Non-human animals capable of DH-DH rearrangement in immunoglobulin heavy chain coding sequences

Abstract

To provide non-human animals capable of DH-DH rearrangement in immunoglobulin heavy chain coding sequences.SOLUTION: Non-human animals and methods and compositions of making and using the same are provided, wherein the non-human animals have a genome comprising engineered or recombinant diverse clusters within an immunoglobin heavy chain variable region, wherein the engineered or recombinant diverse clusters each comprise an insertion of one or more DH segments operably linked to 23mer recombination signal sequences. Methods of generating antibodies from non-human animals are also provided, wherein the antibodies optionally contain human variable regions and rodent, e.g., constant regions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(3) of U.S. Provisional Patent Applications Nos. 62 / 685,203 (filed June 14, 2018), 62 / 702,206 (filed July 23, 2018), and 62 / 812,580 (filed March 1, 2019), each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII-formatted Sequence Listing under the file name "10347_ST25.txt," which was created on June 13, 2019, is approximately 50 kilobytes in size, and is being prepared concurrently with the specification. The Sequence Listing contained in this ASCII-formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Monoclonal antibody products have revolutionized the biopharmaceutical industry and have provided significant advances in the treatment of several diseases. Many of these monoclonal antibody products have exploited the natural properties of antibody molecules (i.e., traditional immunoglobulin gene segments) and, in some cases, incorporated other properties, such as labels (e.g., pegylation, radiolabeling) or conjugation with other drugs. At current approval rates, approximately 70 monoclonal antibody products are expected to be launched by 2020. Despite these advances and the knowledge gleaned from the use of monoclonal antibodies for therapeutic use, diseases remain that are bound by and / or associated with targets that are difficult for monoclonal antibodies to access, highlighting the need for different approaches to developing effective treatments. Summary of the Invention

[0004] The present invention encompasses the recognition that it is desirable to engineer non-human animals (e.g., rodents (e.g., rats, e.g., mice)) to establish additional in vivo systems for identifying and developing new antibody-based therapies, and in some embodiments, antibody agents (e.g., monoclonal antibodies and / or fragments thereof), that can be used for the treatment of various diseases. Furthermore, the present invention also provides methods for the identification and development of antibodies containing complementarity-determining regions 3 (CDR3s) characterized by longer amino acid lengths and diversity compared to wild-type (or reference) CDR3s, diversity that, in some embodiments, directs binding to specific antigens. H and D H One or more D engineered to be operably linked to a recombination signal sequence that allows for the reconstitution of H heavy chain diversity (D) engineered into an immunoglobulin heavy chain variable region (e.g., a heterologous immunoglobulin heavy chain variable region, e.g., a human immunoglobulin heavy chain variable region), including a segment H ) cluster (or manipulated D H In some embodiments, non-human animals described herein provide in vivo systems for the development of antibodies and / or antibody-based therapeutics for administration to humans.

[0005] As used herein, at least one immunoglobulin heavy chain diversity (D) operably linked to a 23-mer recombination signal sequence (RSS) is H ) a nucleotide molecule comprising a gene segment is described, optionally H The gene segment can be, for example, an engineered D of an immunoglobulin heavy chain variable region (e.g., a human or humanized immunoglobulin heavy chain variable region). H Thus, in some embodiments, the nucleotide molecules described herein may be located within at least one D operably linked to a 23mer (RSS). H Engineered immunoglobulin heavy chain variable diversity (D) gene segments containing H In some embodiments, the engineered D HThe region comprises (i) at least one D operably linked to a 23-mer RSS. H gene segment, and (ii) an unrearranged D flanked by a 12-mer RSS at one end and another 12-mer RSS at the other end. H a gene segment, (i) at least one D operably linked to a 23-mer RSS; H gene segment, and (ii) germline D flanked by a 12-mer RSS at one end and another 12-mer RSS at the other end. H The gene segments (i) and (ii) are D according to the 12 / 23 rule. H -D H They are operably linked so as to be able to participate in a recombination event. Also provided are targeting vectors, non-human animals (e.g., rodents (e.g., rats or mice)), and non-human animal cells (e.g., rodent cells (e.g., rat cells or mouse cells)) comprising the nucleotide molecules described herein, methods of using the nucleotide molecules described herein, and the like.

