segment is selected from a VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1- 69, VH2-5, , VH2-70, VH3-7, VH3-9, VH3-11, VH3-13, VHS-15, VH3-16, VHS-20, VHS-21, VH3-23, VH3-30, VH33, -5, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VHS-64, , VH3-72, VH3-73, VH3-74, VH4-4, , VH41, VH42, VH44, VH4- 31, VH4-34, , VH4-59, VH4-61, VH5-51, VHS-1, VH71, and a VH7-81 segment. In one embodiment, the single functional human V segment is a Vin-69 segment; in a speciﬁc embodiment, the single functional human V segment is t in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polymorphic forms found in the human population. In one embodiment, the single functional human V segment is a VH1-2 t; in a speciﬁc embodiment, the single functional human V segment is present in 1, 2, 3, 4, or 5 polymorphic forms found in the human population.

In one embodiment, the heavy chain immunoglobulin locus is a modiﬁed locus of a non-human animal. In one embodiment, the modiﬁed non-human immunoglobulin heavy chain locus is present in the non-human animal at a position in the genome in which the corresponding unmodified man locus is found in the wild-type man animal. In one embodiment, the modified non-human immunoglobulin heavy chain locus is present on a transgene in a non-human animal.

In one embodiment, the single functional human V gene segment is a Vin-69 gene segment. In one ment, the VH1-69 gene segment comprises SEQ ID NO: 34. In one embodiment, the VH1-69 gene segment is derived from SEQ ID NO: 34. In one embodiment, the VH1-69 gene segment is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 34.

In one embodiment, the single functional human V gene segment is encoded by the nucleotide sequence of SEQ ID NO: 34.

In one ment, the single functional human V gene segment is a VH1-2 gene segment. In one embodiment, the VH1-2 gene segment comprises SEQ ID NO: 60. In one ment, the VH1-2 gene segment is derived from SEQ ID NO: 60. In one embodiment, the VH1-2 gene segment is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 60.

In one embodiment, the single onal human V gene segment is encoded by the nucleotide sequence of SEQ ID NO: 60.

In one embodiment, the single functional human V segment is operably linked to one or more D segments and one or more J segments, or one or more J ts. In one embodiment, the V segment and one or more D and/or J segments are operably linked to an immunoglobulin heavy chain constant region sequence. In one embodiment the immunoglobulin heavy chain constant region sequence is selected from a CH1, a hinge, 3 CH2, a CH3 sequence, and a combination thereof. In one embodiment, the CH1, hinge, CH2, CH3, or combination thereof are each non-human endogenous constant sequences. In one embodiment, at least one of the CH1, hinge, CH2, CH3, or combination thereof is a human sequence. In a specific embodiment, the CH1 and/or hinge are human sequences.

In one aspect, a modiﬁed endogenous non-human immunoglobulin heavy chain locus is provided, comprising a replacement of all functional V gene segments with a single human V gene segment (or a single human V gene segment present in multiple polymorphic forms or copy number), wherein the man immunoglobulin heavy chain locus is incapable of rearrangement to form a heavy chain le gene that is derived from a V gene segment other than the single human V gene segment (or one of the polymorphic forms or copies).

In one ment, the single human V gene segment is . In one embodiment, the single human V gene segment is VH1-2.

In one embodiment, the locus comprises at least one human or non-human DH gene segment, and one human or non-human JH gene segment. In a ic embodiment, the locus comprises a human DH gene t and a human JH gene t. In a speciﬁc embodiment, the locus comprises a human JH gene segment. In another speciﬁc embodiment. the locus comprises a human Val-69 gene segment (present as a single copy or multiple copies of different polymorphic variants), all functional human DH gene segments. and all functional human JH gene segments. In another specific embodiment, the locus comprises a human VH1-2 gene segment (present as a single copy or multiple copies of different polymorphic , all functional human DH gene ts, and all functional human JH gene segments. In one embodiment, the human V, D, and J gene segments (or V and J gene segments) are operably linked to a mouse constant region gene at an endogenous mouse heavy chain locus. In a speciﬁc ment, the mouse heavy chain locus comprises a wild- type oire of mouse immunoglobulin constant region sequences.

In one aspect, a genetically modiﬁed non-human animal is ed, n the only functional globulin heavy chain V gene segment of the non-human animal is selected from a human VH1-2,VH1-3,VH1-8,VH1-18,VH1-24,VH1-45,VH1-46,VH1-58,VH1-69, VH2-5, VH2-26, VH2-70, VH3-7, VHS-9, VH3-11, VH3-13, VH3-15, VH3-16, , VHS-21, VHS-23, VH3-30, VH3'30-3, -5, VH3-33, VH3-35, VH3-38, VHS-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VHS-72, , VH3-74, VH4-4, VH4-28, VH4-3O-1, VH42, -4, VH4-31, VH4- 34, VH4-39, VH4-59, VH4-61, VHS-51, VH6-1, VH71, and VH7-81 gene t. In one embodiment, the heavy chain V gene segment is a human VH1-69 gene t. In one embodiment, the heavy chain V gene segment is a human VH1-2 gene segment.

In one aspect, a genetically modiﬁed non-human animal is provided, wherein the non-human animal comprises a single functional human VH gene segment (present as a single copy or multiple copies of different polymorphic , and wherein the non-human animal is substantially incapable of forming a rearranged immunoglobulin heavy chain variable domain gene that lacks the single functional human VH gene segment (or one of the polymorphic forms or copies).

] In one aspect, a genetically modiﬁed non-human animal is provided, wherein the only immunoglobulin heavy chain variable region expressed in the non-human animal is derived from one of a human segment selected from a human VHl-2, VH1-3, VH1-8, VH1-18, VH1-24, VH‘I-45, VH1-46, , VH1-69, VH2-5, VH2-26. VH2-70, VHS-7, VH3-9, VH3-11, VH3-13, VHS-15, VH3-16, VH3-20, VH3-21, VHS-23, VH3-30, VH33, VH35, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, , VH3-72. , VH3-74, VH4-4, VH4-28, VH4- -1, VH42, VH44, VH4-31, VH4-34, VH4-39, , VH4~61, VHS-51, VHS-1, VH71, and VH7-81 gene segment. In one embodiment, the human segment is a VH1-69 segment. In one embodiment, the human segment is a VH1-2 segment. In one embodiment, the only immunoglobulin heavy chain variable region expressed by the mouse is d from a single V segment family member, and in one embodiment the only immunoglobulin heavy chain variable region is derived from a polymorphic variant of the single V segment family member.

In one aspect, a non-human animal comprising a restricted immunoglobulin heavy chain V gene segment repertoire is provided, wherein the man animal further comprises one or more human globulin x light chain variable segments (Vic). In one ment, the one or more VK segments are operably linked to one or more human J segments. In a speciﬁc embodiment, the J segments are human JK ts. In another specific embodiment, the non-human animal does not express an immunoglobulin A light chain. In another c embodiment, the non-human animal does not comprise a functional human or functional endogenous immunoglobulin A light chain variable locus.

In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is a mouse.

In one embodiment, the non-human animal comprises a ement at the endogenous non-human immunoglobulin VK locus of all or substantially all functional endogenous VK segments with one or more functional human VK segments. In a further specific embodiment, the replacement is with all or substantially all functional human immunoglobulin VK segments.

In one embodiment, the non-human animal comprises a replacement at the nous non-human immunoglobulin VK locus of all or substantially all functional endogenous Vx gene segments with human Vx gene segments ed from Vx4-1, VK5-2, VK7-3,VK2'4,VK1-5,VK1-6.VK3-7,VK1-8,VK1-9,VK2-10,VK3-11,VK1-12,VK1-13,VK2-14, Vx3-15, Vx1-16, Vx1-17, Vx2-18, , Vx3-20, Vx6-21, VK1-22, VK1-23, Vx2-24, Vx3-25, Vx2-26, , VK2-28, Vx2-29, VK2-30, Vx3-31, Vx1-32, Vx1-33, Vx3-34, Vx1-35, Vx2-36, VK1-37, Vic2-38, VK1-39, Vx2-40, and a combination thereof.

In one embodiment, the non-human animal comprises a replacement at the endogenous non-human immunoglobulin JK locus of all or substantially all functionaI nous non-human immunoglobulin JK ts with one or more onal human immunoglobulin JK segments. In a further speciﬁc embodiment, the replacement is with all or substantially all functional human immunoglobulin JK segments.

In one ment, the non-human animal comprises a replacement at the endogenous non—human immunoglobulin JK locus of all or substantially all functional endogenous non-human immunoglobulin Jx gene segments with human Jx gene segments selected from JK1, JK2, Jx3, JK4, JKS. and a combination thereof.

In a speciﬁc embodiment, the non-human animal comprises an immunoglobulin heavy chain variable region locus that comprises a repertoire of V segments ting essentially of a single V segment and/or polymorphic variants thereof. In one embodiment, the single immunoglobulin heavy chain V segment is a human VH1-69 segment, and the non- human animal further comprises a replacement of all functional non-human DH segments with all functional human DH segments, and further comprises a replacement of all functional non- human JH segments with all onal human JH segments, and wherein the immunoglobulin heavy chain variable region locus is operably linked to a human or non-human constant region gene sequence. In a speciﬁc embodiment, the constant region gene sequence is an endogenous non-human constant region gene sequence. In a speciﬁc embodiment, the non- human animal rearranges segments at the non-human immunoglobulin heavy chain locus to form a gene encoding heavy chain variable region sing a human Vin-69 sequence, a human DH sequence, a human JH sequence, and a mouse constant region ce.

In a speciﬁc embodiment, the non-human animal ses an immunoglobulin heavy chain variable region locus that comprises a oire of V segments consisting essentially of a single V segment and/or polymorphic variants thereof. in one ment, the single immunoglobulin heavy chain V segment is a human VH1-2 segment, and the non-human animal r comprises a replacement of all functional man DH segments with all functional human DH segments, and further comprises a replacement of all onal non- human JH segments with all functional human JH segments, and wherein the immunoglobulin heavy chain variable region locus is operably linked to a human or man constant region gene sequence. In a specific embodiment, the constant region gene sequence is an endogenous non-human constant region gene sequence. In a specific embodiment, the non- human animal rearranges segments at the non-human immunoglobulin heavy chain locus to form a gene encoding heavy chain variable region comprising a human VH1-2 sequence, a human DH sequence, a human JH sequence, and a mouse constant region sequence.

In one embodiment, a B cell is provided that comprises the rearranged gene. In a speciﬁc embodiment, the B cell is from a mouse as described that has been zed with an n of st, and the B cell encodes an antibody that speciﬁcally binds the antigen of interest. In one embodiment, the antigen of interest is a pathogen. In a speciﬁc embodiment, the pathogen is selected from an inﬂuenza virus, a hepatitis virus (e.g., hepatitis B or hepatitis C virus), and a human immunodeﬁciency virus. In a speciﬁc embodiment, the B cell encodes a somatically mutated, high afﬁnity (e.g., about 10‘9 KB or lower) antibody comprising a human light chain variable region (e.g., a human K light chain variable region) that speciﬁcally binds the antigen of interest.

In one aspect, a non-human animal comprising a restricted immunoglobulin heavy chain V segment repertoire is provided, wherein the man animal ses one or more human 7» light chain variable (VA) segments. In one embodiment, the one or more human V)» segments are operably linked to one or more human J segments. In a ic embodiment, the J segments are human J)» segments. In r speciﬁc embodiment, the non-human animal does not express a K light chain. In r speciﬁc ment. the non-human animal does not comprise a functional human or non-human x light chain variable locus.

In one embodiment, the non-human animal comprises a replacement of all or substantially all functional non-human immunoglobulin VA segments with one or more onal human immunoglobulin VA segments. In a further specific embodiment, the replacement is with all or substantially all functional human immunoglobulin VA segments.

In one embodiment, the non-human animal comprises a replacement of all or substantially all functional non-human VA segments with a fragment of cluster A of the human A light chain locus. In a speciﬁc embodiment, the fragment of cluster A of the human A light chain locus comprises human VA gene segments VA3-27 through VA3-1.

In one embodiment, the man animal comprises a replacement of all or substantially all functional non-human VA segments with a fragment of cluster B of the human A light chain locus. In a specific embodiment, the nt of cluster B of the human A light chain locus comprises human VA gene segments VA5-52 h VA1-40.

In one embodiment, the non-human animal comprises a replacement of all or substantially all functional non-human VA segments with a fragment of cluster A and a fragment of cluster B of the human A light chain locus, wherein as a result of the replacement comprise human VA gene segments VA5-52 through VA3-1.

In one embodiment. the non-human animal comprises a replacement of all or substantially all functional non-human VA segments with at least 12 human VA gene segments, at least 28 human VA gene segments, or at least 40 human VA gene segments.

In one embodiment, the non-human animal comprises a replacement of all or ntially all onal non-human immunoglobulin JA gene segments with one or more functional human immunoglobulin JA gene ts. In a further ic ment, the replacement is with all or substantially all functional human immunoglobulin JA gene segments.

In various embodiments, the functional human JA gene segments include JA1, JA2, JA3 and JA7.

In a c embodiment, the non-human animal comprises an immunoglobulin heavy chain variable (VH) region locus that comprises only a single VH segment, wherein the single VH segment is a human VH1-69 segment or a human VH1-2 segment, and further ses a replacement of all onal non-human DH segments with all functional human DH segments, and further comprises a replacement of all functional non-human JH ts with all functional human JH segments, and wherein the VH region locus is operably linked to a human or non-human constant region gene sequence. In a specific embodiment, the constant region gene ce is a non-human constant region gene sequence, e.g., an endogenous non-human constant gene sequence. In a speciﬁc embodiment, the non-human animal rearranges segments at the man immunoglobulin heavy chain locus to form a gene encoding an immunoglobulin heavy chain variable region comprising a human VH1-69 ce (or a human VH1-2 sequence), a human DH sequence, a human JH sequence, and an endogenous non-human constant region sequence.

In one embodiment, a B cell is provided that comprises the rearranged gene. In a specific embodiment, the B cell is from a non-human animal as described that has been zed with an antigen of interest, and the B cell encodes an antibody that speciﬁcally binds the antigen of interest. In one embodiment, the antigen is a human protein ed from a ligand, a cell surface receptor and an intracellular protein. In one embodiment, the antigen of interest is a pathogen. In a speciﬁc embodiment, the pathogen is selected from an inﬂuenza virus, a hepatitis virus (e.g., tis B or tis C virus), and a human immunodeﬁciency virus. In a speciﬁc embodiment, the B cell encodes a somatically mutated, high affinity (e.g., about 10'9 Kg or lower) antibody comprising a human light chain variable region (e.g., a human A light chain variable region) that specifically binds the antigen of interest.

In one aspect, a non-human animal comprising a cted immunoglobulin VH segment repertoire is provided, wherein the non-human animal comprises a human VH1-69 segment (or a human VH1-2 segment) on a transgene, wherein the human VH1-69 segment is operably linked on the transgene to a human or non-human DH segment, and/or a human or non-human J segment, and the transgene further comprises a human or non-human constant region gene, or a chimeric human/non-human constant region (e.g., a CH1, hinge, CH2, CH3 or combination thereof wherein at least one ce is non-human, e.g., selected from hinge, CH2, and CH3 and/or hinge). In one ment, the non—human animal is a mouse or rat and the non-human D, J, and/or constant region gene is a mouse or rat gene or chimeric human/mouse or rat.

In one embodiment, the non-human animal ses a ene that comprises an globulin light chain variable region locus that comprises one or more human immunoglobulin V?» gene ts and J)» gene segments, or one or more human immunoglobulin Vx gene segments and JK gene segments, and a human immunoglobulin K or 1 light chain nt region gene, such that the transgene rearranges in the non-human animal to form a rearranged immunoglobulin K or k light chain gene. In various embodiments, the human VK and JK gene segments are those bed herein. In various embodiments, the human VA and J)» gene segments are those described herein.

In a speciﬁc embodiment, the non-human animal comprises a transgene having an immunoglobulin heavy chain variable locus that comprises a single V segment that is a human VH1-69 segment (or a human VH1-2 segment), one or more human D segments, one or more human J segments, and a human constant gene operably linked to the heavy chain variable locus, such that the mouse ses from the transgene a fully human antibody derived from the VH1-69 segment (or the VH1-2 segment). In one embodiment, the non-human animal does not comprise a functional endogenous immunoglobulin heavy chain variable region locus. In a speciﬁc embodiment, the non-human animal comprises a nonfunctional endogenous immunoglobulin heavy chain variable region locus that comprises a deletion of an endogenous non-human DH and/or endogenous non-human JH segment, such that the non-human animal is incapable of rearranging the endogenous immunoglobulin heavy chain le region locus to form a rearranged non-human antibody gene. In a speciﬁc embodiment, the non-human animal comprises a deletion of a switch sequence operably linked to an endogenous mouse heavy chain constant . In a c embodiment, the switch sequence is a non-human (e.g., mouse) u switch sequence. In r embodiment, the non-human animal further comprises a lack of a onal endogenous light chain variable locus selected from an globulin K locus and an immunoglobulin k locus. In a ic ment, the nonhuman animal comprises a deletion of a JK and/or a J)» sequence, such that the non-human animal is incapable of rearranging an endogenous non-human immunoglobulin x light chain and/or an endogenous non-human immunoglobulin h light chain variable region to form a rearranged endogenous non-human immunoglobulin x light chain and/or a rearranged endogenous man immunoglobulin A light chain gene.

In one embodiment, the non-human animal comprises a deletion of an endogenous non-human globulin x light chain sequence that results in a functional knockout of the endogenous non-human immunoglobulin x light chain. In one embodiment, the non-human animal comprises a deletion of an endogenous non-human immunoglobulin x light chain sequence that results in a functional knockout of the endogenous non-human immunoglobulin A light chain.

] In one aspect, the non-human animal comprises a functionally silenced endogenous immunoglobulin heavy chain variable gene locus, and comprises a restricted repertoire of human heavy chain variable gene ts (9.9., no more than one, or no more than two). In one embodiment, the functional silencing comprises a modification of an endogenous non- human heavy chain variable gene locus selected from a deletion, an insertion, an inversion, and a combination thereof.

In one aspect, a rodent is provided that comprises an immunoglobulin VH oire derived from no more than one human VH segment or one or more polymorphs thereof, from a D t selected from a oire of one or more D segments, and from a J segment derived from a repertoire of one or more J segments. In one embodiment, the rodent rearranges the human VH segment. a human D segment, and a human J segment and forms a rearranged human heavy chain sequence that is operably linked to a human or a rodent constant region sequence. In one embodiment, the human and/or rodent constant region sequence is selected from a CH1, a hinge, a CH2, a CH3, and a combination thereof. In one embodiment, the rodent expresses an immunoglobulin light chain that comprises a human variable domain, wherein the light chain is cognate with a human heavy chain domain derived from the rearranged human heavy chain sequence. In one embodiment, the rodent does not express a polypeptide sequence selected from a non-human heavy chain variable domain, a non-human light chain variable domain, and a combination thereof.