[0006] In some embodiments, a D operably linked to the 23mer RSS H The gene segment is a human D operably linked to a 23-mer RSS. H In some embodiments, the human D H The gene segment comprises at least 19 nucleotides and / or encodes two cysteines. H The gene segment comprises at least 20 nucleotides and / or encodes two cysteines. H The gene segment comprises at least 23 nucleotides and / or encodes two cysteines. H The gene segment comprises at least 28 nucleotides and / or encodes two cysteines. HThe gene segment comprises at least 31 nucleotides and / or encodes two cysteines. H In some embodiments, the gene segment comprises at least 37 nucleotides and / or encodes two cysteines. H The gene segment is a human D operably linked to a 23-mer RSS. H In some embodiments, the D H In some embodiments, the gene segment comprises at least 30 nucleotides and / or encodes two cysteines. H The gene segment is human D H 3-3 gene segment, D H 3-9 gene segment, D H 3-10 gene segments, D H 3-16 gene segment, D H 3-22 gene segment, human D H 2-2 gene segment, human D H 2-8 gene segments, or human D H D selected from the group consisting of 2-15 gene segments H In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H 3-3 gene segment, human D H 2-2 gene segment, human D H 2-8 gene segments, and human D H Human D selected from the group consisting of 2-15 gene segments H In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H 3-3 gene segment, human D H 2-2 gene segment, human D H 2-8 gene segments, and human D HHuman D selected from the group consisting of 2-15 gene segments H In some embodiments, the human D H The gene segment is human D H 3-3 gene segments. In some embodiments, the human D H The gene segment is human D H 2-2 gene segment. In some embodiments, the human D H The gene segment is human D H 2-8 gene segments. In some embodiments, the human D H The gene segment is human D H Contains 2-15 gene segments.

[0007] In some embodiments, a D operably linked to the 23mer RSS H The gene segment comprises (a) a human D operably linked to a 23-mer RSS; H 3-3 gene segments, possibly 23mer RSS, are used for human D H (b) a human D operably linked to a 23-mer RSS adjacent to the 5' end of the 3-3 gene segment; H 2-2 gene segments, possibly 23mer RSS, are H (c) a human D operably linked to a 23-mer RSS adjacent to the 3' end of the 2-2 gene segment H 2-8 gene segments, optionally 23mer RSSs, are used to identify human D H (d) a human 2-15 gene segment operably linked to a 23-mer RSS adjacent to the 3' end of the 2-8 gene segment, optionally the 23-mer RSS being a human D H 2-15 adjacent to the 3' end of the gene segment, or (e) any combination of (a) to (d).

[0008] In some embodiments, a nucleotide molecule described herein (e.g., a D operably linked to a 23-mer RSS) is provided. H Gene segment, engineered D HThe region (e.g., immunoglobulin heavy chain variable region) comprises a nucleotide sequence comprising the sequence set forth as SEQ ID NO:52.

[0009] In some embodiments, a nucleotide molecule described herein (e.g., a D operably linked to a 23-mer RSS) is provided. H Gene segment, engineered D H The region (e.g., immunoglobulin heavy chain variable region) comprises a nucleotide sequence comprising the sequence set forth as SEQ ID NO:61.

[0010] In some embodiments, a nucleotide molecule described herein (e.g., a D operably linked to a 23-mer RSS) is provided. H Gene segment, engineered D H The region (e.g., immunoglobulin heavy chain variable region) comprises a nucleotide sequence comprising the sequence set forth as SEQ ID NO:70.

[0011] In some embodiments, a nucleotide molecule described herein (e.g., a D operably linked to a 23-mer RSS) is provided. H Gene segment, engineered D H The region (e.g., immunoglobulin heavy chain variable region) comprises a nucleotide sequence comprising the sequence set forth as SEQ ID NO:71.

[0012] In some embodiments, a nucleotide molecule described herein (e.g., a D operably linked to a 23-mer RSS) is provided. H Gene segment, engineered D H The region (e.g., immunoglobulin heavy chain variable region) comprises a nucleotide sequence comprising the sequence set forth as SEQ ID NO:72.

[0013] In some embodiments, a D operably linked to the 23mer RSS H The gene segment is composed of a 23-mer RSS and a D H It contains a gene segment, e.g., a 23-mer RSS from 5' to 3', (human) D HA gene segment and a 12-mer RSS, for example, a 23-mer RSS, H adjacent to the 5' end of the gene segment, e.g., D H The gene segment is operably linked to a 23-mer RSS at the 5' end. In some embodiments, a D H The gene segment is a human D operably linked to a 23-mer RSS at the 5' end. H 3-3 gene segments, e.g., the nucleotide molecule includes a 23-mer RSS from 5' to 3', human D H 3-3 gene segments and a 12-mer RSS.

[0014] At least one D H An engineered D gene segment containing a 23-mer RSS at the 5' end. H The region is an unrearranged D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H It may further comprise a gene segment (e.g., an unrearranged D gene flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS). H The gene segment is germline D H A gene segment, e.g., D in its germline configuration H (including gene segments, etc.), unrearranged D H The gene segment comprises at least one D operably linked to the 23-mer at the 5' end. H In some embodiments, the unrearranged D is flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS. H The gene segment is an unrearranged human D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H Contains gene segments.