In one embodiment, the human VH segment is present in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, or 19 or more polymorphic variants. wherein each polymorphic variant is operably linked to a D and/or J segment such that each rphic variant is capable for nging and forming a rearranged heavy chain variable domain with any of the one or more D segments and any of the one or more J ts. In one embodiment, the rodent is a mouse or a rat. In one embodiment, the oire of D segments comprises two or more D ts. In one embodiment, the repertoire of J segments comprises two or more J segments. In one embodiment, the D and/or J segments are human segments.

In one aspect, a nucleic acid construct is provided that comprises a sequence encoding a single human immunoglobulin VH segment and/or polymorphic variants thereof and one or more DH and one or more J sequences, n the construct comprises at least one gy arm homologous to a non-human immunoglobulin heavy chain variable locus, or a recombinase recognition site (e.g., a lox site). In one embodiment, the V segment is a VH1-69 segment or a VH1-2 segment.

In one aspect, a nucleic acid construct is provided; comprising a nucleic acid sequence encoding a single human immunoglobulin heavy chain V segment, wherein the single VH segment is a VH1-69 (or VH1-2) segment. In one embodiment, the construct comprises a site-speciﬁc inase recognition site. In one embodiment, the construct comprises a first mouse homology arm upstream of the VH1-69 (or VH1-2) t and a second mouse homology arm downstream of the VH1-69 (or VH1-2) segment, and wherein the first mouse homology arm is homologous to a region of a mouse chromosome immediately upstream of a mouse immunoglobulin heavy chain variable region but not ing a functional mouse immunoglobulin heavy chain le segment. In one embodiment, the construct comprises SEQ ID NO: 3. In one ment, the construct comprises SEQ ID NO: 70.

In one aspect, the restricted single VH segment is in a man animal, or the restricted VH segment is at a non-human immunoglobulin heavy chain locus (e.g., in situ or in a transgene), and the non-human animal or non-human globulin heavy chain locus is ed from a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep. goat, chicken, cat, dog, ferret, primate (e.g.. et, rhesus monkey) locus or animal. In a specific embodiment, the non-human animal or locus is a mouse or a rat locus.

In one aspect, a cell or tissue is provided, wherein the cell or tissue is derived from a non-human animal as described herein, and comprises a restricted VH t repertoire. In one ment, the VH segment repertoire is restricted to a single VH segment family member and/or polymorphic variants thereof. In a speciﬁc embodiment, the single VH segment is a human VH1-69 segment or a human VH1-2 segment. In one embodiment, the cell or tissue is d from spleen, lymph node or bone marrow of the non—human animal.

In one embodiment, the cell is an ES cell. In one embodiment, the cell is a B cell.

In one embodiment, the cell is a germ cell.

In one embodiment. the tissue is selected from connective, muscle, nervous and lial tissue. In a speciﬁc embodiment, the tissue is reproductive tissue.

] In one embodiment, the cell and/or tissue derived from a mouse as described herein are isolated for use in one or more ex vivo assays. In various embodiments, the one or more ex vivo assays include measurements of physical, thermal, electrical, mechanical or optical properties, a surgical procedure, measurements of interactions of different tissue types, the development of imaging techniques, or a combination f.

In one embodiment, the man animal is a mouse.

In one aspect, a non—human embryo is provided comprising a restricted heavy chain VH segments as described herein. In one embodiment, the embryo comprises an E3 donor cell that ses the restricted VH segment, and host embryo cells.

In one embodiment, the non-human animal is a mouse.

In one aspect, a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell ses the chromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a nucleus derived from a non-human animal as described herein is provided. In one ment, the nucleus is from a diploid cell that is not a B cell.

In one aspect, a pluripotent, d pluripotent, or totipotent cell derived from a non-human animal as bed herein is provided. In a specific embodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, a non-human induced pluripotent cell comprising a restricted VH segment repertoire is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

In one aspect, a hybridoma or ma is provided, derived from a cell of a nonhuman animal as described herein. In one embodiment, the non-human animal is a mouse or rat.

] In one aspect, a lymphocyte of a non-human animal as described herein is provided.

In one embodiment, the lymphocyte is a B cell.

In one aspect, mouse cells and mouse s are provided, including but not limited to ES cells, pluripotent cells, and induced pluripotent cells, that comprise genetic modiﬁcations as described . Cells that are XX and cells that are XY are provided. Cells that se a nucleus containing a modiﬁcation as described herein are also provided, e.g., a modification introduced into a cell by pronuclear ion.

In one aspect, an antibody variable domain sequence made in a non-human animal as described herein is provided.

In one , a human therapeutic is provided, comprising an antibody variable domain comprising a sequence derived from a non-human animal as described herein.

In one , a method of ing an antibody variable region sequence from a man animal is provided, wherein the antibody variable region sequence is derived from a human VH‘l-69 segment or a VHI-Z segment, n the method comprises (a) immunizing a non-human animal with an antigen of interest, wherein the non-human animal comprises a replacement at the endogenous immunoglobulin heavy chain locus of all or substantially all non-human variable segments with a single human variable segment, wherein the single human variable segment is a VH1-69 segment or a VH1-2 t, and wherein the non-human animal is substantially incapable of forming a immunoglobulin heavy chain le region sequence that is not derived from a human VH1-69 segment or a VH1-2 segment; (b) allowing the non-human animal to mount an immune response with respect to the antigen of interest; and, (0) identifying or isolating an immunoglobulin heavy chain variable region sequence of the non-human animal, wherein the antibody binds the n of interest.

In one embodiment, the single human variable segment is a VH1-69 segment.

] In one embodiment, the antibody variable region sequence is derived from SEQ ID NO: 34. In one embodiment. the antibody variable region ce is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 34. In one embodiment, the dy variable region sequence comprises SEQ ID NO: 34.

In one embodiment, the single human variable segment is a VH1-2 segment.

In one embodiment. the antibody variable region sequence is derived from SEQ ID NO: 60. In one embodiment, the antibody variable region sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 60. In one embodiment, the antibody variable region sequence comprises SEQ ID NO: 60.

In one ment, the immune response to the antigen is characterized by an antibody titer that is about I3x104 to about 5x105 times greater than two times background as determined in an ELISA assay. In a speciﬁc embodiment, the antibody titer is about 1x105 to about 2x105 times greater than two times background as determined in an ELISA assay. In a speciﬁc embodiment, the antibody titer is about 1.5x105 times greater than two times background as determined in an ELISA assay. In one embodiment, the antigen is a human cell surface receptor.

In one aspect, a method for generating a repertoire of human antibody variable regions in a non-human animal is provided, wherein the human heavy chain variable regions of the repertoire are derived from the same VH gene family member and one of a ity of DH segments and one of a ity of JH segments, wherein the repertoire is characterized by having heavy chain immunoglobulin FR1 (framework 1), CDR1, FR2, CDR2, and FR3 sequences from a single VH gene family member. In one ment, the repertoire is further characterized by having a plurality of different CDR3 + FR4 sequences.

In one embodiment, the single VH gene family is ed from VH family 1, 2, 3, 4, 5, 6, and 7. In a specific ment, the single VH gene family is VH family 1. In one embodiment, the single VH gene family member is selected from VH1-2, VH1-69, VH2-26, VH2— 70, and VHS-23. In a speciﬁc embodiment, the single VH gene family member is VH1-69. In a specific embodiment, the single VH gene family member is VH1-2.

In one embodiment, the repertoire ses heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences derived from a VH1-69 segment. In a specific embodiment. the repertoire comprises heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences derived from SEQ ID NO: . In a specific embodiment, the repertoire comprises heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences of SEQ ID NO: 35.

In one ment, the repertoire comprises heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences derived from a VH1-2 segment. In a c embodiment, the repertoire comprises heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences derived from SEQ ID NO: 61. In a speciﬁc ment, the repertoire comprises heavy chain FR1, CDR1, FR2, CDR2 and FR3 sequences of SEQ ID NO: 61.

In one , a biological (i.e., in vivo) system is provided for generating a plurality of different human CDR3 sequences reflecting a plurality of rearrangements of a single human VH gene segment with a plurality of human D and J segments, wherein the system generates human heavy chain variable domains characterized by having human FR1-CDR1-FR2-CDR2- FR3 sequences that are identical but for somatic hypermutations, wherein the heavy chain variable domains are characterized by being somatically hypermutated and derived from a single human VH gene segment and a plurality of human D and J segments; wherein the system comprises a genetically modified man animal (e.g., a rodent, e.g., a mouse or rat) as described herein.

In one embodiment, the single human VH gene segment is selected from VH1-2, VH1-69, VH2-26, , and VHS-23. In one embodiment, the single human VH gene segment is VH1-69. In one ment, the single human VH gene segment is VH1-2. In one ment, the single human VH gene segment is identiﬁed in Table 1. In one embodiment, the single human VH gene segment is identiﬁed in Table 2. In one embodiment. the single human VH gene segment is identiﬁed in Table 3.

In one aspect, an in vivo method for generating a plurality of heavy chain CDR sequences d from rearrangements of a single human VH gene segment with a plurality of human D and J segments is provided, wherein the method generates human heavy chain variable domains terized by having human FR1-CDR1-FR2-CDR2-FR3 sequences that are identical but for somatic hypermutations, wherein the heavy chain variable domains are characterized by being somatically hypermutated and derived from a single human VH gene segment and a plurality of human D and J segments; wherein the system comprises a genetically modiﬁed non-human animal (6.9., a rodent, e.g., a mouse or rat) as bed herein.

In one embodiment, the method comprises exposing a non-human animal as described herein to an antigen of interest, allowing the man animal to develop an immune response to the antigen, wherein the immune response generates the plurality of heavy chain CDR sequences derived from ngements of the single human VH gene segment with one of the human D and one of the human J segments, and identifying a set of heavy chain CDRs that bind the n. In one embodiment, the method ses isolating from the animal a nucleic acid sequence that encodes a human VH domain that comprises the heavy chain CDRs.

In one embodiment, the heavy chain CDR sequences are derived from a rearrangement of a human VH1-69 gene segment. In one ment, the heavy chain CDR sequences are d from a rearrangement of a human VH1-2 gene segment.

In one aspect, a method for generating a plurality of different CDR3 and FR4 sequences in a non-human animal is provided, comprising exposing a man animal that comprises an immunoglobulin heavy chain variable gene locus with a VH segment repertoire restricted to a single VH segment family member to an antigen of interest, allowing the non- human animal to develop an immune response to the n, wherein the immune response generates a B cell repertoire whose heavy chain variable domains are each derived from the single VH segment family member and that comprise a plurality of different CDR3 and FR4 sequences.

In one embodiment, the singe VH t family member is human. In one embodiment, the non-human animal is selected from a mouse, a rat, and a rabbit. In one embodiment, the antigen of interest is selected from a ligand, a or, an intracellular protein and a secreted protein. In one embodiment, the antigen of interest is a human pathogen as described herein.

In one embodiment, the single human VH gene family member is selected from VH1- 2, VH1-69, VH2-26, VH2-70, and VH3-23. In one embodiment, the single human VH gene family member is VH1-69. In one embodiment, the single human VH gene family member is VH1-2. In one embodiment, the single human VH gene family member is identified in Table 1. In one embodiment, the single human VH gene family member is identiﬁed in Table 2. In one embodiment, the single human VH gene family member is identiﬁed in Table 3.

In one aspect, a nucleotide sequence encoding an immunoglobulin variable region made in a non-human animal as described herein is provided.

In one aspect, an globulin heavy chain or globulin light chain variable region amino acid sequence of an antibody made in a non-human animal as described herein is provided.

In one , an immunoglobulin heavy chain or immunoglobulin light chain variable region nucleotide sequence encoding a variable region of an antibody made in a non- human as bed herein is provided.

] In one aspect, an antibody or n-binding fragment thereof (e.g., Fab, F(ab)2, scFv) made in a non-human animal as described herein is provided.

In one aspect, a mouse having a restricted immunoglobulin heavy chain locus characterized by the ce of a single human VH gene segment, one or more human DH gene segments, and one or more human JH gene segments is provided, wherein the single human VH gene segment is at an endogenous mouse locus and the VH gene segment is operably linked to the one or more human DH gene segments, the one or more human JH gene segments, and to an endogenous immunoglobulin heavy chain constant gene.

In one embodiment, the mouse further comprises a humanized immunoglobulin light chain locus comprising one or more human VL gene segments. and one or more human JL gene segments, wherein the human VL gene segments and the human JL gene segments are operably linked to a man immunoglobulin light chain constant region gene. In a speciﬁc embodiment, the human VL and JL gene segments are at an endogenous mouse light chain locus, and wherein the non-human immunoglobulin light chain constant region gene is a mouse gene.

In one embodiment, the humanized immunoglobulin light chain locus is on a transgene, and the constant region gene is ed from mouse, rat. and human. 9] In one embodiment, the human VL and JL gene segments are VK and JK gene segments. In one embodiment, the human VL and JL gene segments are VA and J)» gene segments In one aspect, a man animal is provided, wherein the non-human animal has a B cell repertoire that expresses immunoglobulin heavy chain variable s derived from a single V segment family member. ln one embodiment, at least 10%, at least 20%, at least %, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, or at least 95% of the B cell repertoire of the non-human animal immunoglobulin heavy chain variable domain expressed in the B cell repertoire is derived from the same V segment family member. In a specific embodiment, the percentage is at least 90%. In one embodiment, the B cell repertoire consists essentially of peripheral (blood) 8 cells. In one embodiment, the B cell repertoire consists essentially of splenic B cells. In one embodiment, the B cell repertoire consists ially of bone marrow B cells. In one embodiment, the B cell repertoire consists essentially of peripheral B cells, splenic B cells, and bone marrow B cells.

In one aspect, a genetically modified non-human animal is provided, wherein more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more than 90% of the B cells of the non- human animal that express a heavy chain immunoglobulin variable domain express a heavy chain immunoglobulin variable domain d from a single VH gene segment family member.

In one embodiment, at least 75% of the B cells of the non-human animal that s an immunoglobulin heavy chain variable domain express an immunoglobulin heavy chain variable domain derived from the single VH gene segment family member. In a c embodiment, the tage is at least 90%. In one embodiment, all of the B cells that express a heavy chain domain that is derived from the single VH gene family member.

In one aspect, a genetically modified mouse is provided that makes an antigen- specitic B cell population in response to immunization with an antigen of interest, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more than 90%, of said antigen-speciﬁc B cell population ses immunoglobulin heavy chains that are all d from the same VH gene segment. In one embodiment, at least 75% of the antigen-speciﬁc B cell population expresses globulin heavy chains derived from the same V” gene segment. In one embodiment, all of the antigen-specific B cells express a heavy chain that is d from the same V... gene segment.

In one aspect, a non-human animal comprising a restricted VH gene segment repertoire is provided, wherein the restriction is to a human VH1-69 gene segment or a VH1-69 gene segment that is at least about 75.5%, 76.5%, 86.7%, 87.8%, 94.9%, 96.9%, 98%, or 99% identical to a VH1-69*01 gene segment. In a speciﬁc ment, the restricted repertoire is selected from one or more of the VH1-69 variants of .

In one aspect, a non-human animal sing a restricted VH gene segment oire is provided, wherein the restriction is to a human VH1-2 gene segment or a VH1-2 gene segment that is at least about 94.9%, 95.9%, 96.9%, 98%, or 99% identical to a VH1-2 gene segment. In a speciﬁc embodiment, the restricted repertoire is selected from one or more of the VH1-2 variants of .

In one ment. the non-human animal is a mouse.

In one embodiment, the mouse exhibits an immunophenotype having a characteristic of a higher ratio of mature B cells to immature B cells as ed to a wild type mouse. In a speciﬁc embodiment, the ratio is calculated from B cells harvested from spleen. In one embodiment, the mouse exhibits a population of mature B cells of about 1x107. In one embodiment, the mouse exhibits a population of immature B cells of about 0.5x107. In one embodiment, the mouse exhibits a ratio of mature B cells to immature B cells in the spleen of the mouse that is about 1.5-fold to about 2-fold higher than exhibited by a wild type mouse.

In one embodiment, the ratio is calculated from B cells harvested from bone marrow.

In a speciﬁc embodiment, the mouse exhibits a population of mature B cells of about 3x105. In one ment, the mouse exhibits a population of immature B cells of about 7x105. In one embodiment, the mouse exhibits a ratio of mature B cells to immature B cells in the bone marrow of the mouse that is about 3-fold, or about 3.3-fold higher than exhibited by a wild type mouse.

In one embodiment, the mouse exhibits an phenotype having a characteristic of a higher number of pro B cells in the bone marrow as compared to a wild type mouse. In a specific embodiment, the mouse exhibits a population of pro 8 cells in the bone marrow of the mouse that is about 2.5-fold to about 3-fold higher than exhibited in the bone marrow of a wild type mouse. In a c embodiment, the mouse exhibits a population of pro B cells in the bone marrow of the mouse that is about 2.75-fold higher than ted in the bone marrow of a wild type mouse.

In one embodiment, the mouse exhibits an immunophenotype having a teristic selected from the group consisting of a CD19+ splenic B cell population that is about 80% of a wild-type B cell, a CD3+ splenic T cell population that is about the same as a wild type mouse, and a combination thereof.

In one embodiment, the mouse comprises a cyte population whose % CD19+ B cells in spleen are about the same as a ype mouse. In one embodiment, the number of CD19’ B cells per spleen of the mouse is at least about 50% of the number of CD19+ B cells per spleen of a wild—type mouse.

In one ment, the non-human animal comprises at least about 75% to about 80% of CD19+ B cells in bone marrow as compared with a wild-type mouse.

In one ment, the total number of CD19+ bone cells per femur of the mouse is non less than about 30%, 40%, 50%, 60%, or 75% of the total number of CD19+ bone marrow cells in a wild-type mouse.

In one ment, the mouse expresses lgD and IgM at about the same level as observed in a wild-type mouse.

In one aspect, a mouse comprising a restricted human VH segment repertoire is provided, further comprising a humanized immunoglobulin light chain variable segment locus, wherein the ratio of A to x light chains expressed in the mouse is about the same as in a wild- type mouse.

In one aspect, a mouse is provided, comprising a restricted globulin heavy chain locus characterized by the presence of a single VH gene segment, one or more DH gene ts, and one or more JH gene segments, wherein the single VH gene segment is a polymorphic V... gene segment.

In one embodiment, the polymorphic VH gene segment is a human VH gene segment that is associated with a high copy number in human populations. In one embodiment, the human VH gene t is ed from VH1-2, , VH2-26, VH2-70, VH3-23, or a polymorphic variant thereof. In a specific embodiment, the human VH gene segment is a VH1-69 gene segment. In another specific embodiment, the human VH gene segment is a VH1-2 gene segment. 7] In one embodiment, the single VH gene segment is operably linked to a human, mouse, or ic human/mouse immunoglobulin constant region gene. In a specific embodiment, the immunoglobulin constant region gene is a mouse constant region gene. In one embodiment, the immunoglobulin constant gene comprises a human sequence selected from a human CH1, a human hinge, a human CH2, a human CH3, and a combination thereof. In one embodiment, the mouse constant gene is at an endogenous immunoglobulin heavy chain locus.