[0015] In some embodiments, a D operably linked to the 23mer RSS HThe gene segment is D from 5' to 3' H A gene segment and a 23-mer RSS, e.g., a 12-mer RSS from 5' to 3', (human) D H The gene segment and the 23-mer RSS, for example, H adjacent to the 3' end of the gene segment, e.g., D H The gene segment is operably linked to a 23-mer RSS at its 3' end, etc. In some embodiments, the D H The gene segment is a human D operably linked to a 23-mer RSS at the 3' end. H Contains two gene segments, D H 2 gene segments are human D H 2-2 gene segment, human D H 2-8 gene segments, and human D H In some embodiments, the RSS is selected from the group consisting of 2-15 gene segments. H The gene segment is operably linked from 5' to 3' to a 23-mer RSS at the 3' end of the human D H 2-2 gene segment, human D operably linked to a 23-mer RSS at the 3' end H 2-8 gene segment and human D operably linked to a 23-mer RSS at the 3' end H In some embodiments, the RSS comprises a 23-mer D operably linked to the 3' end of the RSS. H The gene segment contains, from 5' to 3', a 12-mer RSS, human D H 2-2 gene segment, and the first flanking nucleotide sequence containing a 23-mer RSS, a 12-mer RSS, human D H 2-8 gene segment, and a second flanking nucleotide sequence containing a 23-mer RSS, and a 12-mer RSS, human D H 2-15 gene segment, and a third flanking nucleotide sequence containing a 23-mer RSS.

[0016] At least one D operably linked to the 23-mer RSS at the 3' end H engineered D containing gene segments H The region is an unrearranged D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H It may further comprise a gene segment (e.g., an unrearranged D gene flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS). H The gene segment is germline D H A gene segment, e.g., D in its germline configuration H a 12-mer RSS at one end and another 12-mer RSS at the other end), H The gene segment comprises at least one D operably linked to the 23-mer at the 3' end. H In some embodiments, the unrearranged D is flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS. H The gene segment is an unrearranged human D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H Contains gene segments.

[0017] In some embodiments, the engineered D H The region comprises (i) one or more unrearranged human D H Gene segment, human D H Each of one or more of the gene segments is flanked at its 5' and 3' ends by a 12-mer RSS, and (ii) a human D gene segment operably linked at its 5' end to a 23-mer RSS. H Gene segments (e.g., human D H 3-3 gene segment) and at least one D operably linked to a 23mer RSS. HIn some embodiments, the engineered D-H regions described herein comprise, from 5' to 3', (i) at least one D-H region operably linked to a 23-mer RSS. H a gene segment, e.g., at least one human D operably linked at its 3' end to a 23-mer RSS; H 2 gene segments, optionally at least one human D operably linked at its 3' end to a 23-mer RSS. H The 2 gene segment is a human D gene operably linked at its 3' end to a 23-mer RSS. H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-15 gene segments, or any combination thereof; and (ii) one or more human D H gene segment, one or more human D H Each of the gene segments contains a 12-mer RSS flanked at its 5' and 3' ends.

[0018] In some embodiments, the engineered D H The region is human D H Contains only gene segments.

[0019] In some embodiments, the nucleotide molecules described herein (e.g., engineered D H region, immunoglobulin heavy chain variable region, etc.) is operably linked to the 23mer RSS H A gene segment and an unrearranged D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H D comprising a gene segment and operably linked to a 23-mer RSS H A gene segment and an unrearranged D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H A gene segment can be (i) a separate D Hgene segment, (ii) V H gene segment, (iii) J H In some embodiments, the nucleotide molecules described herein have not undergone recombination with (i) another D gene segment, or (iv) any combination thereof. H gene segment, (ii) V H gene segment, (iii) J H one or more D recombined with a gene segment, or any combination thereof H engineered gene segments containing D H Molecules containing gene regions include, for example, nucleotide molecules containing rearranged VDJ or VDDJ coding sequences that encode immunoglobulin heavy chain variable regions.

[0020] Thus, in some embodiments, the engineered D H The nucleotide molecules described herein, comprising a region, are operably linked to (a) an engineered D H upstream of and operably linked to the region, at least one unrearranged immunoglobulin heavy chain variable (V H ) gene segments (e.g., unrearranged human V H 6-1 gene segment), (b) engineered D H upstream of and operably linked to the J region, at least one unrearranged immunoglobulin heavy chain joining (J H ) gene segments (e.g., unrearranged human J H 6) Contains a gene segment or a combination of (a) and (b).