In one embodiment, the mouse r comprises a human immunoglobulin VL gene segment operably linked to a J gene segment and a light chain constant gene. In a c embodiment, the VL gene segment and/or the J gene segment are selected from a human x gene segment and a human k gene segment. In one embodiment, the V._ and/or J gene segments are human x gene segments.

In various embodiments, the mouse comprises a deletion of all or substantially all endogenous VH gene segments.

In various embodiments, the non-human animal comprises an inactivated endogenous heavy chain variable gene locus. In various embodiments, the inactivated endogenous heavy chain variable gene locus is not operably linked to an endogenous heavy chain constant region gene.

In one aspect, a mouse is provided, wherein the mouse is characterized by the expression of serum immunoglobulin, wherein greater than 80% of the serum immunoglobulin ses a human heavy chain variable domain and a cognate human light chain variable domain, wherein the human heavy chain variable domain is derived from a VH gene segment repertoire consisting ially of a single human VH gene segment and/or rphic variants thereof.

In one embodiment, the single human VH gene segment is a human VH1-69 gene segment and/or polymorphic variants thereof. In one embodiment, the single human VH gene segment is a human VH1-2 gene segment and/or polymorphic variants thereof.

In one aspect, a mouse is provided, comprising, in its germiine, a replacement at an endogenous immunoglobulin heavy chain locus of all or substantially all endogenous VH gene segments with a single human VH gene segment and/or polymorphic variants thereof. In one embodiment, the single human VH gene segment is a human VH1-69 gene segment and/or polymorphic variants thereof. In one ment, the single human VH gene segment is a human VH1-2 gene segment and/or polymorphic variants thereof.

In one embodiment, the mouse further comprises a replacement at an endogenous immunoglobulin light chain locus of all or substantially all endogenous VL gene segments with one or more human VL gene segments. In a speciﬁc embodiment, the mouse further comprises one or more human JL gene segments operably linked to the human VL gene ts.

In one aspect, use of a mouse as described herein to make an immunoglobulin variable region nucleotide sequence is provided. In one embodiment, the ce comprises a rearranged VH1-69 gene t. In one embodiment, the sequence comprises a rearranged VH1-2 gene segment. in one embodiment, the globulin variable region nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with a human VH1- 69 gene segment. In a speciﬁc ment, the immunoglobulin variable region nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with SEQ ID NO: 34. In various embodiments, the human VH1-69 gene segment is identiﬁed from Table 2.

In one embodiment, the immunoglobulin variable region nucleotide sequence s an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with SEQ ID NO: 35.

In one embodiment, the immunoglobulin variable region nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with a human VH1- 2 gene t. In a specific embodiment, the immunoglobulin le region nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with SEQ ID NO: 60. In s embodiments, the human VH1-2 gene segment is identiﬁed from Table 3.

In one embodiment, the immunoglobulin variable region nucleotide ce s an amino acid ce that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical with SEQ ID NO: 61.

In one aspect, use of a mouse as described herein to make a fully human Fab or a fully human F(ab)2 is provided. In one embodiment, the fully human Fab or fully human F(ab)2 comprises a heavy chain variable region that comprises a rearranged human VH1-69 gene segment. In one embodiment, the fully human Fab or fully human F(ab)2 comprises a heavy chain variable region that comprises a rearranged human VH1-2 gene segment.

In one aspect, use of a mouse as described herein to make an immortalized cell line is provided.

In one , use of a mouse as described herein to make a hybridoma or quadroma is provided.

In one , use of a mouse as described herein to make a phage library containing human heavy chain le regions and human light chain variable regions is In one embodiment, the human heavy chain variable regions are derived from a human VH1-69 gene segment that comprises a sequence selected from SEQ ID NO: 34. SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 58.

In one embodiment, the human heavy chain variable s are derived from a human VH1-69 gene segment that comprises a sequence selected from SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55. SEQ ID NO: 57 and SEQ ID NO: 59.

In one embodiment, the human heavy chain variable regions are all derived from a human VH1-2 gene segment that comprises a sequence selected from SEQ ID NO: 60. SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 and SEQ ID NO: 68. 7] In one embodiment, the human heavy chain variable regions are derived from a human VH1-2 gene segment that ses a sequence selected from SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69.

In one aspect, use of a mouse as described herein to generate a variable region sequence for making a human antibody is provided, comprising (a) immunizing a mouse as described herein with an n of interest, (b) isolating a cyte from the immunized mouse of (a), (c) exposing the lymphocyte to one or more labeled antibodies, (d) identifying a lymphocyte that is capable of binding to the antigen of interest, and (e) amplifying one or more variable region nucleic acid sequence from the lymphocyte y generating a variable region sequence.

In one embodiment, the lymphocyte is derived or isolated from the spleen of the mouse. In one embodiment, the lymphocyte is d or isolated from a lymph node of the mouse. In one embodiment, the lymphocyte is derived or isolated from the bone marrow of the mouse. In one embodiment, the lymphocyte is derived or isolated from the blood of the mouse.

In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, the one or more fluorophore-conjugated antibodies are selected from an lgM, an lgG, and/or a combination thereof. in one embodiment, the lymphocyte is a B cell. in one embodiment, the one or more variable region nucleic acid sequence comprises a heavy chain variable region sequence. In one embodiment, the one or more le region nucleic acid sequence comprises a light chain variable region sequence. In a specific embodiment, the light chain variable region sequence is an immunoglobulin K light chain variable region sequence. In one embodiment, the one or more variable region nucleic acid ce comprises a heavy chain and a K light chain variable region sequence.

In one embodiment, use of a mouse as described herein to generate a heavy and a K light chain le region sequence for making a human antibody is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating the spleen from the immunized mouse of (a), (c) exposing B lymphocytes from the spleen to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of st, and (e) amplifying a heavy chain variable region nucleic acid sequence and a x light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the heavy chain and K light chain variable region sequences.

In one embodiment, use of a mouse as described herein to generate a heavy and a x light chain le region sequence for making a human dy is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating one or more lymph nodes from the zed mouse of (a), (c) exposing B lymphocytes from the one or more lymph nodes to one or more labeled dies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a heavy chain le region nucleic acid sequence and a K light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the heavy chain and x light chain variable region sequences.

In one embodiment, use of a mouse as described herein to generate a heavy and a K light chain variable region ce for making a human antibody is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) ing bone marrow from the immunized mouse of (a), (c) exposing B lymphocytes from the bone marrow to one or more labeled dies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a heavy chain variable region nucleic acid sequence and a x light chain variable region nucleic acid sequence from the B lymphocyte thereby ting the heavy chain and x light chain variable region sequences. In various embodiments, the one or more labeled antibodies are selected from an lgM, an lgG, and/or a combination thereof.

In various ments, the antigen of interest is a pathogen that afflicts human subjects including, e.g., a viral antigen. Exemplary viral pathogens include, e.g., mainly those of the families of Adenoviridae, bacteria Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, iridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, viridae, and Togaviridae. Such exemplary viruses typically range between 20-300 nanometers in . In various embodiments, the antigen of interest is a viral antigen selected from a hepatitis virus (e.g., HCV, HBV, etc.), a human immunodeficiency virus (HIV), or an inﬂuenza virus (e.g., H1 N1).

In various embodiments, use of a mouse as described herein to generate a heavy and x light chain variable region ce for making a human antibody is provided, further comprising fusing the ampliﬁed heavy and light chain variable region sequences to human heavy and light chain constant region sequences, expressing the fused heavy and light chain sequences in a cell, and recovering the expressed heavy and light chain sequences thereby generating a human antibody.

In various embodiments, the human heavy chain constant regions are selected from lgM, IgD, lgA, IgE and lgG. In various speciﬁc embodiments, the lgG is selected from an [961, an lgG2, an lgGB and an lgG4. In various embodiments, the human heavy chain constant region comprises a CH1, a hinge, a CH2, a CH3, a CH4, or a combination thereof. In various embodiments, the light chain constant region is an immunoglobulin K constant region. In various embodiments, the cell is selected from a HeLa cell, a DU145 cell, a anap cell, a MCF- 7 cell, a MDA-MB-438 cell, a PC3 cell, a T47D cell, a THP-1 cell, a U87 cell, a SHSY5Y (human neuroblastoma) cell, a Saos-2 cell, a Vero cell, a CHO cell, a GH3 cell, a PC12 cell, a human retinal cell (e.g., a PER.C6TM cell), and a MC3T3 cell. In a speciﬁc embodiment, the cell is a CHO cell.

In one , a method for generating a e-chimeric rodent-human antibody specific against an antigen of st is provided, comprising the steps of zing a mouse as described herein with the antigen, isolating at least one cell from the mouse producing a e-chimeric mouse-human antibody speciﬁc against the n, culturing at least one cell producing the reverse—chimeric mouse-human antibody speciﬁc against the antigen, and obtaining said antibody.

In one ment, the reverse-chimeric mouse-human antibody comprises a human heavy chain variable domain fused with a mouse or rat heavy chain constant gene, and a human light chain le domain fused with a mouse or rat or human light chain constant gene. In a speciﬁc embodiment, the human heavy chain variable domain contains a rearranged human VH1-69 or human VH1-2 gene segment.

In one embodiment, culturing at least one cell producing the reverse-chimeric -human dy specific against the n is performed on at least one hybridoma cell generated from the at least one cell isolated from the mouse.

In one embodiment, the antigen of interest is a pathogen that afﬂicts human subjects as described herein.

In one aspect, a method for ting a fully human antibody speciﬁc against an antigen of interest is provided, sing the steps of immunizing a mouse as described herein with the antigen, isolating at least one cell from the mouse producing a e-chimeric rodent-human antibody speciﬁc against the antigen, generating at least one cell ing a fully human antibody derived from the reverse-chimeric rodent-human antibody c against the n, and ing at least one cell producing the fully human antibody, and obtaining said fully human antibody.

In various embodiments, the at least one cell isolated from the mouse producing a reverse-chimeric rodent-human antibody c against the antigen is a splenocyte or a B cell.

In s embodiments, the antibody is a monoclonal antibody.

In various embodiments, the antibody comprises a heavy chain variable domain that contains a rearranged human VH1-69 or human VH1-2 gene segment.

In various ments, immunization with the antigen of interest is carried out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen. In one embodiment, the antigen of interest is a pathogen that afﬂicts human subjects as described herein.

In one aspect, use of a mouse as described herein to make a nucleic acid ce encoding an immunoglobulin variable region or fragment thereof is provided. In one embodiment, the c acid sequence is used to make a human antibody or antigen-binding fragment thereof. In one embodiment, the mouse is used to make an antigen-binding protein selected from an antibody, a multi-speciﬁc antibody (e.g., a bi-specific antibody), an scFv, a bi- specific scFv, a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, a F(ab), a F(ab)2, a DVD (i.e., dual variable domain antigen-binding protein), a an SVD (i.e., single variable domain antigen—binding n), or a bispeciﬁc T—cell engager (BiTE).

In one , a method for making a human antigen-binding protein is provided, comprising exposing a genetically modiﬁed man animal as described herein to an antigen of st, allowing the genetically modiﬁed non-human animal to mount an immune response to the antigen, obtaining from the genetically modiﬁed non-human animal a heavy chain variable domain nucleic acid sequence encoding a human heavy chain variable domain that speciﬁcally binds the antigen of interest, cloning the heavy chain variable domain nucleic acid sequence to a human constant region sequence, and expressing in a mammalian cell an antibody comprising the human heavy chain variable domain sequence and the human constant region sequence. In one ment, the mammalian cell is a CHO cell. ln one embodiment the genetically modiﬁed non-human animal comprises a human VH gene segment repertoire that consists essentially of a single human VH gene segment, ally present in two or more polymorphic variants thereof, operably linked to one or more human D and/or J segments. In one embodiment, the human VH gene segment repertoire is at an endogenous man VH segment locus. In one embodiment, the human VH gene segment repertoire is at a locus that is not an endogenous VH segment locus. In one embodiment, the human VH gene segment rearranges with a human D segment and a human J segment to form a rearranged human VDJ gene operably linked to a constant region sequence, wherein the constant region sequence is selected from a human sequence and a rodent sequence (9.9., a mouse or rat or hamster sequence). In one embodiment, the constant region sequence comprises a sequence selected from a CH1, a hinge, a CH2, a CH3, and a ation thereof; in a speciﬁc embodiment, the constant region sequence ses a CH1, a hinge, a CH2, and a CH3. In one embodiment, the human variable domain and the constant sequence are expressed in the mammalian cell with a cognate human light chain variable domain obtained from the same mouse (e.g., sequence obtained from the same B cell as the human variable domain sequence); in one embodiment the sequence encoding the human light chain variable domain obtained from the mouse is then fused with a sequence encoding a human light chain nt sequence, and the light chain sequence and the heavy chain sequence are expressed in the mammalian cell.

In one embodiment, the n of interest is a pathogen that afﬂicts human subjects as described .

In one aspect, a method for making an antibody heavy chain variable domain that binds an antigen of interest is provided, sing expressing in a single cell (a) a ﬁrst VH sequence of an zed non-human animal as described herein, wherein the ﬁrst VH sequence is fused with a CH gene sequence; and (b) a VL gene sequence of an immunized non-human animal as described herein, wherein the VL gene sequence is fused with a human CL gene sequence; ining the cell under conditions sufﬁcient to express an antibody; and, isolating the antibody heavy chain variable domain. In one embodiment, the VL gene sequence is e with the ﬁrst VH sequence.

In one embodiment, the cell comprises a second VH gene sequence of an immunized man animal as described herein, wherein the second VH gene ce is fused with a CH gene sequence, n the ﬁrst VH gene sequence encodes a VH domain that specifically binds a ﬁrst epitope, and the second VH gene sequence encodes a VH domain that specifically binds a second epitope, wherein the first epitope and the second epitope are not identical.

In one embodiment, the constant region sequences are all human constant region sequences. In one embodiment, the antigen of interest is a pathogen that afﬂicts human subjects as described herein.

In one . a method for making a human bispeciﬁc antibody is provided, comprising making the ific antibody using human variable region gene ces of B cells of a non-human animal as described .

In one embodiment, the method comprises (a) identifying a ly selected lymphocyte of the non-human animal, wherein the non-human animal has been exposed to an antigen of interest and allowed to develop an immune response to the antigen of interest, and wherein the lymphocyte expresses an antibody that speciﬁcally binds the antigen of interest, (b) obtaining from the lymphocyte or the antibody a nucleotide sequence that encodes a human heavy chain variable region that specifically binds the n of interest, and (c) employing the nucleotide sequence that encodes the human heavy chain variable region that speciﬁcally binds the n of interest in making the bispeciﬁc antibody. In a speciﬁc embodiment, the human heavy chain variable region comprises a rearranged VH1-2 or VH1-69 gene t.

In one embodiment, steps (a) through (c) are performed a ﬁrst time for a ﬁrst antigen of interest to generate a ﬁrst human heavy chain variable region sequence, and steps (a) h (c) are performed a second time for a second antigen of interest to generate a second human heavy chain le region sequence, and wherein the ﬁrst human heavy chain variable region sequence is expressed fused with a ﬁrst human heavy chain constant region to form a ﬁrst human heavy chain, the second human heavy chain variable region sequence is expressed fused with a second human heavy chain constant region to form a second human heavy chain, wherein the ﬁrst and the second human heavy chains are expressed in the presence of a single human light chain expressed from a nged human VK1-39 or a human Vx3-20 gene segment. in a speciﬁc embodiment, the single human light chain comprises a ne sequence.

In one embodiment, the method comprises (a) cloning heavy chain variable regions from B cells of a non-human animal as described herein which has been exposed to a ﬁrst antigen of interest, and the same non-human , or a different non-human animal which is genetically the same and has been exposed to a second antigen of interest; and (b) expressing in a cell the heavy chain variable regions of (a) with the same heavy chain constant region and the same light chain to make a bispecific dy.

In one , a use of a non-human animal as described herein is provided, to obtain a nucleic acid sequence that encodes a human heavy chain variable domain. in one embodiment, the heavy chain variable domain comprises a rearranged human VH gene segment selected from VH1-2 and VH1-69.

In one aspect, a use of a non-human animal as described herein is provided, to obtain a cell that encodes a human heavy chain variable domain. In one embodiment, the heavy chain variable domain comprises a rearranged human V... gene segment selected from VH1-2 and VH1-69.

In one , use of a non-human animal as described herein to make a human dy variable domain is provided. In one ment, the variable domain comprises a rearranged human VH gene segment selected from VH1-2 and VH1-69.

In one aspect, use of a non-human animal as described herein to make a human antibody is provided, comprising making the antibody using human variable region gene sequences of B cells of a non-human animal as described herein. In one embodiment, the human dy is a human bispecific antibody. In a speciﬁc embodiment, the bispeciﬁc antibody ses one heavy chain variable domain derived from a rearranged human VH1-2 or VH1—69 gene segment. In one embodiment, the human le region gene sequences comprise a rearranged human VH1-2 or VH1-69 gene t.

In one aspect, use of a non-human animal as described herein is provided to select a human immunoglobulin heavy chain variable domain. In one embodiment, the heavy chain variable domain comprises a rearranged human VH gene segment selected from VH1-2 and VH1-69.

In one , use of the mouse as described herein for the manufacture of a medicament (9.9., an antigen-binding protein), or for the manufacture of a sequence encoding a variable sequence of a medicament (e.g., an antigen—binding protein), for the treatment of a human disease or disorder is provided. In one embodiment, the variable sequence of a medicament comprises a polymorphic human VH gene segment. In one embodiment, the variable sequence of a ment comprises a human VH1-69 gene segment. In one embodiment, the variable sequence of a medicament comprises a human VH1-2 gene segment.

In one aspect, a nucleic acid construct ng an immunoglobulin variable domain made in a mouse as described herein is provided. In one embodiment, the variable domain is a heavy chain variable domain. In a speciﬁc embodiment, the heavy chain variable domain comprises a rearranged human VH gene segment selected from VH1-2, , VH2-26, VH2- 70, or . In another specific embodiment, the heavy chain variable domain comprises a rearranged human VH1-2 gene segment. In another specific embodiment, the heavy chain le domain comprises a rearranged human Vin-69 gene segment.

In one embodiment, the variable domain is a light chain le domain. In a c embodiment, the variable domain is a x light chain variable domain that is cognate with a human heavy chain variable domain that comprises a rearranged human VH1—69 gene segment. In a speciﬁc embodiment, the variable domain is a K light chain variable domain that is cognate with a human heavy chain variable domain that comprises a rearranged human VH1- 2 gene segment.

In one aspect, use of a mouse as described herein to make a nucleic acid construct encoding a human immunoglobulin variable domain is provided. in one embodiment, the le domain is a light chain variable domain. in one embodiment, the variable domain is a K light chain variable domain that comprises a rearranged human VK gene segment selected from VK4-1. Vx5-2, Vx7-3, Vx2-4, Vx1-5, Vx1-6, Vx3-7, VK1-8, VK1-9, Vx2-10, Vx3-11, Vx1-12, Vx1-13, , VK3-15, Vx1-16, , Vx2-18, Vic2-19, VK3-20, Vx6-21, Vx1-22, VK1-23, Vx2-24, Vx3-25, , Vx1-27, Vx2-28. Vx2-29, Vx2-30, , Vx1-32, VK1-33, Vx3-34, Vx1-35, Vx2-36, Vx1—37, Vx2-38, Vx1-39, and Vx2-40.

In one embodiment, the variable domain is a heavy chain variable domain. In a ic embodiment, the heavy chain variable domain comprises a rearranged human VH gene segment selected from VH1-2, , VH2-26, VH2-70, or VHS-23. in a specific embodiment, the heavy chain variable domain comprises a rearranged human VH1-69 gene t. In a ic embodiment, the heavy chain variable domain comprises a rearranged human VH1-2 gene segment. in one aspect, use of a mouse as described herein to make a human immunoglobulin variable domain is provided. in one embodiment, the variable domain is a light chain variable domain. In one embodiment, the variable domain is a x light chain variable domain that comprises a rearranged human VK gene segment selected from VK4-1, Vx5-2, VK7-3. Vx2-4, VK1-5, Vx1-6, VK3-7, VK1-8, Vi<1-9, VK2-10, Vx3-11, Vic1-12, Vx1-13, Vx2-14, Vx3-15, 6, Vx1-17, Vx2-18, VK2-19, VK3-20, Vx6-21, Vx1-22, Vx1-23, Vx2-24, Vx3-25, Vx2-26, Vx1-27, VK2-28, Vx2—29, Vx2-30, Vx3-31, Vx1-32, Vx1-33, VK3-34. VK1-35, VK2-36, Vx1—37, VK2—38, Vx1-39, and VK2-40.

In one embodiment, the variable domain is a heavy chain variable domain. In a ic embodiment, the heavy chain variable domain comprises a rearranged human VH gene segment selected from VH1-2, VH1-69, VH2-26, VH2-70, or . in a speciﬁc embodiment, the heavy chain variable domain comprises a rearranged human VH1-69 gene segment. In a speciﬁc embodiment, the heavy chain variable domain comprises a rearranged human VH1-2 gene segment.

In one aspect, use of a non-human animal as described herein to make a nucleic acid sequence encoding a human heavy chain le domain is provided. In one embodiment, the human heavy chain variable domain is characterized by having human FR1- CDR1-FR2-CDR2-FR3 sequences that are derived from a polymorphic human VH gene segment. In a speciﬁc embodiment, the human VH gene t is selected from a human VH1-2, VH1-69, VH2-26, VH2-70, or VH3-23 gene segment. In one embodiment, the human VH gene segment is a human VH1-69 gene segment. in one embodiment, the human VH gene segment is a human VH1-2 gene segment.

In one aspect, a method for making a c acid sequence encoding a human VH domain is provided, the method comprising immunizing a non-human animal as described herein with an antigen of interest, ng the non-human animal to mount an immune response to the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human VH domain that binds the n of interest. In one embodiment, the method further comprises making a c acid sequence encoding a human VL domain that is cognate with the human VH domain, comprising isolating a B cell encoding the human VH domain and the human VL domain, and obtaining therefrom the sequence of the heavy and light chain variable domains. In various embodiments, the human VH domain is derived from a rearranged human VH1-69 or human VH1-2 gene segment. In various embodiments, the human VL domain is ed from a human VK or a human V)» domain.

In one aspect, use of a non-human animal as described herein to make a human therapeutic is provided, comprising immunizing the non-human animal with an antigen of interest, ng the non-human animal to mount an immune response, and obtaining from the animal a nucleic acid sequence encoding an immunoglobulin variable domain that binds the antigen of interest, and employing the immunoglobulin variable domain in a human therapeutic. ln one embodiment, the variable domain is a heavy chain variable domain. In a speciﬁc embodiment, the heavy chain variable domain is derived from a rearranged human VH1-69 or a human VH1-2 gene segment. In one embodiment, the variable domain is a light chain variable domain. In a specific ment, the light chain le domain is derived from a rearranged human VK or human V)» gene segment. in one , a method for making a human therapeutic is provided, comprising zing a non-human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response, and obtaining from the animal a nucleic acid sequence encoding an immunoglobulin variable domain that binds the antigen of interest, and employing the immunoglobulin variable domain in a human eutic. In one embodiment, the variable domain is a heavy chain variable . In a speciﬁc embodiment, the heavy chain variable domain is d from a rearranged human VH1-69 or a human VH1-2 gene segment. In one embodiment, the le domain is a light chain variable domain. In a specific embodiment, the light chain variable domain is derived from a rearranged human Vx or human VA gene segment.

In one aspect, a method for making a human antigen-binding protein is provided, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the animal to mount an immune response, obtaining from the mouse a nucleic acid sequence encoding an immunoglobulin variable domain that speciﬁcally binds the antigen of interest, cloning the nucleic acid sequence in a vector suitable for expression of the nucleic acid, n the nucleic acid sequence is cloned in frame with a nucleic acid sequence encoding a human immunoglobulin constant region or functional fragment thereof, and inserting the vector in a mammalian cell, and maintaining the cell under ions suitable for expressing an antigen-binding protein that comprises the immunoglobulin variable domain and the immunoglobulin constant region or functional fragment thereof. In one embodiment, the antigen-binding protein is a human antibody. In a specific embodiment, the antibody comprises a heavy chain variable domain and a light chain variable domain obtained from a mouse as described herein. In a speciﬁc embodiment, the antibody ses a heavy chain variable domain obtained from a mouse as described herein. In s embodiments, the heavy chain le domain is derived from a rearranged human VH1—69 or a human VH1-2 gene segment.

] In one , a nucleic acid sequence encoding a human n-binding domain made in a non-human animal as described herein is provided. In one ment, the nucleic acid sequence encodes a human immunoglobulin VH domain. In one embodiment, the nucleic acid sequence encodes a human immunoglobulin VH domain and a cognate human VL domain.

In various embodiments, the human VH domain is derived from a nged human VH1-69 or a human VH1-2 gene segment.

In one aspect, a method for preparation of a human antibody is provided, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the man animal to mount an immune se, harvesting a cyte (e.g., a B cell) from the immunized animal, fusing the lymphocyte with a myeloma cell to form a hybridoma cell, obtaining from the hybridoma cell a nucleic acid sequence that encodes a human VH domain and a human VL domain, cloning the nucleic acid sequence in frame (i.e., in operable linkage) with a human nt region sequence to create an immunoglobulin heavy chain and an immunoglobulin light chain, and expressing the heavy and light chains in a cell capable of expressing the fully human antibody. In one embodiment, the cell is a CHO cell. In s embodiments, the human VH domain is derived from a rearranged human VH1-69 gene segment or a human VH1-2 gene segment.

In one aspect, a method for preparation of a human antibody is provided, comprising immunizing a non-human animal as described herein with an n of interest, allowing the non-human animal to mount an immune response, harvesting a lymphocyte (e.g., a B cell) from the immunized animal, obtaining from the lymphocyte a nucleic acid sequence that encodes a human VH domain and a human VL domain, cloning the nucleic acid sequence in frame (i.e., in le e) with a human constant region sequence to create an immunoglobulin heavy chain and an immunoglobulin light chain, and expressing the heavy and light chains in a cell capable of expressing the fully human antibody. In one embodiment, the lymphocyte is derived from the spleen of the non-human animal. In one embodiment, the cell is a CHO cell. In various embodiments, the human VH domain is derived from a rearranged human VH1-69 gene segment or a human VH1-2 gene segment. 8] In various aspects, the antigen of interest is a pathogen that afflicts human subjects as described herein. In various aspects, the antigen of interest is a virus that is capable of infecting a human. Exemplary ns that can be employed in the methods and uses described herein include microbes or microorganisms such as a virus, bacterium, prion, or fungus or any other pathogen that causes disease in humans. A person of skill, upon reading the disclosure, will appreciate those human pathogens that will be applicable for the methods and uses described herein. The various aspects and embodiments are capable of use together, unless expressly noted otherwise or the context clearly prohibits use together. [000198A] In one aspect, there is ed a rat or mouse having in its germline genome a restricted endogenous immunoglobulin heavy chain locus characterized by the presence of a single ranged human V H gene segment, one or more unrearranged human DH gene segments, and one or more unrearranged human J H gene segments ly linked to a nonhuman constant region gene ce comprising a non-human IgM gene, wherein the rat or mouse further comprises a diverse repertoire of rearranged human immunoglobulin heavy chain variable region genes, each of which is linked to the nonhuman constant region gene sequence comprising a man IgM gene and is derived from the restricted immunoglobulin heavy chain locus. [000198B] In one aspect, there is provided a cell or tissue derived from the rat or mouse according to the present disclosure. [000198C] In one aspect, there is ed a method of making a nucleic acid sequence that s a human immunoglobulin heavy chain variable domain comprising ying a nucleic acid comprising a rearranged human immunoglobulin heavy chain variable region gene from a lymphocyte of a rat or mouse according to the present disclosure, or a hybridoma produced from the lymphocyte, wherein the lymphocyte comprises one of the diverse repertoire of rearranged human immunoglobulin heavy chain variable region genes. [000198D] In one aspect, there is provided use of a rat or mouse according to the present disclosure to make a nucleic acid sequence encoding a human heavy chain variable domain. 8E] In one aspect, there is provided an antigen binding protein made in a rat or mouse according to the present sure. [000198F] In one aspect, there is provided a c acid sequence comprising a ce that encodes the human immunoglobulin heavy chain variable domain made according to the present sure. [000198G] In one aspect, there is provided a totipotent mouse cell for making a rat or mouse that produces a human heavy chain variable domain comprising in its genome a restricted immunoglobulin heavy chain locus characterized by the presence of a single unrearranged human VH gene segment, one or more ranged human DH gene segments, and one or more unrearranged human J H gene segments ly linked to a non-human heavy chain constant region gene sequence comprising a non-human IgM gene. [000198H] In one aspect, there is provided a method of making a human immunoglobulin heavy chain variable domain comprising expressing in a host cell the nucleic acid made or the c acid according to the present disclosure.

In one aspect, there is provided a non-human host cell sing the nucleic acid made or the nucleic acid ing to the present disclosure. [000198J] In one aspect, there is provided a method of obtaining a cell that expresses a human immunoglobulin heavy chain variable domain comprising harvesting a lymphocyte from a non-human animal that comprises in its germline genome a restricted immunoglobulin heavy chain locus characterized by the ce of a single human unrearranged V H gene segment, one or more human unrearranged DH gene segments, and one or more human unrearranged JH gene segments operably linked to a non-human immunoglobulin constant region comprising a non-human IgM gene, wherein the lymphocyte expresses a rearranged human immunoglobulin VH region gene derived from the restricted globulin heavy chain locus. [000198K] In one aspect, there is provided a method of making a human immunoglobulin heavy chain le domain comprising culturing the cell obtained or the hybridoma according to the present disclosure.

BRIEF DESCRIPTION OF FIGURES shows a general illustration, not to scale, of a series of targeting and molecular ering steps employed to make a targeting vector for construction of a modified heavy chain locus containing a single human V H 1-69 gene segment, twenty-seven human DH and six human JH gene segments at an endogenous immunoglobulin heavy chain locus. shows a general illustration, not to scale, of a series of ing and molecular engineering steps employed to make a targeting vector for construction of a modified heavy chain locus ning a single human V H 1-2 gene segment, twenty-seven human DH and six human J H gene segments at an endogenous immunoglobulin heavy chain locus. shows contour plots of splenocytes gated on single lymphocytes and d for CD19 (B cell) and CD3 (T cell) from a wild type mouse (WT) and a mouse homozygous for a single human VH gene segment, -seven human DH and six human JH gene ts at the endogenous immunoglobulin heavy chain locus (1hV H HO). shows, on the left, the percent of CD19+ B cells in spleens harvested from wild type mice (WT) and mice homozygous for a single human V H gene segment, twenty-seven human DH and six human J H gene segments at the endogenous immunoglobulin heavy chain locus (1hV H HO). On the right, the number of CD19÷ B cells per spleen is shown for both wild type mice (WT) and mice homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hVH HO). shows, on the left, the percent of CD19+ B cells in bone marrow harvested from femurs of wild type mice (WT) and mice homozygous for a single human V H gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hV H HO). On the right, the number of CD19+ B cells per femur is shown for both wild type mice (WT) and mice homozygous for a single human V H gene segment, twenty-seven human DH and six human JH gene segments at the nous immunoglobulin heavy chain locus (1hVH HO). shows contour plots of splenocytes gated on CD19+ B cells and stained for lgM and |gr<+ expression from a wild type mouse (WT) and a mouse homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hVH HO). ] shows contour plots of splenocytes gated on CD19+ B cells and stained for immunoglobulin D (lgD) and immunoglobulin M (lgM) from a wild type mouse (WT) and a mouse homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVH HO). shows the total number of transitional B cells (CD19’lthilgDin‘), mature B cells (CD19‘lgM‘mlgDhi), and the ratio of mature to re B cells in harvested spleens from wild type mice (WT) and mice homozygous for a single human VH gene segment, -seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVH HO). shows contour plots of bone marrow gated on singlets stained for immunoglobulin M (lgM) and 8220 from a wild type mouse (WT) and a mouse homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hVH HO). shows the total number of immature (Bzzo‘n‘lgm and mature (3220"‘igM*) B cells in bone marrow ed from the femurs of wild type mice (WT) and mice gous for a single human VH gene segment, twenty-seven human DH and six human JH gene ts at the endogenous immunoglobulin heavy chain locus (1 hVH HO). shows contour plots of bone marrow gated on CD19’ B cells and stained for ckit+ and CD43+ from a wild type mouse (WT) and a mouse homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous globulin heavy chain locus (1 hVH HO). 0] A shows the percent of CD19‘ cells in populations of pro B (CD19‘CD43’ckit’) and pre B (CD19’CD43’ckit‘) cells in bone marrow ted from the femurs of wild type mice (WT) and mice homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVH HO). 8 shows the absolute number of cells per femur in populations of pro 8 (CD19‘CD43’ckit‘) and pre B (CD19‘CD43'ckit’) cells in bone marrow harvested from wild type mice (WT) and mice homozygous for a single human V” gene t. twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVH HO). shows the relative mRNA expression (y-axis) in puriﬁed splenic B cells of VH1derived heavy chains in a quantitative PCR assay using a probe specific for the human VH1-69 gene t in mice homozygous for a replacement of the endogenous heavy chain VH, DH, JH, and a replacement of the endogenous light chain VIC and JK gene segments with human VH. DH. JH, VK and JK gene segments (HK), wild type mice (WT), mice heterozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVH HET) and mice homozygous for a single human VH gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hVH HO). Signals are normalized to expression of mouse CK. shows the nucleotide alignment of the second exon for each of en reported alleles for the human VH1-69 gene. Lower case bases indicate germline nucleotide differences among the alleles. Complementary determining regions (CDRs) are indicated with boxes around the sequence. Dashes te artificial gaps for proper sequence alignment. *01 (SEQ ID NO: 34); VH1-69*02 (SEQ ID NO: 36); VH1-69*03 (SEQ ID NO: 38); VH1- 69*04 (SEQ ID NO: 40); VH1-69*05 (SEQ ID NO: 42); VH1-69*06 (SEQ ID NO: 44); VH1-69*07 (SEQ ID NO: 46); VH1-69*08 (SEQ ID NO: 48); VH1-69*09 (SEQ ID NO: 50); VH1-69*10 (SEQ ID NO: 52); VH1-69*11 (SEQ ID NO: 54); VH1-69*12 (SEQ ID NO: 56); VH1-69*13 (SEQ ID NO: 58). shows the protein alignment of the mature heavy chain variable gene ce for each of thirteen reported alleles for the human VH1-69 gene. Lower case amino acids indicate germline differences among the alleles. Complementary determining regions (CDRs) are ted with boxes around the sequence. Dashes indicate artificial gaps for proper sequence alignment. VH1-69*O1 (SEQ ID NO: 35); VH1-69*02 (SEQ ID NO: 37); VH1- 69*03 (SEQ ID NO: 39); VH1-69*04 (SEQ ID NO: 41); *05 (SEQ ID NO: 43); VH1-69*06 (SEQ ID NO: 45); VH1-69*07 (SEQ ID NO: 47); *08 (SEQ ID NO: 49); VH1-69*09 (SEQ ID NO: 51); VH1-69*10 (SEQ ID NO: 53); VH1-69*11 (SEQ ID NO: 55); VH1-69*12 (SEQ ID NO: 57); *13 (SEQ ID NO: 59). shows a percent identity/percent similarity matrix for the aligned protein sequences of the mature variable gene for each of thirteen reported alleles for the human VH1- 69 gene. Percent identity among the VH1-69 s is indicated above the shaded boxes and percent similarity is indicated below the shaded boxes. Scores for percent identity and percent rity were scored by a CIustalW (v1.83) alignment tool using MacVector software (MacVector, Inc, North Carolina). shows the nucleotide alignment of the second exon for each of five reported alleles for the human VH1-2 gene. Lower case bases indicate germline nucleotide differences among the alleles. Complementary determining s (CDRs) are ted with boxes around the sequence. Dashes indicate artificial gaps for proper sequence alignment. VH1-2*01 (SEQ ID NO: 60); VH1-2*02 (SEQ ID NO: 62); VH1-2*03 (SEQ ID NO: 64); VH1—2*O4 (SEQ ID NO: 66); VH1-2*05 (SEQ ID NO: 68). shows the protein alignment of the mature heavy chain variable gene sequence for each of ﬁve reported s for the human VH1-2 gene. Lower case amino acids indicate germline differences among the alleles. Complementary determining regions (CDRs) are indicated with boxes around the sequence. Dashes indicate artiﬁcial gaps for proper sequence alignment. VH1-2*01 (SEQ ID NO: 61); VH1-2*02 (SEQ ID NO: 63); VH1-2*03 (SEQ ID NO: 65); 04 (SEQ ID NO: 67); VH1-2*05 (SEQ ID NO: 69). 8] shows a percent identity/percent similarity matrix for the d protein sequences of the mature variable gene for each of five reported alleles for the human VH1-2 gene. Percent identity among the VH1-2 alleles is indicated above the shaded boxes and percent similarity is indicated below the shaded boxes. Scores for percent identity and percent similarity were scored by a ClustalW (v1.83) alignment tool using tor software (MacVector, Inc., North Carolina). shows the antibody titer from mice homozygous for human heavy and human K light chain variable gene loci (HK; n=4) and mice homozygous for a single human VH1- 69 gene segment. twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1 hVHHO; n=10) that were immunized with a human cell surface receptor (Antigen A). 0] shows the antibody titer from mice homozygous for human heavy and human K light chain variable gene loci (HK; n=5) and mice homozygous for a single human VH1- 69 gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus (1hVHHO; n=5) that were immunized with two different influenza vaccines. 1] shows the percentage (y-axis) of lgM-primed heavy chains having a specified amino acid length for the VH CDR3 region (x-axis) from mice homozygous for a single human VH1-69 gene segment, twenty-seven human DH and six human JH gene segments at the endogenous immunoglobulin heavy chain locus and homozygous for a replacement of the endogenous K light chain variable loci with human x light chain variable loci that were immunized with a human cell surface receptor (Antigen A). shows the percentage (y-axis) of IgG-primed heavy chains having a ied amino acid length for the VH CDR3 region (x-axis) from mice homozygous for a single human VH1-69 gene segment, -seven human DH and six human JH gene ts at the endogenous immunoglobulin heavy chain locus and homozygous for a replacement of the nous K light chain variable loci with human K light chain variable loci that were immunized with a human cell surface receptor (Antigen A).