[0021] In some embodiments, at least one unrearranged V H The gene segments are unrearranged human V H 3-74 and unrearranged human V H Functional unrearranged human V spanning and including gene segments 1-6 H In some embodiments, at least one unrearranged VH The gene segments are in germline configuration and are unrearranged human V H 3-74 and unrearranged human V H Functional unrearranged human V spanning and including gene segments 1-6 H In some embodiments, the full repertoire of gene segments includes at least one unrearranged J H The gene segments are unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H In some embodiments, the at least one unrearranged J H The gene segments are unrearranged human J H 1 gene segment, unrearranged human J H 2 gene segments, unrearranged human J H 3 gene segments, unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H In some embodiments, the at least one unrearranged J H The gene segments are in germline configuration and are unrearranged human J H 1 gene segment, unrearranged human J H 2 gene segments, unrearranged human J H 3 gene segments, unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H It contains 6 gene segments.

[0022] In some embodiments, the nucleotide molecules described herein are selected from the group consisting of engineered D H immunoglobulin heavy chain variable (V) domain H) region, e.g., 5' to 3' in operable linkage: (a) at least one unrearranged immunoglobulin heavy chain variable (V H ) gene segments, (b) at least one D operably linked to the 23-mer RSS; H engineered D containing gene segments H region 、 (c) at least one unrearranged immunoglobulin heavy chain (J H ) gene segments, including

[0023] In some embodiments, (a) at least one unreconstructed V H The gene segment is (i) Unrearranged human V H 6-1 gene segment, (ii) unrearranged human V H 2-1 gene segment and unrearranged human V H 6-1 gene segment, and / or (iii) unrearranged human V H Human V unreconstructed from 3-74 H All functional unrearranged human Vs spanning the 6-1 gene segment and containing them H Gene segments, e.g., all functional unrearranged human V in germline configuration H The gene segment, possibly the rodent Adam6 gene, is a non-rearranged human V H 2-1 and V H 6-1, including substituting a pseudogene between gene segments; (b) Manipulated D H The region, from 5' to 3': (i) one or more, e.g., multiple, unrearranged human D H Gene segments, multiple unrearranged human D HEach of the gene segments is flanked at its 5' and 3' ends by a 12-mer RSS and operably linked at its 5' end to a 23-mer RSS. H gene segments, possibly multiple unrearranged human D H The gene segments are in germline configuration and are unrearranged human D H 1-1 gene segment and unrearranged human D H 1-26, including unrearranged human D H The human gene segment comprising the gene segment and / or operably linked at its 5' end to a 23-mer RSS is H 3-3 gene segments, e.g., engineered D H is operably linked to the 23-mer RSS at the 5' end of D H Unrearranged human D substituted at 3-3 H Unrearranged human D in germline configuration, except for the 7-27 gene segment H Contains a complete repertoire of gene segments; (ii) at least one human D operably linked at its 3' end to a 23-mer RSS; H gene segments, and one or more, e.g., multiple, human D H gene segment, one or more human D H Each gene segment is flanked at its 5' and 3' ends by a 12-mer RSS and, optionally, at its 3' end by at least one human D operably linked to a 23-mer RSS. H The gene segment is operably linked at its 3' end to a 23-mer RSS. H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-15 gene segments and / or one or more human D H The gene segments are unrearranged human D H1-1 gene segment and unrearranged human D H D spanning between 7-27 gene segments and including them H Gene segments, optionally engineered D H is unrearranged human D H 2-2, D H 2-8, and D H The 2-15 gene segment is operably linked at its 3' end to a 23-mer RSS. H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment and a human D operably linked at its 3' end to a 23-mer RSS. H Unrearranged human D in germline configuration, except for the 2-15 gene segment H Contains a complete repertoire of gene segments; (iii) or a combination of (b)(i) and (b)(ii); (c) at least one unreconstructed J H The gene segment is (i) Unrearranged human J H 6 gene segments, (ii) unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H 6 gene segments, and / or (iii) unrearranged human J H A complete repertoire of gene segments, e.g., unrearranged human J H 1 gene segment, unrearranged human J H 2 gene segments, unrearranged human J H 3 gene segments, unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H 6 gene segments, possibly unrearranged human JH 1. J H 2. J H 3. J H 4. J H 5, and J H In some embodiments, the immunoglobulin V gene segments are in a germline configuration. H Region (at least one functional V H Gene segment, engineered V H region, and at least one functional J H The gene segments are human immunoglobulin V H region, e.g., D operably linked to a 23mer RSS H Each gene segment (e.g., each V H , D H , and J. H gene segments) are human (V H , D H , or J H ) gene segment.

[0024] In some embodiments, the immunoglobulin V described herein H The nucleotide molecules comprising the region are operably linked 5' to 3' (a) At least one unrearranged human V H 6-1 gene segment, e.g., unrearranged human V H 3-74 and unrearranged human V H All unrearranged human V sequences spanning and including the 6-1 gene segment H All or part of a gene segment, for example, optionally two unrearranged human V H Between gene segments, there is a rodent Adam6 gene, e.g., (e.g., the rodent Adam6 gene is similar to human V H 1-2 gene segment and human V H functional unrearranged human V (located between the 6-1 gene segments) H Complete repertoire of gene segments (b) Unrearranged human D in germline configurationH 1-1 gene segment and unrearranged human D H 5' to 3' unrearranged human D spanning and including gene segments 1-26 H Human-engineered D containing gene segments H region, and unrearranged human D operably linked to the 23-mer RSS at the 5' end. H 3-3 gene segment, e.g., unrearranged human D operably linked to a 23-mer RSS at the 5' end H Unrearranged human D replaced by 3-3 gene segments H Unrearranged human D in germline configuration, excluding the 7-27 gene segment H a complete repertoire of gene segments, and (c) at least unrearranged human J H 6 gene segments, including

[0025] In some embodiments, the immunoglobulin V described herein H The nucleotide molecule comprising the region is operably linked, 5' to 3': (a) At least one unrearranged human V H 6-1 gene segment, e.g., unrearranged human V H 3-74 and unrearranged human V H All unrearranged human V sequences spanning and including the 6-1 gene segment H All or part of a gene segment, for example, optionally two unrearranged human V H Between gene segments, there is a rodent Adam6 gene, e.g., (e.g., the rodent Adam6 gene is similar to human V H 1-2 gene segment and human V H functional unrearranged human V (located between the 6-1 gene segments) H Complete repertoire of gene segments (b) Unrearranged human D in germline configuration H1-1 gene segment and unrearranged human D H 5' to 3' unrearranged human D spanning and including gene segments 1-26 H Human-engineered D containing gene segments H region, and unrearranged human D operably linked to the 23-mer RSS at the 5' end. H 3-3 gene segment, e.g., unrearranged human D operably linked to a 23-mer RSS at the 5' end H Unrearranged human D replaced by 3-3 gene segments H Unrearranged human D in germline configuration, excluding the 7-27 gene segment H a complete repertoire of gene segments, and (c) Unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J H 6 gene segments, including

[0026] In some embodiments, the immunoglobulin V described herein H The nucleotide molecules comprising the region are operably linked 5' to 3' (a) At least one unrearranged human V H 6-1 gene segment, e.g., unrearranged human V H 3-74 and unrearranged human V H All unrearranged human V sequences spanning and including the 6-1 gene segment H All or part of a gene segment, for example, optionally two unrearranged human V H Between gene segments, there is a rodent Adam6 gene, e.g., (e.g., the rodent Adam6 gene is similar to human V H 1-2 gene segment and human V H functional unrearranged human V (located between the 6-1 gene segments) H Complete repertoire of gene segments (b) 5' to 3', unrearranged human D H 1-1 gene segment, D operably linked at its 3' end to a 23-mer RSS H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-15 gene segment, and unrearranged human D H 3-16 gene segment and unrearranged human D H Unrearranged human D spanning and including gene segments 7-27 H Human-engineered D containing gene segments H Area, possibly manipulated D H is unrearranged human D H 2-2, D H 2-8, and D H The 2-15 gene segments each comprise a D operably linked at their 3' ends to a 23-mer RSS. H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment and a human D operably linked at its 3' end to a 23-mer RSS. H Unrearranged human D in germline configuration except for the replacement of the 2-15 gene segment H containing the complete repertoire of gene segments, and (c) unrearranged human J H A complete repertoire of gene segments, e.g., unrearranged human J H 1 gene segment, unrearranged J H 2 human gene segments, unrearranged J H 3 human gene segments, unrearranged J H 4 human gene segments, unrearranged J H 5 human gene segments, and unrearranged J H 6 human gene segments, optionally unrearranged human JH 1. J H 2. J H 3. J H 4. J H 5, and J H 6 gene segments are in germline configuration, including