DETAILED DESCRIPTION This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the ology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

Unless d otherwise, all terms and s used herein include the meanings that the terms and phrases have ed in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. gh any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described. All publications ned are hereby incorporated by nce. [000224A] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of ts, rs or steps.

The phrase "substantial" or "substantially" when used to refer to an amount of gene segments (e.g., "substantially all" V gene segments) includes both functional and non functional gene segments and include, in various embodiments, e.g., 80% or more, 85% or more, 90% or more, 95% or more 96% or more, 97% or more, 98% or more, or 99% or more of all gene segments; in s embodiments, "substantially all" gene segments includes, e.g., at least 95%, 96%, 97%, 98%, or 99% of functional (i.e., non-pseudogene) gene segments.

The term "replacement" includes n a DNA sequence is placed into a genome of a cell in such a way as to replace a sequence within the genome with a heterologous sequence (e.g., a human sequence in a mouse), at the locus of the genomic sequence. The DNA sequence so placed may include one or more regulatory sequences that are part of source DNA used to obtain the sequence so placed (e.g., promoters, enhancers, 5'- or 3'- untranslated regions, appropriate recombination signal sequences, etc.). For example, in various embodiments, the replacement is a substitution of an nous sequence for a heterologous sequence that results in the production of a gene product from the DNA sequence so placed (comprising the logous sequence), but not expression of the nous sequence; the replacement is of an endogenous genomic sequence with a DNA sequence that encodes a protein that has a r function as a protein encoded by the endogenous c sequence (e.g., the endogenous genomic sequence encodes an immunoglobulin gene or domain, and the DNA fragment encodes one or more human immunoglobulin genes or domains). In various embodiments, an endogenous gene or fragment thereof is ed with a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of, a homolog of, or is substantially identical or the same in structure and/or function, as the endogenous gene or fragment thereof that is replaced.

A precise, in situ replacement of six megabases of the le regions of the mouse heavy chain immunoglobulin loci (VH-DH-JH) with a restricted human globulin heavy chain locus was performed, while leaving the ﬂanking mouse ces intact and functional within the hybrid loci, including all mouse constant chain genes and locus transcriptional control regions (and . Specifically, a single human V“, 27 DH, and six JH gene segments were introduced through chimeric BAC targeting s into mouse ES cells using VELOCIGENE® genetic engineering logy (see, e.g., US Pat. No. 6,586,251 and Valenzuela et a/., 2003, High-throughput engineering of the mouse genome coupled with high- resolution expression analysis, Nat Biotechnol 21:652-659).

Non-Human Animals With Restricted lmmunoglobulin VH Gene Segments Non-human animals comprising immunoglobulin loci that comprise a restricted number of VH genes, and one or more D genes and one or more J genes, are provided, as are methods of making and using them. When immunized with an antigen of interest, the non- human animals generate B cell populations with dy variable s d only from the restricted, pre-selected VH gene or set of VH genes (e.g., a pre-selected VH gene and variants thereof). In various embodiments, non-human animals are provided that generate B cell populations that express human antibody variable domains that are human heavy chain variable domains, along with cognate human light chain variable domains. In various embodiments, the man s rearrange human heavy chain variable gene ts and human light chain variable gene segments from modiﬁed endogenous mouse immunoglobulin loci that comprise a replacement or insertion of the non-human unrearranged variable region sequences with human unrearranged variable region sequences.

Early work on the organization, structure, and function of the immunoglobulin genes was done in part on mice with disabled endogenous loci and engineered to have transgenic loci (randomly ) with partial human immunoglobulin genes, e.g., a partial repertoire of human heavy chain genes linked with a human constant gene, randomly inserted into the genome, in the ce or absence of a human light chain transgene. Although these mice were somewhat less than optimal for making useful high afﬁnity antibodies, they facilitated certain functional analyses of immunoglobulin loci. Some of these mice had as few as two or three, or even just a single, heavy chain variable gene.

Mice that express fully human immunoglobulin heavy chains d from a single human VHS-51 gene and 10 human DH genes and six human JH genes, with human 1.). and 71 constant genes, on a ly inserted transgene (and disabled endogenous immunoglobulin loci) have been reported (Xu and Davis, 2000, Diversity in the CDR3 Region of VH ls Sufﬁcient for Most Antibody Speciﬁcities, Immunity 45). The fully human immunoglobulin heavy chains of these mice are mostly expressed with one ofjust two fully mouse x light chains derived from the endogenous mouse A light chain locus (VM-JM or VA2-JA2 only). and can express no K light chain (the mice are ng"‘). These mice exhibit severely abnormal dysfunction in B cell development and antibody expression. 8 cell numbers are reportedly 5-10% of wild- type, lgM levels 540% of wild-type, and lgG1 levels are only 0.1-1% of wild-type. The observed lgM repertoire revealed highly restricted junctional diversity. The fully human heavy chains display largely cal CDR3 length across antigens, the same JH (JH2) usage across antigens, and an initial junctional Q residue, thus reflecting a certain lack of CDR3 diversity.

The fully mouse 7» light chains nearly all had a W96L substitution in JM as initial junctional residue. The mice are reportedly unable to generate any antibodies against bacterial polysaccharide. e the human variable domains couple with mouse light chains, the utility of the human variable regions is highly limited.

Other mice that have just a single human VH3-23 gene, human DH and JH genes, and mouse light chain genes have been reported, but they exhibit a limited diversity (and thus a limited usefulness) due in part to mispairing potential between human VH and mouse VL domains (see, e.g., Mageed et al., 2001, Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region, Clin. Exp.

Immunol. 123:1-5). Similarly, mice that bear two VH genes (3-23 and 6-1) along with human DH and JH genes in a transgene containing the human 0 constant gene (Bruggemann et al., 1991, Human antibody production in enic mice: expression from 100kb of the human lgH locus, Eur. J. ol. 21 :1323-1326) and express them in human lgM chains with mouse light chains may t a oire limited by mispairing (Mackworth-Young et a/., 2003, The role of antigen in the selection of the human V3-23 immunoglobulin heavy chain variable region gene, Clin. Exp. Immunol. 134:420-425).

Other transgenic mice that express VH-restricted fully human heavy chains from a human transgene randomly inserted in the genome, with a limited human A repertoire expressed from a fully human randomly inserted transgene, have also been reported (see, e.g., Taylor et al., 1992, A enic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins, Nucleic Acids Res. 20(23):6287-6295; Wagner et al., 1994, Antibodies generated form human immunoglobulin ci in transgenic mice, Nucleic Acids Res. 1389-1393). However, transgenic mice that express fully human dies from transgenes randomly ated into the mouse genome, and that comprise damaged endogenous loci, are known to exhibit ntial differences in immune response as compared with wild-type mice that affect the diversity of the antibody variable s obtainable from such mice.

Useful non-human animals that generate a diverse tion of B cells that express human antibody variable domains from a restricted VH gene repertoire and one or more D genes and one or more J genes will be capable of generating, preferably in some ments. oires of rearranged variable region genes that will be sufﬁciently diverse.

In various embodiments, diversity includes junctional diversity, somatic hypermutation, and polymorphic ity in VH gene sequence (for embodiments where VH genes are present in rphic forms). Combinatorial diversity occurs in the pairing of the VH gene with one of a plurality of cognate human light chain variable domains (which, in various embodiments, se junctional diversity and/or somatic utations).

Non-human s comprising a restricted human VH gene repertoire and a complete or substantially complete human VL gene repertoire will in various embodiments generate populations of B cells that reﬂect the various sources of diversity, such as junctional ity (e.g., VDJ, VJ joining, P ons, N additions), atorial ity (e.g., cognate VH-restricted human heavy, human light), and somatic hypermutations. In embodiments comprising a restriction of the VH repertoire to one human VH gene. the one human VH gene can be t in two or more variants. In various embodiments, the presence of two or more polymorphic forms of a VH gene will enrich the diversity of the variable domains of the B cell population.

Variations in the germline ces of gene segments (e.g., V genes) contribute to the diversity of the antibody response in humans. The relative contribution to diversity due to V gene sequence differences varies among V genes. The degree of polymorphism varies across gene families, and is reﬂected in a plurality of haplotypes (stretches of sequence with coinherited polymorphisms) capable of generating further diversity as observed in VH haplotype differences between related and unrelated individuals in the human population (see, e.g., Souroujon et al., 1989, rphisms in Human H Chain V Region Genes from the VHIII Gene Family, J. Immunol. 143(2):706-711). Some have suggested. based on data from particularly polymorphic human VH gene families, that haplotype diversity in the germline is a major contributor to VH gene heterogeneity in the human population, which is reﬂected in the large diversity of different germline VH genes across the human population (see, Sasso et al., 1990, Prevalence and rphism of Human VH3 Genes, J. Immunol. 145(8):2751-2757).

Although the human population displays a large diversity of haplotypes with respect to the VH gene repertoire due to widespread polymorphism. n polymorphisms are reﬂected in prevalent (i.e., conserved) s observed in the human population (Sasso et al., 1990). VH polymorphism can be described in two principle forms. The first is variation arising from allelic variation associated with differences among the nucleotide sequence between alleles of the same gene segment. The second arises from the numerous duplications, insertions, and/or deletions that have occurred at the immunoglobulin heavy chain locus. This has resulted in the unique situation in which VH genes derived by ation from identical genes differ from their tive alleles by one or more tide substitutions. This also directly inﬂuences the copy number of VH genes at the heavy chain locus.

Polymorphic alleles of the human immunoglobulin heavy chain variable gene segments (VH genes) have largely been the result of insertion/deletion of gene segments and single nucleotide differences within coding s, both of which have the potential to have functional uences on the immunoglobulin molecule. Table 1 sets forth the functional VH genes listed by human VH gene family and the number of identiﬁed alleles for each VH gene in the human immunoglobulin heavy chain locus. There are some ﬁndings to suggest that polymorphic VH genes have been implicated in susceptibility to certain diseases such as, for e, rheumatoid arthritis. whereas in other cases a linkage between VH and disease has been less clear. This ambiguity has been attributed to the copy number and presence of s alleles in different human populations. In fact, several human VH genes demonstrate copy number variation (e.g., VH1-2, VH1-69, VH2-26, VH2-70, and VHS-23). In s embodiments, humanized mice as described herein with restricted VH repertoires comprise multiple polymorphic variants of an individual VH family member (e.g., two or more polymorphic variants of VH1-2, VH1-69, V32-26, VH2-70, or VH3-23, replacing all or substantially all functional mouse VH segments at an endogenous mouse locus). In a speciﬁc embodiment, the two or more polymorphic variants of mice described herein are in number up to and including the number ted for the corresponding VH family member in Table 1 (e.g., for , 13 variants; for VH1-2, ﬁve variants; etc.).

Commonly observed variants of particular human VH genes are known in the art.

For example, one of the most complex polymorphisms in the V“ locus belongs to the VH1-69 gene. The human VH1-69 gene has 13 reported alleles (Sasso et al., 1993, A fetally expressed immunoglobulin VH1 gene s to a complex set of alleles, Journal of Clinical Investigation 91:2358—2367; Sasso et al., 1996, Expression of the immunoglobulin VH gene 51 p1 is proportional to its germline gene copy , Journal of Clinical Investigation 97(9):2074- 2080) and exists in at least three haplotypes that carry duplications of the VH1-69 gene, which results in multiple copies of the VH gene at a given locus. These polymorphic alleles include differences in the complementarity determining regions (CDRs), which may dramatically inﬂuence antigen speciﬁcity. Table 2 sets for the reported alleles for human VH1-69 and the SEQ ID NOs for the DNA and protein sequences of the mature heavy chain variable regions.

Table 3 sets forth the reported alleles for human VH1-2 genes and the SEQ lD NOs for the DNA and protein sequences of the mature heavy chain le regions. 9] entative genomic DNA and full-length protein sequences of a VH1-69 gene are set forth in SEQ lD NO: 1 and SEQ ID NO: 2, respectively. and set forth DNA and protein alignments of en reported VH1-69 alleles, respectively. Representative DNA and protein sequences of a VH1-2 gene are set forth in SEQ ID NO: 60 and SEQ ID NO: 61, respectively. and set forth DNA and protein alignments of ﬁve reported VH1-2 alleles, respectively. and set forth a percent identity/similarity matrix for aligned protein sequences corresponding to thirteen reported human VH1-69 alleles and ﬁve reported human VH1-2 alleles, respectively. In various embodiments, the modiﬁed locus of the invention comprises a VH gene selected from Table 1, present in two or more copy number, wherein the copy number es up to and including the number of alleles shown in Table 1.

In one embodiment, the modified locus of the invention comprises a VH1-69 gene selected from Table 2, present in two or more copy number, wherein the copy number includes up to and including the number of alleles shown in Table 1. In one embodiment, the modified locus of the invention comprises a VH1-2 gene selected from Table 3, present in two or more copy number, n the copy number es up to and including the number of s shown in Table 1.

Although embodiments employing a restricted human VH repertoire in a mouse are extensively discussed, other non-human animals that express a restricted human VH repertoire are also provided. Such non-human animals include any of those which can be genetically modified to express a restricted human VH repertoire as disclosed herein, including, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, o), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. For example, for those non-human animals for which suitable genetically modiﬁable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modiﬁcation.

Such methods include, e.g., modifying a non-ES cell genome (e.g., a ﬁbroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modiﬁed genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modiﬁed oocyte) in a non-human animal under suitable conditions to form an embryo. s for modifying a non-human animal genome (e.g., a pig, cow, rodent, chicken, etc. ) include, e.g., employing a zinc finger nuclease (ZFN) or a transcription activator-like effector se (TALEN) to modify a genome to include a restricted human VH repertoire. Thus, in one embodiment a method is provided for editing a non-human animal genome to include a restricted human VH oire, comprising a step of g the genome employing a ZFN or a TALEN to e no more than one, or no more than two, human VH gene ts (or rphic variants thereof), wherein the no more than one or no more than two human VH gene segments are operably linked to an immunoglobulin constant gene sequence. In one embodiment, the constant gene sequence is selected from a human heavy chain constant ce and a non-human heavy chain constant sequence. In one ment, the constant ce is non-human and the no more than one or no more than two human VH gene segments are operably linked to non~human constant gene sequence at an endogenous non-human immunoglobulin locus.

In one aspect, the man animal is a small , e.g., of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically ed animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one ment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modiﬁed animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), e (true mice and rats, gerbils, spiny mice, crested rats), idae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In a speciﬁc embodiment, the genetically d rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a d rat.

In one embodiment, the genetically modiﬁed mouse is from a member of the family Muridae, In one embodiment, the non-human animal is a rodent that is a mouse of a CS7BL strain. In one embodiment, the C57BL strain is ed from C57BL/A, C57BL/An, C57BL/GrFa, CS7BL/KaLwN, CS7BL/6, CS7BL/6J, CS7BL/6ByJ, CS7BL/6N, C57BL/6NJ, CS7BL/10, CS7BL/108c8n, CS7BL/10Cr, and C57BL/Ola. In another ment, the mouse is a 129 strain. In one embodiment, the 129 strain is selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 12981 (e.g., 12981/8V, 12981/Svlm), 12982, 12984, 12985, 12989/8vaH, 12986 (129/8vaTac), 12987, 12988, 129T1, 129T2 (see, e.g., Festing etal. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al. (2000) ishment and Chimera Analysis of 129/Sva- and CS7BL/6- Derived Mouse Embryonic Stem Cell Lines). In one embodiment, the genetically modiﬁed mouse is a mix of an aforementioned 129 strain and an aforementioned CS7BL strain (e.g., a C57BL/6 strain). In r embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned CS7BL/6 strains. In one embodiment, the 129 strain of the mix is a 12986 (129/8vaTac) strain. In another embodiment, the mouse is a mix of a 129/8va- and a CS7BL/6-derived strain. In a speciﬁc embodiment, the mouse is a mix of a 129/Sva- and a CS7BL/6-derived strain as described in Auerbach et al. 2000 hniques 29:1024-1032. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In another embodiment, the mouse is a mix of a BALB strain (e.g., BALB/c strain) and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more of a strain selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

Table 1 VH Family VH Gene Alleles VH Family VH Gene Alleles 1-2 4-4 1-3 4-28 1-8 41 1-18 42 VH1 1—24 44 1-45 4-31 1-46 4-34 .—l.—I.oooxnwooum-smxl 1-58 4-39 1-69 amww—xwwmm 4-59 _\ 4-61 VH2 2-26 *8 VH5 5-51 5 2-70 .3 (A) VH6 6-1 2 71 5 7-81 ._L 1 wNNAmAmh-NN—‘m—‘NamhAthb-Nw Table 2 SEQ ID NO: lgHV1-69 Allele ion Number sDNA/Proteinz lg HV1 -69*01 L22582 34/35 lg HV1 -69*02 Z27506 36/37 lgHV1-69*03 X92340 38/39 lgHV1-69*04 M83132 40/41 lg HV1 -69*05 X67905 42/43 lg HV1 -69*06 L22583 44/45 lgHV1-69*07 Z29978 46/47 lgHV1-69*08 Z14309 48/49 lgHV1-69*09 Z14307 50/51 lgHV1-69*10 214300 52/53 lgHV1-69*11 Z14296 54/55 lgHV1-69*12 Z14301 56/57 lgHV1-69*13 Z14214 58/59 Table 3 SEQ ID NO: lgHV1-2 Allele Accession Number SDNA/Proteinz lg HV1 -2*01 X07448 60/61 lgHV1-2*02 X62106 62/63 lgHV1-2*03 X92208 64/65 lgHV1-2*04 Z12310 66/67 2*05 HM855674 68/69 n-Dependent VH Gene Usage Antigen-dependent preferential usage of VH genes can be exploited in the pment of human therapeutics ing clinically significant antigens. The y to generate a repertoire of antibody le domains using a particular VH gene can provide a signiﬁcant advantage in the search for high-afﬁnity antibody variable domains to use in human therapeutics. Studies on naive mouse and human VH gene usage in antibody variable s reveal that most heavy chain variable domains are not derived from any particular single or dominantly used VH gene. On the other hand, studies of antibody response to certain antigens reveal that in some cases a particular antibody response displays a biased usage of a particular VH gene in the B cell repertoire following immunization.

] Although the human VH repertoire is quite diverse, by some estimates the expected frequency of usage of any given VH gene, assuming random selection of VH genes, is about 2% (Brezinschek et al., 1995, Analysis of the Heavy Chain Repertoire of Human Peripheral B Cells Using Single-Cell Polymerase Chain Reaction. J. Immunol. 155:190-202). But VH usage in peripheral B cells in humans is skewed. In one study. functional V gene abundance followed the pattern VH3 > VH4 > VH1 > VH2 > VH5 > VH6 (Davidkova et al., 1997, Selective Usage of VH Genes in Adult Human Lymphocyte Repertoires, Scand. J. Immunol. 45:62-73). One early study ted that VH3 family usage frequency was about 0.65, whereas VH1 family usage frequency was about 0.15; these and other observations suggest that the germline complexity of the human VH repertoire is not precisely reﬂected in the peripheral B cell compartment in humans that have a normal germline VH repertoire, a situation that is similar to that observed in the mouse—Le, VH gene expression is non-stochastic (Zouali and These, 1991, Probing VH Gene-Family Utilization in Human Peripheral B Cetls by In Situ Hybridization, J. l. 146(8):2855-2864). According to one report, VH gene usage in humans, from greatest to least, is VH3 > VH4 > VH1 > VH5 > VH2 > VH6; rearrangements in peripheral B cells reveal that VH3 family usage is higher than to be expected based on the relative number of germline VH3 genes (Brezinschek et al., 1995). According to another report VH usage in humans s the pattern VH3 > VH5 > VH2 > VH1 > VH4 > VH6, based on analysis of pokeweed n-activated peripheral small immunocompetent B cells (Davidkova et al., 1997, Selective Usage of VH Genes in Adult Human B Lymphocyte Repertoires, Scand. J. Immunol. 45:62-73). One report asserts that among the most frequently used VH3 family members are 3-23, 3-30 and 3-54 (Brezinschek et al., 1995). In the VH4 , member 4-59 and 4-4b were found relatively more frequently (ld.), as well as 4-39 and 4-34 (Brezinscheck et al., 1997, Analysis of the Human VH Gene Repertoire, J. Clin. Invest. 99(10):2488-2501). Others postulate that the activated heavy chain repertoire is skewed in favor of high VH5 expression and lower VH3 sion (Van Dijk- Hard and ist, 2002, Long-term kinetics of adult human antibody repertoires, Immunology 107:136-144). Other studies assert that the most commonly used VH gene in the adult human repertoire is VH4-59, followed by VHS-23 and VH3-48 (Arnaout et‘ al., 2001, High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans, PLoS ONE 6(8):108). Although usage studies are based on relatively small sample numbers and thus t high ce, taken together the studies suggest that V gene expression is not purely stochastic. indeed, studies with ular ns have ished n certain cases—the deck is ﬁrmly stacked against certain usages and in favor of others.

Over time, it became apparent that the observed repertoire of human heavy chain le domains generated in response to certain antigens is highly restricted. Some antigens are associated almost exclusively with neutralizing dies having only certain particular V... genes, in the sense that effective neutralizing antibodies are derived from essentially only one VH gene. Such is the case for a number of ally important human pathogens. -derived heavy chains have been observed in a variety of antigen-speciﬁc antibody repertoires of therapeutic significance. For instance, VH1-69 was frequently observed in heavy chain transcripts of an lgE repertoire of peripheral blood lymphocytes in young children with atopic disease (Bando et al., 2004, Characterization of VH2 gene expressed in PBL from children with atopic diseases: detection of homologous VH1-69 derived transcripts from three unrelated patients. logy Letters 94:99-106). -derived heavy chains with a high degree of somatic hypermutation also occur in B cell lymphomas (Perez et al., 2009, Primary cutaneous B-cell lymphoma is associated with somatically utated immunoglobulin variable genes and frequent use of VH1-69 and VH4—59 segments. British Journal of Dermatology 162:611-618), s some -derived heavy chains with essentially ne sequences (i.e., little to no somatic hypermutation) have been observed among autoantibodies in patients with blood disorders (Pos et al., 2008, VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura, Journal of Thrombosis and Haemostasis 72421-428).

Further, neutralizing antibodies against viral antigens such as HIV, inﬂuenza and hepatitis C (HCV) have been found to utilize germline and/or somatically mutated VH1 derived sequences (Miklos et al., 2000, Salivary gland mucosa-associated id tissue ma immunoglobulin VH genes show frequent use of V1-69 with distinctive CDR3 features. Blood 95(12):3878—3884; Kunert etal., 2004, Characterization of lar features, antigen-binding, and in vitro properties of lgG and lgM variants of 4E10, an anti-HIV type I neutralizing monoclonal antibody, Aids Research and Human Retroviruses 20(7):?55-762; Chan et al., 2001, VH1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen, Blood 97(4):1023-1026; ari et al., 2005, Hepatitis C virus drives the unconstrained monoclonal ion of VH1- 69-expressing memory B cells in type II cryoglobulinemia: A model of infection-driven lymphomagenesis, Journal of Immunology 174:6532-6539; Wang and Palese, 2009, Universal epitopes of inﬂuenza virus hemagglutinins?, Nature Structural & Molecular Biology 16(3):233- 234; Sui et al., 2009, Structural and functional bases for spectrum neutralization of avian and human za A viruses, Nature Structural & Molecular Biology 16(3):265-273; a et al., 2001, lmmunoglobulin Gene Mutations and Frequent Use of VH1-69 and VH4-34 Segments in Hepatitis C Virus-Positive and Hepatitis C Virus-Negative Nodal Marginal Zone B- Cell Lymphoma, Am. J. Pathol. 159(1):253-261).

VH usage bias is also observed in the humoral immune se to Haemophilus influenzae type b (Hib P3) in humans. Studies suggest that the VHIII family (the VHlllb subfamily in particular, VH9.1) ively characterizes the human humoral response to Hib P8, with diverse D and J genes (Adderson et al., 1991, Restricted lg H Chain V Gene Usage in the Human Antibody Response to Haemophilus nzae Type b Capsular Polysaccharide, J.

Immunol. 147(5):1667-1674; Adderson et al., 1993, Restricted lmmunoglobulin VH Usage and VDJ Combinations in the Human Response to Haemophilus influenzae Type b Capsular Polysaccharide, J. Clin. Invest. 91 :2734-2743). Human JH genes also display biased usage; JH4 and JH6 are observed at about 38-41% in peripheral B cells in humans (Brezinschek et al., 1995).

VH usage in HIVinfected humans is reportedly biased against VH3 usage and in favor of VH1 and VH4 gene families (Wisnewski et al., 1996, Human Antibody Variable Region Gene Usage in HIV-1 Infection, J. Acquired Immune Deﬁciency Syndromes & Human Retrovio/ogy 11(1):31-38). However, cDNA analysis of bone marrow from affected patients’ revealed signiﬁcant VH3 usage not expressed in the functional B cell repertoire, where Fabs reﬂecting the VH3 usage exhibited effective in vitro neutralization of HIV-1 (Id.). It might be postulated that the humoral immune response to HIV-1 infection is possibly ated due to the VH restriction; modiﬁed non-human animals as described herein (not infectabIe by HIV-1) might thus be useful for generating neutralizing antibody domains derived from particular VH genes present in the genetically d animals described herein, but derived from different VH genes than those ed in the restricted repertoire of affected humans.

Thus, the ability to generate high affinity human antibody variable domains in VH- restricted mice, e.g., (restricted. e.g., to a VH3 family member and polymorph(s) thereof) immunized with HIV-1 might provide a rich resource for designing effective HIVneutralizing human therapeutics by thoroughly mining the restricted (e.g., restricted to a VH3 family member or variant(s) thereof) repertoire of such an immunized mouse.

Restriction of the human antibody response to certain pathogens may reduce the likelihood of obtaining antibody variable regions from ed humans that can serve as springboards for designing high affinity neutralizing antibodies against the pathogen. For e, the human immune response to HIV-1 infection is ly restricted throughout HIV-1 infection and into AIDS progression (Muller et al., 1993, B-cell alities in AIDS: stable and clonally restricted antibody response in HIV-1 infection, Scand. J. Immunol. 38:327-334; Wisnewski et al., 1996). Further, VH genes are in l not present in all polymorphic forms in any particular individual; n individuals in certain populations possess one variant, s individuals in other populations possess a ent variant. Thus, the availability of a biological system that is restricted to a single VH gene and its variants will in various embodiments e a to unexploited source of diversity for generating antibody variable regions (e.g., human heavy and light cognate domains) based on a cted VH gene. Thus, in one aspect. a genetically modiﬁed non-human animal is provided that comprises a plurality of polymorphic variants of no more than one, or no more than two, human VH gene segment family member. In one embodiment, the no more than one, or no more than two. human VH gene ts are ly linked to one or more human DH gene segments, one or more human JH gene segments, and a human or non-human constant region gene segment. In one embodiment the constant region is at an endogenous non-human immunoglobulin constant gene locus. In one embodiment. the man animal further comprises a nucleic acid sequence derived from a human VL sequence, e.g., a rearranged or unrearranged human VL gene segment or a rearranged human VL/JL ce. In one embodiment. the nucleic acid sequence derived from the human VL sequence is at an endogenous non-human VL gene locus; in one embodiment, the nucleic acid sequence derived form the human VL sequence is on a transgene. In a speciﬁc embodiment, the man animal is incapable of expressing an immunoglobulin light chain variable domain that itself comprises an endogenous VL or JL gene segment, and comprises no more than one, or no more than two, light chain genes that encode rearranged human VL domains (i.e., from no more than one, or no more than two, rearranged human VL/JL sequences).

Genetically modiﬁed mice that express human heavy chain variable regions with restricted VH gene segment usage are useful to generate a relatively large repertoire of junctionally diverse, combinatorially diverse, and somatically mutated high afﬁnity human immunoglobulin heavy chain variable regions from an otherwise restricted repertoire. A restricted repertoire, in one embodiment, refers to a predetermined tion in the number and/or identity of germline genes that results in the mouse being unable to form a rearranged heavy chain gene that is derived from any V gene other than a ected V gene. In embodiments that employ a preselected V gene but not a ected D and/or J gene, the repertoire is restricted with t to the identity of the V gene but not the D and/or J gene (e.g., the repertoire consists ially of no more than one, or no more than two, V... gene segments (and/or polymorphs thereof); and a plurality of D gene segments and a plurality of J gene segments». The identity of the preselected V gene (and any preselected D and/or J genes) is not limited to any particular V gene.

Designing a mouse so that it rearranges a single VH gene (present as a single segment or a set of variants) with a variety of human D and J gene ts (e.g., DH and JH segments) provides an in vivo junctional ity/combinatorial diversity/somatic hypermutation permutation machine that can be used to iterate mutations in resulting rearranged heavy chain variable region ces (e.g., V/D/J or V/J, as the case may be). In such a mouse. the clonal selection process operates to select suitable variable regions that bind an antigen of interest that are based on a single preselected VH gene (or variants thereof). Because the mouse's clonal ion components are dedicated to selection based on the single preselected VH gene segment, background noise (e.g., a wide variety of non antigen-binding VH domains derived from many germline gene segments) is largely eradicated. With judicious selection of the VH gene segment, a relatively larger number of clonally selected, antigen- specific antibodies can be screened in a shorter period of time than with a mouse with a large diversity of V ts.

] Preselecting limited repertoire and restricting a mouse to a single V segment provides a system for permuting V/D/J junctions at a rate that is in various embodiments higher than that observed in mice that ise have up to 40 or more V segments to ine with D and J s. Removal of other V segments frees the locus to form more V/D/J combinations for the ected V segment than ise observed. The increased number of transcripts that result from the recombination of the ected V with one of a plurality of D and one of a plurality of J segments will feed those transcripts into the clonal selection system in the form of pre-B cells, and the clonal selection system is thus ted to cycling 8 cells that express the preselected V region. In this way, more unique V region rearrangements derived from the preselected V segment can be screened by the organism than would othenrvise be possible in a given amount of time.

In various aspects, mice are described that enhance the junctional diversity of V/D/J recombinations for the preselected V region, because all or substantially all recombinations of the immunoglobulin heavy chain variable locus will be of the preselected V t and the D and J segments that are placed in such mice. Therefore, the mice provide a method for generating a diversity of CDR3 segments using a base, or restricted VH gene repertoire. 7] In one aspect, a non-human animal is provided, wherein the B cell tion of the non-human animal expresses immunoglobulin heavy chains that are derived from no more than one, or no more than two human VH gene segments. In one embodiment, each of the no more than one, or no more than two, human VH gene segments are t in two or more polymorphic forms. In one embodiment, the human VH gene segment is present in three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19 or 20 polymorphic forms. In one embodiment, the non-human animal expresses a human light chain variable domain derived from a human VL gene segment.

In one aspect, a method is provided for generating a B cell population in a nonhuman , wherein the B cell population expresses human heavy chains derived from a single germline human VH gene segment and two or more human D gene segments and two or more human J gene segments; the method comprising a step of immunizing a non-human animal as described herein with an antigen of st, and allowing the non-human animal to mount an immune response to the antigen of interest, wherein the immune response comprises expressing the human heavy chains on the surface of B cells in the B cell population In one embodiment, the non-human animal is a rodent (e.g., a mouse or rat). In one embodiment, the human VH gene segment, human DH segment, and human JH t are operably linked to a non-human constant region gene. In one embodiment, the non-human animal further comprises a nucleic acid sequence encoding a human VL domain. In one ment, the nucleic acid sequence encoding the human VL domain is linked to a non-human light chain constant region gene sequence.

In one aspect, a method for making a non-human animal that expresses an immunoglobulin population characterized by the immunoglobulins having heavy chains that are derived from a plurality of rearrangements of a single human VH gene t (or sing human VH gene family member) and one of a ity of DH gene segments and one of a plurality of JH gene segments, is provided. In one embodiment, the human VH gene segment is a human VH1-69 gene segment. In one embodiment, the human VH gene segment is a human VH1-2 gene segment.

In one aspect. a method is provided for ting a population of human immunoglobulin heavy chain le domains whose CDR1 and CDR2 are derived from the same germline VH gene segment, and whose CDR3 are derived from the germline gene segment and two or more human D ts, and two or more human J ts; the method comprising zing a non-human animal as described herein with an n of interest, and allowing the non-human animal to mount an immune response to the antigen of interest, wherein the immune se comprises expressing the human heavy chain variable domains in the context of a light chain variable domain. In one embodiment, the non-human animal is a rodent (9.9., a mouse or rat). In one embodiment, the human VH gene segment, human D segment, and human J segment are operably linked to a non-human constant region gene. In one embodiment, the non-human animal further comprises a nucleic acid sequence encoding a human VL domain. In one embodiment, the nucleic acid sequence encoding the human VL domain is linked to a non-human light chain constant region gene sequence.

In one aspect, a genetically modiﬁed non-human animal is ed, wherein the man animal is incapable of expressing a man VH , and wherein each immunoglobulin heavy chain of the heavy chain population expressed in the animal ses a human VH domain comprising a CDR1 and a CDR2 that are identical but for one or more somatic hypermutations, and wherein the heavy chain population comprises a plurality of CDR3 sequences derived from a ity of rearrangements with a plurality of D and J gene segments.

In one aspect, a biological system for generating variation in CDR3 identity and length is provided, comprising a genetically modified non-human animal as described herein, n the non-human animal comprises no more than or no more than two human VH gene segments, and two or more D gene segments and one or more J gene segments, wherein the non-human animal further comprises a humanized immunoglobulin light chain locus. In various embodiments, the non-human animal in se to immunization with an antigen of interest generates an immune se that comprises expressing an immunoglobulin heavy chain population characterized by each heavy chain having CDR1s and CDRZS that differ only by somatic hypermutation, and CDR3s that differ by rearrangement and somatic hypermutation.

In one embodiment, the biological system is a mouse that is genetically modiﬁed as described herein. In one embodiment, the human VH gene segment and the human VL gene segment are at endogenous mouse heavy and light immunoglobulin loci, respectively. In one embodiment, one or more of the human VH gene segment and the human VL gene segment are on transgenes (i.e., at a locus other than an endogenous immunoglobulin locus).

EXAMPLES The following examples are provided so as to describe to those of ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, ature is indicated in Celsius, and pressure is at or near atmospheric. In the ing Examples, when the use of kits agents from various suppliers is indicated, all procedures were carried out according to manufacturer's speciﬁcations.

Example 1 Construction of Restricted Heavy Chain Loci A uniquely engineered human heavy chain locus containing a single human VH gene t d upstream of all the human DH and JH gene segments was d by a series of homologous ination reactions in bacterial cells (BHR) using Bacterial Artiﬁcial Chromosome (BAC) DNA. Several targeting constructs for creation of a single VH containing heavy chain locus were constructed using GENE® genetic engineering technology (see, e.g.. US Pat. No. 6,586,251 and Valenzuela, D.M. et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnology 21 (6): 652-659).

Construction of a Human VH1-69 Restricted Heavy Chain Locus. Brieﬂy, four modifications were performed using human BAC DNA to create a targeting uct containing a human VH1-69 gene segment with all the human DH and JH segments (. In the ﬁrst modiﬁcation, a modiﬁed human BAC containing multiple distal (5’) human VH gene segments, including VH1-69, an upstream hygromycin selection cassette and a 5’ mouse homology arm was targeted with a second spectinomycin cassette, which also contained a modiﬁed recombination signal sequence (RSS; BHR 1, top left). This d recombination signal sequence (RSS) uced two point mutations (T to A and G to A) in the 3’ RSS region of the human VH1-69 gene changing the RSS nonamer to the optimal consensus sequence.

Thus, the ﬁrst modiﬁcation (BHR 1) created a human genomic fragment containing the human VH1-69 gene t with a modified 3’ RSS, a unique AsiSl restriction site about 180 bp downstream of the RSS and a spectinomycin cassette ( middle left).

The second modification (BHR 2) included the use of a neomycin (Neo) cassette flanked by Frt sites to delete the hygromycin cassette and 5’ human VH gene segments upstream of the VH1-69 gene segment. This modiﬁcation was targeted 5’ to the human VH1-69 gene t to leave intact about 8.2 kb of the promoter region of human VH1-69 and the 5’ mouse homology arm ( bottom left).