[0027] In some embodiments, the nucleotide molecules described herein comprise, from 5' to 3', (a) at least one (human) V H (Human) immunoglobulin heavy chain variable (V) gene segments H ) region, the (human) engineered D H region, and (b) a heavy chain immunoglobulin constant region (C H ) or a portion thereof; H gene segments, optionally C H is a rodent C gene comprising a rodent intronic enhancer region, a rodent IgM gene, a rodent IgD gene, a rodent IgG gene, a rodent IgA gene, a rodent IgE gene, or any combination thereof. H In some embodiments, the nucleotide molecules described herein comprise, from 5' to 3', (a) at least one human V H Gene segments, human engineered D H region, and (b) at least a rodent intronic enhancer region, optionally including a rodent IgM gene. H at least one human J domain operably linked to the H Human immunoglobulin heavy chain V, including gene segments H In some embodiments, the nucleotide molecules described herein comprise, from 5' to 3', (a) at least one human V H Gene segments, human engineered D H region, and (b) at least a rodent intronic enhancer region, optionally including a rodent IgM gene. H at least one human J domain operably linked to the HHuman heavy chain V containing gene segments H In some embodiments, the nucleotide molecules described herein comprise, from 5' to 3', (a) at least one human V H Gene segments, human engineered D H area, and (b) endogenous rodent C H at least one human J region operably linked to, e.g., an endogenous rodent immunoglobulin heavy chain locus; H Human V, including gene segments H In some embodiments, the rodent may be a rat. In some embodiments, the rodent may be a mouse.

[0028] In some embodiments, the nucleotide molecules described herein further comprise a rodent Adam6 gene. In some embodiments, the rodent Adam6 gene is a human V H 2-1 and V H Located between the 6-1 gene segment, e.g., in germline configuration, human V H 2-1 and V H The human Adam6 gene located between the 6-1 gene segment is replaced. In some embodiments, the rodent may be a rat. In some embodiments, the rodent may be a mouse.

[0029] In some embodiments, the nucleotide molecules described herein comprise one or more drug selection cassettes, e.g., a drug resistance gene flanked by one or more site-specific recombination sites, e.g., a neomycin drug resistance gene flanked by loxP site-specific recombination recognition sites, wherein at least one of the one or more drug resistance cassettes optionally comprises at least one D operably linked to a 23-mer RSS. H Immediately upstream of the gene segment. In some embodiments, the nucleotide molecule comprises the sequence illustrated in FIG.

[0030] Additionally, the genome (e.g., germline genome) of a non-human animal (e.g., a rodent such as a rat or mouse) can be transformed with the engineered D H Also described herein are targeting vectors for modification to include regions. Generally, the targeting vectors described herein include any of the nucleotide molecules described herein, and optionally include 5' and 3' homology arms for homologous recombination within an immunoglobulin heavy chain variable region, and optionally the immunoglobulin heavy chain variable region is a human or humanized immunoglobulin heavy chain variable region. In some embodiments, the targeting vectors described herein include unrearranged human gene segments, e.g., V H 5' homology arm containing the 6-1 gene segment, and / or rodent (e.g., mouse) C H A region or part thereof, e.g., rodent (mouse) C H It contains an intronic enhancer region and / or a 3' homology arm containing the rodent (mouse) IgM gene.

[0031] In some embodiments, the targeting vector comprises a nucleotide molecule described herein and 5' and 3' homology arms positioned to allow homologous recombination with an immunoglobulin heavy chain sequence, optionally located at an endogenous rodent immunoglobulin heavy chain locus and / or comprising a human or humanized immunoglobulin heavy chain variable region. In some embodiments, the targeting vector comprises an unrearranged human gene segment (e.g., a V H 6-1 gene segment), human-engineered D H region, at least one unrearranged human J H The 5' homology arm containing the gene segment, as well as the rodent (mouse) C HThe targeting vectors described herein, which include 3' homology arms containing an intronic enhancer region and / or rodent (mouse) 3' homology arms, optionally contain a functional ADAM6 gene to preserve their reproductive potential, a modified endogenous heavy chain constant region gene sequence, including an intact endogenous IgM gene and another endogenous modified constant region gene (e.g., IgG) for the production of a reverse chimeric non-IgM antibody lacking a functional CH1 domain, a sequence encoding a reverse chimeric humanized common light chain, a sequence encoding a reverse chimeric humanized kappa light chain, a sequence encoding a reverse chimeric humanized lambda light chain, a sequence encoding a hybrid kappa / lambda or kappa / lambda light chain, or a modified endogenous heavy chain constant region gene (e.g., IgG) for the production of a reverse chimeric non-IgM antibody lacking a functional CH1 domain. and a rodent comprising a humanized immunoglobulin heavy chain locus, e.g., a mouse comprising a substitution of mouse immunoglobulin variable sequences with human immunoglobulin variable sequences, e.g., a VELOCIMMUNE® mouse, which may comprise unrearranged germline human heavy chain gene segments and / or rearranged (or unrearranged) germline light chain gene segments modified with histidine codons for expression of variable domains having histidine amino acids and capable of exerting terminal deoxynucleotidyl transferase (TdT) activity for pH-sensitive antigen binding and / or increased antigen receptor diversity. HIt may be useful to manipulate regions, e.g., see U.S. Patent Nos. 9,035,128; 9,066,502; 9,163,092; 9,150,662; 9,334,333; 9,850,462; 9,844,212; 9,029,628; 9,006,511; 9,394,373; 9,206,261; 9,206,262; 9,206,263; 9,226,484; 9,399,683; 9,540,452; 9,012,717; 9,796,788; 9,697,940; 9,796,788; 54,287; 9,334,334; 9,801,362; 9,332,742; 9,969,814; U.S. Patent Publication Nos. 2011 / 0195454, 2012 / 0021409, 2012 / 0192300, 2013 / 0185821, 2013 / 0302836, 2013 / 0045492, and 2018 / 0125043; International Patent Application Publication Nos. 2017210586, and 2019 / 113065, each of which is incorporated by reference herein in its entirety.