The third modiﬁcation (BHR 3) included another spectinomycin cassette ﬂanked by uniquely engineered 5’ Pl-Scel and 3’ AsiSI sites ed to a human genomic fragment containing the ﬁrst three functional human VH gene segments and all the human DH and JH gene segments ( middle right). The human genomic fragment was previously targeted with a neomycin te and contained 5’ and 3’ homology arms containing the mouse genomic sequence 5’ and 3’ of the endogenous heavy chain locus including the 3’ intronic enhancer and the lgM gene. This modiﬁcation deleted the 5’ mouse genomic sequence and human VH gene segments, leaving about 3.3 kb of the VH-DH intergenic region upstream of the human DH1-1 gene segment, all of the human DH and JH ts, and the 3’ mouse genomic fragment containing the 3’ ic enhancer and the lgM gene ( bottom right).

The fourth modification was ed by employing the unique PI-Scel and AsiSl sites (described above) to Iigate the two modified BACs from BHR 2 and BHR 3 ( bottom center), which yielded the final targeting construct. The ﬁnal targeting construct for the creation of a modified heavy chain locus containing a single human VH gene segment and all the human DH and JH gene segments in ES cells contained, from 5’ to 3’, a 5’ homology arm containing about 20 kb of mouse genomic sequence upstream of the endogenous heavy chain locus, a 5’ Frt site, a in cassette, a 3’ Fri site, about 8.2 kb of the human VH1-69 promoter, the human VH1-69 gene segment with a modified 3’ RSS, 27 human DH gene segments, six human JH segments, and a 3’ homology arm containing about 8 kb of mouse genomic sequence downstream of the mouse JH gene segments ing the 3’ intronic enhancer and lgM gene ( bottom). The Human VH1-69 Targeting Vector (SEQ ID NO: 3) was linearized and electroporated into mouse ES cells heterozygous for a deletion of the endogenous heavy chain locus.

Construction of a Human VH1-2 Restricted Heavy Chain Locus. Using the steps described above, other polymorphic VH gene segments in the context of mouse heavy chain constant regions are employed to construct a series of mice having a restricted number globulin heavy chain V segments (e.g., 1, 2, 3, 4, or 5), wherein the V segments are polymorphic variants of a V gene family member. Exemplary polymorphic VH gene segments are derived from human VH gene segments including, e.g., VH1-2, VH2-26, VH2-70 and VH3-23.

Such human VH gene segments are obtained, e.g., by de novo synthesis (e.g., Blue Heron hnology, Bothell, WA) using ces available on hed databases. Thus, DNA fragments encoding each VH gene are, in some embodiments, ted independently for incorporation into targeting vectors, as bed herein. In this way, multiple modiﬁed immunoglobulin heavy chain loci comprising a restricted number of VH gene segments are engineered in the t of mouse heavy chain constant regions. An exemplary targeting strategy for creating a restricted humanized heavy chain locus containing a human VH1-2 gene segment, 27 human DH gene segments, and six human JH gene segments is shown in Brieﬂy, a modified human BAC clone containing three human V... gene ts (VHS-1, VH1-2, VH1-3), 27 human DH gene segments, and six human JH gene segments (see USSN 13/404,075; ﬁled 24 February 2012, herein incorporated by reference) is used to create a restricted humanized heavy chain locus containing a human VH1-2 gene segment. This modified BAC clone functionally links the aforementioned human heavy chain gene segments with the mouse intronic enhancer and the [9M constant region. The restricted human VH1-2 based heavy chain locus is achieved by two homologous recombinations using the modified human BAC clone described above.

For the ﬁrst homologous recombination, 205 bp of the human VHS-1 gene segment (from about 10 bp upstream (5’) of the VHS—1 start codon in exon 1 to about 63 bp ream (3') of the beginning of exon 2) in the modiﬁed human BAC clone is d by bacterial homologous recombination using a spectinomycin (aadA) cassette d by unique Pl-Scel restriction sites ( BHR 1). This allows for subsequent removal of the aadA cassette without ting other human gene segments within the restricted heavy chain locus.

For the second homologous ination, the 5’ end of the modified human BAC clone including the entire human VH1-3 gene segment and about 60 bp downstream (3’) of the gene segment is d by homologous recombination using a hygromycin cassette containing ﬂanking 5' AsiSl and 3’ Ascl restriction sites ( BHR 2). As described above, the spectinomycin cassette is optionally d after confirmation of the final targeting vector including deletion of the two human VH gene segments ﬂanking the human VH1-2 gene segment ( bottom). An exemplary human VH1-2 targeting vector is set forth in SEQ ID NO: 70. ing polymorphic VH gene segments in a cted immunoglobulin heavy chain locus represents a novel approach for generating antibodies, populations of antibodies, and populations of B cells that express antibodies having heavy chains with diverse CDRs derived from a single human VH gene segment. ting the somatic hypermutation machinery of the host animal along with combinatorial association with rearranged human immunoglobulin light chain variable s results in the engineering of unique heavy chains and unique VHNL pairs that expand the immune repertoire of genetically modiﬁed animals and enhance their usefulness as a next generation platform for making human therapeutics. ally useful as a rm for making neutralizing antibodies specific for human pathogens.

Thus, using the strategy outlined above for incorporation of additional and/or other polymorphic V... gene segments into the mouse immunoglobulin heavy chain locus allows for the generation of novel antibody repertoires for use in neutralizing human pathogens that might othenivise ively evade the host immune system.

Targeted ES cells bed above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (supra). Mice bearing a humanized heavy chain locus containing a single human VH gene segment, all the human DH and JH gene segments operably linked to the mouse immunoglobulin constant region genes were identiﬁed by genotyping using a modiﬁcation of allele assay (Valenzuela et al., supra) that detected the presence of the neomycin cassette, the human VH gene segment and a region within the human DH and JH gene segments as well as endogenous heavy chain sequences.

Table 4 sets forth the primers and probes used in this assay to confirm mice harboring a restricted heavy chain locus containing a single human VH1-69 gene segment, 27 human DH gene segments and six human JH gene segments.

Mice bearing an engineered heavy chain locus that ns a single human VH gene segment can be bred to a FLPe deletor mouse strain (see, e.g., Rodriguez, C.l. et al. (2000) High-efficiency deleter mice show that FLPe is an alternative to Cre-onP. Nature Genetics 25: 139-140) in order to remove any Frt’ed neomycin cassette introduced by the targeting vector that is not removed, e.g., at the ES cell stage or in the embryo. Optionally, the neomycin cassette is retained in the mice.

Pups are genotyped and a pup heterozygous for a humanized heavy chain locus containing a single human VH gene segment, all the human DH and JH ts operably linked to the endogenous mouse immunoglobulin constant genes is selected for characterizing the immunoglobulin heavy chain repertoire.

Table 4 Name sequence (5 '3), , SEQ ID SRegion Detected) NO: hyg Forward: TGCGGCCGAT CTTAGCC 4 (hygromycin Reverse: TTGACCGATT GG 5 cassette) Probe: ACGAGCGGGT TCGGCCCATT C 6 neo Forward: GAGG CTATTCGGC 7 (neomycin Reverse: GAACACGGCG GCATCAG 8 cassette) Probe: TGGGCACAAC AGACAATCGG CTG 9 $31220 Forward: TCCTCCAACG ACAGGTCCC 10 H _ JH Reverse: CTGA CGGGCACAGG 11 99mm Probe: TCCCTGGAAC TCTGCCCCGA CACA 12 sequence) 77n3 Forward: CTCTGTGGAA AATGGTATGG AGATT 13 (human VH1-69 Reverse: GGTAAGCATA GAAGGTGGGT ATCTTT 14 gene segment) Probe: ATAGAACTGT CATTTGGTCC AGCAATCCCA 15 $233370 Forward: TGGTCACCTC CAGGAGCCTC 16 H _ J H Reverse: GGGT GTATCAGGTG c 17 gemm'.c Probe: AGTCTCTGCT rcr GAGC 18 sequence) 8871‘" d- GATGGGAAGA TAAC. ATTTGTAC 19 (mouse 3, VH . e: TATT TCACTCTTTG AGGCTC 20 3:232:26) Probe: CCTCCACTGT GTTAATGGCT GCCACAA 21 mlng10 Forward: GGTGTGCGAT GTACCCTCTG AAC (mouse 5' vH Reverse: TGTGGCAGTT TAATCCAGCT TTATC g: genomic Probe: CTAAAAATGC TGGG GCAAAACACC sequence) TG m'ng2 Forward: GCCATGCAAG C 25 (muse: JH Reverse: AGTTCTTGAG CCTTAGGGTG CTAG 26 22333;) Probe: CCAGGAAAAT GCTGCCAGAG CCTG 27 Example 2 Characterization of Mice Expressing Heavy Chains Derived From a Single Human VH Gene Segment Mice homozygous for a single human VH gene segment at the endogenous heavy chain locus as described in Example 1 were evaluated for expression and B cell development using flow cytometry. 9] Briefly, spleens and bone marrow was harvested from wild type (n=3 per group; six weeks old, male and female) and mice homozygous for a single human VH gene segment, all human DH and JH gene segments operably linked to mouse heavy chain constant regions. Red blood cells from spleens were lysed with ACK lysis buffer (Lonza Walkersville), followed by washing with complete RPMl medium. 0] Flow cytometry. Cells ) were incubated with anti-mouse CD16/CD32 (2.462, BD PHARMINGEN'") on ice for 10 minutes, followed by labeling with the following antibody panels for 30 minutes on ice. Bone marrow panel: anti-mouse FlTC-CD43 (1 B1 1, BioLegend), PE-ckit (288, BlOLEGEND®), PeCy7—IgM (II/41, EBIOSCIENCE®), PerCP-Cy5.5- lgD (11-26c.23, BlOLEGEND®), APC—eFluor 780—8220 (RA3-6BZ, IENCE®), APC- CD19 1, EBIOSCIENCE®). Bone marrow and spleen panel: anti-mouse FITC—lgx (187.1, BD ences), PE-lg)» (RML-42, BlOLEGEND®), PeCy7-lgM (ll/41, EBIOSCIENCE®), PerCP-Cy5.5-lgD c.2a, BlOLEGEND®), Paciﬁc Blue-CD3 (17A2, BlOLEGEND®), AFC-8220 (RA3-682, EBIOSCIENCE®), APC-H7-CD19 (lD3, BD Biosciences). Bone marrow: immature B cells “lgM*), mature B cells (B220hilgM*), pro B cells (CD19‘ckit’CD43’), pre B cells (CD19+ckit’CD43'), immature lgic+ B cells (BZZO‘n‘IgM’ngWgA‘), immature lg)»+ B cells "‘lgM"lgi<‘lg}t’), mature lgx" B cells (8220“ilgM’lgflgA'), mature lg)»+ B cells (8220"‘lgM*lgx‘lgiﬁ). Spleen: B cells (CD19‘), mature B cells (CD19‘lgDh‘lgMin’), transitional/immature B cells (CD19‘IgD‘mlth‘). Bone marrow and spleen: lgx" B cells (CD19‘IgK*lg)C), lg)»+ B cells (CD19‘lgx‘lgA‘). ing staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a LSRIl flow cytometer and analyzed with TM software (Tree Star, lnc.). Results for the splenic compartment are shown in Fle. 3. 4A and 5 — 7.

Results for the bone marrow compartment are shown in FIGS. 48 and 8 — 11B.

Human VH Expression. Expression of the human VH1-69 gene segment was determined for mice heterozygous and homozygous for a human VH1-69 gene segment, all human DH and JH gene segments operably linked to mouse heavy chain constant regions by a tative PCR assay using TAQMAN® probes.

Briefly, CD19+ B cells were ed from the s of groups of mice (n=3 per group) using mouse CD19 microbeads (Miltenyi Biotec) according to manufacturer’s specifications. Total RNA was purified using the RNEASYTM Mini kit (Qiagen) and genomic RNA was d using an RNase-free DNase on-column treatment (Qiagen). About 200 ng mRNA was reverse-transcribed into cDNA using the First Stand cDNA Synthesis kit (lnvitrogen), followed by ampliﬁcation with the TAQMAN® Universal PCR Master Mix ed Biosystems) using the ABI 7900 Sequence Detection System (Applied Biosystems). Unique primer/probe combinations were employed to speciﬁcally determine expression of human VH1- 69-derived heavy chains (Table 5). Relative expression was normalized to the mouse x constant region (mCx). The results are shown in FlG. 12.

Table 5 Name Sequence ) SEQ ID NO: Sense: AACTACGCAC TCCA GG 28 hlgHV1-69 ense: GCTCGTGGAT TTGTCCGC 29 Probe: CAGAGTCACG ATTACC 30 Sense: TGAGCAGCAC CCTCACGTT 31 mCK Antisense: GTGGCCTCAC AGGTATAGCT GTT 32 Probe: ACCAAGGACG AGTATGAA 33 Example 3 Humoral Immune Response in Mice Expressing Heavy Chains Derived From a Single Human VH Gene Segment 4] The humoral immune response was determined for mice homozygous for human heavy and x light chain variable gene loci (Hx) and mice homozygous for a single human VH gene segment, all human DH and JH gene segments operably linked to mouse heavy chain constant regions (1 hVH HO) by comparative immunization using a human cell surface receptor (Antigen A). zation. Serum was collected from groups of mice prior to immunization with the above n. Antigen (2.35 pg each) was administered in an initial priming zation mixed with 10 ug of CpG oligonucleotide (lnvivogen) and 25 ug of Adju-phos (Brenntag) as adjuvants. The immunogen was administered via footpad (f.p.) in a volume of 25 pl per mouse.

Subsequently, mice were boosted via f.p. with 2.3 ug of antigen along with 10 ug CpG and 25 ug Adju-Phos as adjuvants on days 3, 6, 11, 13, 17, and 20 for a total of six boosts. Mice were bled on days 15 and 22 after the fourth and sixth boosts, respectively, and antisera were assayed for antibody titers to Antigen A. 6] Antibody titers were ined in sera of immunized mice using an ELISA assay.

Ninety six-well microtiter plates (Thermo Scientific) were coated with Antigen A (1 ug/ml) in phosphate-buffered saline (PBS, Irvine Scientific) overnight at 4°C. The following day, plates were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T, Sigma- AIdrich) four times using a plate washer (Molecular Devices). Plates were then blocked with 250 pl of 1% bovine serum albumin (BSA, Aldrich) in PBS and incubated for one hour at room ature. The plates were then washed four times with PBS-T. Sera from immunized mice and pre-immune sera were serially diluted ten—fold in 0.1% BSA PBS-T starting at 1:100 and added to the blocked plates in duplicate and incubated for one hour at room temperature.

The last two wells were left blank to be used as secondary antibody control. The plates were again washed four times with PBS-T in a plate . A 1:5000 dilution of goat anti-mouse -Horse Radish Peroxidase (HRP, Jackson research) ated secondary dy was added to the plates and incubated for one hour at room temperature. Plates were again washed eight times with PBS-T and developed using TMB/H202 as substrate. The substrate was incubated for twenty minutes and the reaction stopped with 1 N H2804 (VWR).

Plates were read on a spectrophotometer (Victor, Perkin Elmer) at 450 nm. Antibody titers were calculated using GRAPHPAD PRISMTM (GraphPad Software, Inc).

Serum titer was calculated as serum dilution within experimental titration range at the signal of antigen binding lent to two times above background. Antibody titer for the humoral immune response against a human cell surface receptor (Antigen A) is set forth in FIG.

In a similar experiment, humoral immune responses were determined for mice homozygous for human heavy and K light chain variable gene loci (HK) and mice homozygous for a single human VH gene segment, all human DH and JH gene segments operably linked to mouse heavy chain constant regions (1 hVH HO) by comparative immunization using influenza viral vaccines FLUVIRIN® (Novartis Vaccines) and FLUMIST® (Medlmmune LLC). 9] Brieﬂy, serum was collected from groups of mice prior to immunization with the above antigen (as described above). Mice (n=5) homozygous for a single human VH gene t (VH1-69), all human DH and JH gene segments operably linked to mouse heavy chain constant regions (1 hVH HO) were immunized intra-nasally (i.n.) with FLUMIST® (live attenuated inﬂuenza vaccine) at 1/3 the normal dose/mouse. One normal dose of FLUMIST® contains ‘5'5'7'5 FFU (ﬂuorescent focus units) of live attenuated inﬂuenza vaccine. Therefore, each mouse was primed with 70 pl FLUMIST® on day 1 ed by in boost on days 3, 6, 11, 13. 17, 20 for a total of 6 . No adjuvants were employed in this immunization. The mice were bled on days 15 and 22 after 4th and 6th boosts tively and antiserum assayed for dy titers to T® (as described above).

In a similar manner. in immunizations with FLUVIRIN®, pre-immune serum was collected from mice prior to initiation of immunization. Mice (n=5) homozygous for a single human VH gene segment (VH1-69), all human DH and JH gene segments operably linked to mouse heavy chain constant regions (1 hVH HO) were immunized with IN® (trivalent inactivated nza vaccine) via footpad (f.p.) with 0.75 pg each of hemagglutinin/mouse/boost. Mice were primed on day 1 followed by f.p. boost on days 3, 6, 11, 13, 17, 20 for a total of 6 boosts. No adjuvants were employed in this zation. The mice were bled on days 15 and 22 after 4th and 6th boosts respectively and antiserum assayed for antibody titers to FLUVIRIN® (as described above).

Serum titer was calculated as serum on within experimental titration range at the signal of antigen binding equivalent to two times above background. Antibody titer for the humoral immune response against FLUMIST® and FLUVIRIN® is set forth in .

As shown in this Example, antibody titers generated in 1hVH HO mice were comparable to those generated in mice having a plurality of human VH gene segments (Hx) for both a human cell surface receptor and a viral antigen (e.g., inﬂuenza). Thus, mice having immunoglobulin heavy chain loci restricted to a single VH gene segment are capable of ng a robust immune response to antigen in a manner comparable to mice having immunoglobulin heavy chain loci containing a ity of human VH gene segments (e.g., 8O VH).

Example 4 Analysis of dy Gene Usage and CDR3 Length in Mice Having a Restricted lmmunoglobulin Heavy Chain Locus Splenocytes ted from mice homozygous for a single human VH gene segment at the endogenous heavy chain locus and homozygous for a replacement of the endogenous K light chain variable loci with human x light chain variable loci immunized with a human cell surface receptor (Antigen A) were analyzed for heavy and light chain gene segment usage by e-transcriptase polymerase chain reaction (RT-PCR) on mRNA from splenic B cells.

Brieﬂy, spleens were harvested and homogenized in 1xPBS (Gibco) using glass slides. Cells were pelleted in a centrifuge (500xg for 5 minutes), and red blood cells were lysed in ACK Lysis buffer (Gibco) for 3 minutes. Cells were washed with 1xPBS and d using a 0.7pm cell strainer. B-cells were isolated from spleen cells using MACS magnetic positive selection for CD19 (Miltenyi Biotec). Total RNA was isolated from pelleted B-cells using the RNeasy Plus Kit (Qiagen). PolyA” mRNA was isolated from total RNA using the Oligotex® Direct mRNA mini kit (Qiagen).