[0032] Thus, for example, D in rodents H -D H D for recombination H Methods for manipulating a region are described herein. In some embodiments, the methods include manipulating a region by at least one D operably linked to a 23-mer RSS. H One or more unrearranged D H D containing gene segment H In some embodiments, the D H -D H The method of modifying an immunoglobulin heavy chain variable region to engineer recombination involves the addition of one or more unrearranged D H D containing gene segment H obtaining an immunoglobulin heavy chain variable region comprising an unrearranged D HEach of the gene segments is flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS, and at least one D operably linked to a 23-mer RSS. H D to further include gene segments H In some embodiments, the D H The region contains one or more unrearranged human D H Human D containing gene segment H region, e.g., multiple unrearranged human D H The gene segments are optionally present in, e.g., germline configuration, D H 1-1 and D H All functional unrearranged human D sequences spanning and including gene segments 7-27 H In some embodiments, the modification comprises one or more unrearranged D sequences flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS. H Gene segments, e.g., functional unrearranged human D H One or more of the gene segments are operably linked to a 23-mer RSS by at least one D H In some embodiments, the D H Most 3' unreconstructed Ds are flanked by a 12-mer RSS on one end of the region and another 12-mer RSS on the other end. H The gene segment is operably linked to a 23-mer RSS. H D replaced with a gene segment and operably linked to a 23-mer RSS H The gene segment is composed of a 23-mer RSS, D H In some embodiments, the unrearranged D gene segment comprises a 12-mer RSS flanked on one end by a 12-mer RSS and on the other end by another 12-mer RSS. H The gene segment is a corresponding D engineered to be operably linked to a 23-mer RSS. H In some embodiments, the DH The region is unrearranged human D H 7-27 gene segments (e.g., germline D H 7-27 gene segments) and unrearranged human D H The 7-27 gene segment is a human D gene operably linked to a 23-mer RSS at the 5' end. H gene segments (e.g., unrearranged human D H 3-3 gene segment). H The region is unreconstructed D H 2-2 gene segments, unrearranged D H 2-8 gene segments and / or unrearranged D H Contains 2-15 gene segments (e.g., engineered D H is the unrearranged human D in germline configuration. H containing the complete repertoire of gene segments), unrearranged D H 2-2 gene segments, unrearranged D H 2-8 gene segments and / or unrearranged D H The 2-15 gene segments each comprise a D operably linked at their 3' ends to a 23-mer RSS. H 2-2 gene segment, human D operably linked at its 3' end to a 23-mer RSS H 2-8 gene segment and / or a human D operably linked at its 3' end to a 23-mer RSS. H In some embodiments, the modification is an unrearranged D H Gene segments (e.g., human germline D H In some embodiments, the immunoglobulin heavy chain variable region to be modified comprises replacing one of the two 12-mer RSSs flanking the corresponding gene segment with a 23-mer RSS. H In addition to the area, D H J operably linked to the region H region (possibly human germline J H1 gene segment, human germline J H 2 gene segments, human germline J H 3 gene segments, human germline J H 4 gene segments, human germline J H 5 gene segments, and human germline J H Human germline J containing six gene segments H Contains a complete repertoire of gene segments, optionally including human germline J H 1. J H 2. J H 3. J H 4. J H 5, and J H 6 gene segments in the germline configuration) and is flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H Gene segment substitutions are H At least one unreconstructed J in the region H This includes deleting gene segments, e.g., D H Unrearranged human J adjacent to the region H 1. J H 2. J H 3. J H 4, and / or J H In some embodiments, the deletion of a J gene segment results in a J H At least one germline J in the region H Deleting gene segments results in unrearranged human J H 1. J H 2, and J H 3 gene segment deletion, and optionally, J H 4 and J H In some embodiments, all functional D H Replacing one or more gene segments, e.g., D H The 7-27 gene segment was operably linked to a 23-mer RSS at the 5' end of the D HA gene segment, e.g., a D operably linked to a 23-mer RSS at the 5' end H Replacing the 3-3 gene segment H Unrearranged human J adjacent to the region H 1. J H 2, and J H In some embodiments, the deletion of all functional D H Replacing one or more gene segments, e.g., D H The 7-27 gene segment was operably linked to a 23-mer RSS at the 5' end of the D H A gene segment, e.g., a D operably linked to a 23-mer RSS at the 5' end H Replacing the 3-3 gene segment H Unrearranged human J adjacent to the region H 1. J H 2. J H 3. J H 4, and J H In some embodiments, the deletion of a 5 gene segment results in a D H The region comprises at least one D operably linked to a 23-mer RSS. H at least one D modified to contain a gene segment and operably linked to a 23-mer RSS; H The gene segment comprises (a) a human D operably linked to a 23-mer RSS; H 3-3 gene segments, possibly 23mer RSS, D H (b) a human D operably linked to a 23-mer RSS adjacent to the 5' end of the 3-3 gene segment; H 2-2 gene segments, possibly 23mer RSS, D H (c) a human D operably linked to a 23-mer RSS adjacent to the 3' end of the 2-2 gene segment H 2-8 gene segments, possibly 23mer RSS, D H (d) a human D operably linked to a 23-mer RSS adjacent to the 3' end of the 2-8 gene segment; H2-15 gene segments, possibly 23mer RSS, D H 2-15 adjacent to the 3' end of the gene segment, or (e) any combination of (a) to (d).