] Double-stranded cDNA was prepared from splenic B cell mRNA by 5’ RACE using the SMARTerTM Pico cDNA Synthesis Kit (Clontech) with substitution of the supplied reverse transcriptase and dNTPs with Superscript® II and dNTPs (lnvitrogen). VH and VK antibody repertoires were amplified from the cDNA using primers c for lgM, lgG, or ng constant regions and the SMARTerTM 5’ RACE primer (Table 6). PCR products were puriﬁed using a QlAquick® PCR Puriﬁcation Kit (Qiagen). A second round of PCR was done using the same 5' RACE primer and a nested 3’ primer specific for the lgM, lgG, or lgx constant regions (Table 7).

Second round PCR products were puriﬁed using a SizeSelectTM E-Gel® system (lnvitrogen). A third PCR was performed with primers that added 454 adapters and barcodes. Third round PCR products were ed using Agencourt® AMPure® XP Beads (Beckman Coulter).

Purified PCR products were quantiﬁed by SYBR® qPCR using a KAPA Library Quantiﬁcation Kit (KAPA Biosystems). Pooled libraries were subjected to emulsion PCR (emPCR) using a 454 GS Junior Titanium Series Lib-A emPCR Kit (Roche Diagnostics) and bidirectional sequencing using Roche 454 GS Junior instrument according to manufacturer’s speciﬁcations.

Bioinformatic analysis. The 454 sequences were sorted based on the sample e t match and trimmed for quality. Sequences were annotated based on alignment of rearranged immunoglobulin sequences to human germline V(D)J segment database using local installation of lgblast (NCBI, v2.2.25+). A sequence was marked as ambiguous and d from analysis when multiple best hits with cal score were detected. A set of per! scripts was developed to analyze results and store data in mysql database. CDR3 region was deﬁned between conserved C codon and FGXG motif for light and WGXG motif for heavy chains. CDR3 length was determined using only productive antibodies. From the nucleic acid sequences and predicted amino acid ces of the antibodies, gene usage was identiﬁed for lgM-primed (15,650), lgG-primed (18,967). and IgK- primed 4) sequences. Results are shown in Table 8. Table 9. and . 7] Table 8 sets forth the percentage of observed human DH and JH gene segments used among lgM-primed (15,650 sequences) and lgG-primed (18,967 sequences) VH1-69 derived heavy chain variable region sequences. Human DH4-11 and human DH5-5/DH5- 18 gene segments are ted in Table 8 together due to identical sequence identity between the respective pairs of DH gene segments. Table 9 sets forth the percentage of human VK and JK gene ts observed among light chains (26,804 sequences) cognate with VH1-69 derived heavy chain variable regions. Percentages in Tables 8 and 9 represent rounded values and in some cases may not equal 100% when added together.

Amino acid length of the CDR3 region of imed -derived heavy chains is shown in . Amino acid length of the CDR3 region of lgG-primed VH1derived heavy chains is shown in .

As shown in Tables 8 and 9, mice according to the invention generate antigen- speciﬁc antibodies ning VH1derived heavy chains, which demonstrate a variety of rearrangements of a human VH1-69 gene segment with a variety of human DH segments and human JH segments. r, the antigen-speciﬁc antibodies contain cognate human light chains containing human VK domains resulting from a variety of rearrangements of human VK and JK gene segments.

Table 6 Primer Sequence (5’-3’) 3' 091 outer GGAAGGTGTG GCTG GAC (SEQ ID NO: 71) 3' CgZac outer GGAAGGTGTG CACACCACTG GAC (SEQ ID NO: 72) 3' Cga outer GGAAGGTGTG CACACTGCTG GAC (SEQ ID NO: 73) 3' 093 outer AGACTGTGCG CACACCGCTG GAC (SEQ ID NO: 74) 3' mlgM CH1 outer TCTTATCAGA CAGGGGGCTC TC (SEQ ID NO: 75) 3' mlng outer AAGAAGCACA CGACTGAGGC AC (SEQ ID NO: 76) Table 7 Primer Sequence (5'-3’) 3' mlgG1/2b CH1 inner AGTGGATAGA CWGATGGGGG TG (SEQ ID NO: 77) 3' mlgGZa/Zc CH1 inner AGTGGATAGA CCGATGGGGC TG (SEQ ID NO: 78) 3' mlgG3 CH1 inner AAGGGATAGA CAGATGGGGC TG (SEQ ID NO: 79) 3' mlgM CH1 inner CATT GGAC TG (SEQ ID NO: 80) 3' mngC-Z inner GGAAGATGGA TACAGTTGGT GC (SEQ ID NO: 81) Table 8 Human DH lgM lgG H lgM lgG 1-1 1.2 6.0 1 7.5 1.5 1-7 39.9 9.0 2 3.3 4.2 1-14 0.5 2.3 3 22.2 12.8 1-20 2.3 1.4 4 51.5 36.4 1-26 3.5 5.7 5 10.5 9.5 2-2 1.1 3.2 6 4.9 29.4 2-8 0.7 0.6 2-15 0.3 1.2 2—21 0.7 0.3 3-3 6.3 5.2 3-9 0.6 0.6 3-10 0.9 10.3 3-16 0.9 2.0 3-22 5.1 2.7 4-4/4-11 1.5 4.0 4-17 1.5 4.7 4-23 11.5 2.4 -12 1 1 1.8 -5/5-18 1.3 3.2 -24 0.3 3.3 6-6 1 8 4.5 6-13 6 1 7.4 6-19 3.0 5.1 6-25 0.1 0.6 7-27 3.3 7.3 Table 9 Human Vx % ed Human JK % Observed 1-5 3.4 1 28.1 1-6 1.3 25.3 1-8 0 12.1 1-9 1.3 22.5 1-12 1.0 U'IbOON 11.1 1-13 0 1-16 2.5 1-17 3.6 1-22 0 1-27 0.5 1-32 0 1-33 14.3 1-35 0 1-37 0 1-39 1.6 2-4 0 2-10 0 2-14 0 2-18 0 2-19 0 2-23 0 2-24 0.7 2-26 0 2-28 0 229 0 2-30 1.9 2-36 0 2-38 0 2-40 1.5 3-7 0 3-11 2.7 3-15 3.9 3-20 41.2 3-25 0 3-31 0 3-34 0 4-1 13.2 -2 0.1 6-21 0 7-3 0

Claims

We claim:

1. A rat or mouse having in its germline genome a restricted endogenous immunoglobulin

heavy chain locus terized by the presence of a single unrearranged human V H gene

segment, one or more unrearranged human DH gene segments, and one or more unrearranged

human J H gene segments operably linked to a non-human constant region gene ce

comprising a non-human IgM gene,

wherein the rat or mouse further comprises a diverse repertoire of rearranged human

immunoglobulin heavy chain variable region genes, each of which is linked to the non-human

constant region gene ce sing a non-human IgM gene and is derived from the

restricted immunoglobulin heavy chain locus.

2. The rat or mouse of claim 1, n the rat or mouse comprises a deletion of all or

substantially all endogenous VH, DH, and J H gene segments at the nous immunoglobulin

heavy chain locus.

3. The rat or mouse of claim 1 or 2, wherein the single unrearranged human V H gene

segment is selected from V H 1-2, VH1-69, V H2-26, VH2-70, , or a polymorphic variant of

any of VH1-2, VH 1-69, VH2-26, VH2-70, and VH3-23, capable of rearranging and forming a

rearranged heavy chain variable domain with one or more DH segments and one or more JH

segments.

4. The rat or mouse of any one of claims 1 to 3, n the single unrearranged human

VH gene segment is V H1-69 or a polymorphic t thereof capable of rearranging and

forming a rearranged heavy chain variable domain with one or more DH segments and one or

more J H segments.

5. The rat or mouse of claim 3, wherein the single unrearranged human V H gene segment

is VH1-2 or a polymorphic variant thereof capable of rearranging and forming a rearranged

heavy chain variable domain with one or more DH segments and one or more JH segments.

6. The rat or mouse of any one of claims 1 to 5, wherein the single unrearranged human

VH gene segment is operably linked to a rodent immunoglobulin constant region gene

sequence comprising a rodent IgM gene.

7. The rat or mouse of any one of claims 1 to 6, wherein the immunoglobulin constant

region gene is a rat or mouse constant region gene sequence.

8. The rat or mouse of any one of claims 1 to 7, further comprising one or more

unrearranged human globulin V L gene segments operably linked to one or more

unrearranged human JL gene segments.

9. The rat or mouse of claim 8, wherein the one or more unrearranged human V L gene

segments and/or the one or more unrearranged human J L gene segments are selected from

human lc and human X gene segments.

10. The rat or mouse of claim 8 or 9, wherein the one or more unrearranged human

immunoglobulin V L gene segments and one or more unrearranged human J L gene segments

are operably linked to a man light chain constant gene.

11. The rat or mouse of claim 10, wherein the non-human light chain constant gene is

selected from a mouse or rat x or A, constant region gene.

12. The rat or mouse of any one of claims 1 to 11, wherein all or substantially all

endogenous VH, DH, and J H gene segments at the endogenous immunoglobulin heavy chain

locus is replaced with the single human V H gene segment, the one or more unrearranged

human DH gene segments and the one or more unrearranged human J H gene segments.

13. The rat or mouse of claim 12, r comprising a replacement at an nous

immunoglobulin light chain locus of all or substantially all endogenous V L and J L gene

segments with one or more unrearranged human V L and one or more unrearranged human J L

gene segments.

14. The rat or mouse of claim 12, wherein the unrearranged single human V H gene segment

is selected from a human V H 1-69 and a human VH 1-2 gene segment.

15. A cell or tissue derived from the rat or mouse of any one of claims 1 to 14.

16. A method of making a nucleic acid sequence that s a human immunoglobulin

heavy chain le domain comprising

amplifying a nucleic acid comprising a rearranged human immunoglobulin heavy chain

variable region gene from a lymphocyte of a rat or mouse of any one of claims 1 to 14, or a

hybridoma produced from the lymphocyte,

wherein the lymphocyte comprises one of the diverse repertoire of rearranged human

immunoglobulin heavy chain variable region genes.

17. The method of claim 16, n the unrearranged human immunoglobulin heavy chain

variable region gene is derived from a human V H1-69 gene t that comprises a

sequence at least 75% identical to SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID

NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,

SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58.

18. The method of claim 16, wherein the nged human immunoglobulin heavy chain

variable region gene is derived from a human VH1-69 gene segment that comprises a

sequence comprising SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ

ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,

SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, or a polymorphic t thereof,

wherein the polymorphic variant is capable of rearranging and forming a rearranged heavy

chain le domain with one or more DH segments and one or more JH segments.

1 9.? The method of claim 16, wherein the ranged human immunoglobulin heavy chain

variable region gene comprises a sequence encodes a protein that is at least 75% identical

with SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ

ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,

SEQ ID NO: 57 or SEQ ID NO: 59.

20. The method of claim 16, wherein the rearranged human immunoglobulin heavy chain

variable region gene is derived from a human V H 1-2 gene segment that comprises a sequence

at least 95% identical to SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 or

SEQ ID NO: 68.

21. The method of claim 20, wherein the unrearranged human globulin heavy chain

variable region gene is d from a human V H 1-2 gene segment that comprises a sequence

comprising SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68.

or a polymorphic variant thereof, wherein the polymorphic variant is capable of rearranging and

more JH segments.

22. The method of claim 16, wherein the rearranged human immunoglobulin heavy chain

le region gene comprises a sequence that encodes a protein that is at least 95% identical

with SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 or SEQ ID NO: 69.

23. Use of a rat or mouse according to any one of claims 1 to 14 to make a nucleic acid

sequence encoding a human heavy chain variable domain.

24. Use according to claim 23, wherein the human heavy chain variable domain is

characterized by having human FR1-CDR1-FR2-CDR2-FR3 ces derived from a

polymorphic human VH gene segment capable of rearranging and forming a rearranged heavy

chain variable domain with one or more DH segments and one or more J H segments.

25. Use according to claim 23 or 24, wherein the human VH gene segment is selected from

a human VH 1-2, VH 1-69, VH2-26, V H2-70, or VH3-23 gene segment.

26. Use according to claim 25, wherein the human V H gene segment is V H 1-2.

27. Use according to claim 25, wherein the human V H gene segment is VH 1-69.

28. Use of a rat or mouse according to any one of claims 1 to 14 to make a human

antibody, wherein the human antibody comprises a heavy chain variable domain derived from a

rearranged human V H 1-2 gene segment, human V H 1-69 gene segment, or polymorphic variant

thereof, wherein the polymorphic variant is capable of rearranging and forming a rearranged

heavy chain variable domain with the one or more DH segments and the one or more J H

29. Use according to claim 28, wherein the nged human V H 1-69 gene segment is at

least 75% identical to SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ

SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58, wherein the rearranged human V H1-69

gene t is capable of rearranging and forming a rearranged heavy chain variable domain

with the one or more DH segments and the one or more JH ts.

30. Use ing to claim 28, wherein the rearranged human V H1-2 gene t is at

least 95% cal to SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 or

SEQ ID NO: 68, wherein the rearranged human V H 1-2 gene segment is capable of rearranging

and forming a nged heavy chain variable domain with the one or more DH segments and

the one or more JH segments.

31. An antigen binding protein made in a rat or mouse of any one of claims 1 to 14.

32. The cell of claim 15, wherein the cell is a lymphocyte or a oma produced from the

cyte.

33. The method of any one of claims 16 to 22, further comprising immunizing the rat or

mouse with an antigen of interest before amplifying the nucleic acid.

34. The method of any one of claims 16 to 22 and 33, wherein the rearranged human

immunoglobulin VH region gene sequence comprises at least one somatic hypermutation.

35. The method of any one of claims 16 to 22, 33, and 34, wherein the lymphocyte

specifically binds an antigen of interest.

36. A nucleic acid sequence comprising a ce that encodes the human

immunoglobulin heavy chain variable domain made according to the method of any one of

claims 16 to 22 and 33 to 35.

37. The nucleic acid sequence of claim 36, wherein the sequence that encodes the human

immunoglobulin heavy chain variable domain is linked to a sequence that encodes a human

heavy chain constant domain.

38. A totipotent mouse cell for making a rat or mouse that es a human heavy chain

variable domain sing in its genome a restricted immunoglobulin heavy chain locus

characterized by the ce of a single unrearranged human VH gene segment, one or more

unrearranged human DH gene segments, and one or more unrearranged human JH gene

segments operably linked to a non-human heavy chain constant region gene sequence

comprising a non-human IgM gene.

39. The totipotent rat or mouse cell of claim 38, wherein the non-human heavy chain

constant region gene ce is a rat or mouse heavy chain constant region gene sequence.

40. The totipotent rat or mouse cell of claim 38 or claim 39, wherein the totipotent rat or

mouse cell is an embryonic stem cell.

41. A method of making a human immunoglobulin heavy chain variable domain sing

expressing in a host cell the nucleic acid made according to the method of any one of claims 16

to 22 and 33 to 35 or the nucleic acid of any one of claims 36 or 37.

42. A non-human host cell comprising the nucleic acid made ing to the method of

any one of claims 16 to 22 and 33 to 35 or the nucleic acid of any one of claims 36 or 37.

43. The non-human host cell of claim 42, wherein the cell is a man ian cell.

44. A method of obtaining a cell that expresses a human immunoglobulin heavy chain

variable domain comprising ting a lymphocyte from a non-human animal that comprises

in its germline genome a restricted immunoglobulin heavy chain locus characterized by the

presence of a single human unrearranged V H gene segment, one or more human unrearranged

DH gene segments, and one or more human unrearranged J H gene segments operably linked to

a non-human immunoglobulin constant region comprising a non-human IgNI gene, wherein the

lymphocyte expresses a rearranged human globulin VH region gene derived from the

restricted immunoglobulin heavy chain locus.

45. The method of claim 44, further comprising as last steps obtaining from the lymphocyte,

or a hybridoma produced from the lymphocyte, a first nucleotide sequence that comprises the

rearranged human immunoglobulin VH region gene sequence; and expressing in a host cell a

first c acid comprising a sequence identical to or substantially identical to the first

nucleotide sequence.

46. The method of claim 45, wherein the first nucleic acid is operably linked to a human

heavy chain constant region gene.

47. The method of claim 45 or claim 46, further comprising expressing in the cell a second

nucleic acid operably linked to a human light chain constant region gene, wherein the second

nucleic acid comprises a sequence identical to or substantially identical to a second nucleotide

sequence that encodes a human light chain le (V L) domain that is cognate to the human

immunoglobulin VH .

48. The method of any one of claims 44 to 47, further comprising expressing in the cell a

third nucleic acid operably linked to a human heavy chain nt region gene, wherein the

third nucleic acid encodes a second human immunoglobulin V H domain that is cognate to the

human immunoglobulin V L domain.

49.? The method of any one of claims 45 to 48, n the host cell is selected from a HeLa

cell, a DU145 cell, a Lncap cell, a MCF-7 cell, a MDA-MB-438 cell, a PC3 cell, a T47D cell, a

THP-1 cell, a U87 cell, a SHSY5y cell, a Saos-2 cell, a Vero cell, a CHO cell, a GH3 cell, a

PC12 cell, a human retinal cell, and a MC3T3 cell.

50.? The method of claim 44, further comprising producing a hybridoma from the ted

cyte.

51.? The method of any one of claims 44-50, wherein the single human unrearranged VH

gene segment the one or more human unrearranged DH gene segments, and the one or more

human unrearranged J H gene segments are ly linked to the nonhuman immunoglobulin

constant region at an endogenous heavy chain locus of the nonhuman animal.

52.? The method of any one of claims 44-51, n the non-human animal further

comprises one or more human immunoglobulin V L and one or more human immunoglobulin JL

gene segments operably linked to a non-human light chain constant region.

53.? The method of any one of claims 44-52, wherein the non-human animal comprises:

(a) a deletion of an endogenous immunoglobulin heavy chain variable locus and a

deletion of an endogenous K light chain variable locus, or

(b) a deletion of an endogenous immunoglobulin heavy chain variable locus and a

deletion of an endogenous X light chain variable locus.

54.? The method of any one of claims 44-53, n the non-human animal is a rodent

selected from the group consisting of a hamster, a rat and a mouse.

55.? A method of making a human immunoglobulin heavy chain variable domain comprising

culturing the cell ed according to of any one of claims 44 to 54 or the hybridoma of claim

56.? The rat or mouse according to claim 1, the cell or tissue according to claim 15, the

method according to claim 16, 44 or 55, the use according to claim 23, the antigen binding

protein according to claim 31, the nucleic acid sequence according to claim 36, the mouse cell

according to claim 38, the non-human host cell according to claim 42, substantially as

hereinbefore bed with reference to any of the Examples and/or