[0033] Such methods involve engineered D H The present invention provides a method for the preparation of a nucleotide molecule comprising an immunoglobulin heavy chain variable region, e.g., a (human) immunoglobulin heavy chain variable region as described herein, wherein the (unrearranged) (human) immunoglobulin heavy chain region is a non-human immunoglobulin heavy chain constant region or portion thereof, e.g., a rodent C H The intronic enhancer region and / or rodent IgM gene may optionally be operably linked to a rodent immunoglobulin heavy chain constant region, e.g., optionally at a non-human immunoglobulin heavy chain locus. In some embodiments, upon recombination, such an immunoglobulin heavy chain locus may comprise an immunoglobulin heavy chain variable domain, e.g., a variable domain having a length of V H (D H AD H B)J H , V H D H J H 6, or V H (D H AD H B)J H The immunoglobulin heavy chain variable region coding sequence may be a result of a recombinant human immunoglobulin heavy chain (HCHC) gene. The immunoglobulin heavy chain variable region coding sequence may be a rearranged immunoglobulin heavy chain variable domain having a complementarity determining region 3 (CDR3) amino acid length of more than 20 amino acids. Thus, the length of the CDR3 is at least 20 amino acids. H (D H AD H B)J H , V H (D H )J H 6, or V H (D H AD H B)J HProvided herein are rodents, rodent cells, loci and / or nucleotide molecules comprising the six sequences.

[0034] Also, for example, the genome, e.g., the germline genome, may contain an engineered D H Also described herein are non-human animals, e.g., rodents, that comprise a region, e.g., a nucleic acid, targeting vector, and / or immunoglobulin heavy chain variable locus, described herein. Also described herein are such non-human animal genomes, e.g., rodent genomes. In some embodiments, described herein are rodents whose germline genome, or rodent germline genome, comprises a described immunoglobulin heavy chain variable region, wherein the immunoglobulin heavy chain variable region comprises (i) at least one unrearranged heavy chain variable (V H ) gene segments, (ii) engineered heavy chain variable region diversity (D H ) area, manipulated D H The region consists of one or more unrearranged D regions, each flanked by a 12-mer RSS on one end and a 12-mer RSS on the other end. H A gene segment, and one or more Ds each operably linked to a 23-mer recombination signal sequence (RSS). H and (iii) at least one unrearranged heavy chain joint (J H ) gene segments, (i) to (iii) being operably linked such that, upon recombination, the immunoglobulin heavy chain variable region comprises a rearranged heavy chain variable region sequence encoding an immunoglobulin heavy chain variable domain, and optionally, the rearranged heavy chain variable region sequence is H (D H -D H )J H Formed after a recombination event, optionally one or more Ds each operably linked to a 23-mer recombination signal sequence (RSS) H At least one of the gene segments is V H (D H -D H) During a recombination event, one or more unrearranged Ds, each flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end, H In some embodiments, one or more Ds operably linked to the 23-mer RSS bind to one of the gene segments. H The segment is operably linked to the 3' 23mer RSS. H In some embodiments, the RSS comprises one or more Ds operably linked to the 23-mer RSS. H The segment is operably linked to the 5' 23mer RSS. H In some embodiments, the immunoglobulin heavy chain variable region comprises a human V H , D H , and J. H In some embodiments, the human immunoglobulin heavy chain variable region is operably linked to an immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is, for example, an endogenous immunoglobulin heavy chain constant region of a non-human animal at an endogenous immunoglobulin heavy chain locus or of a non-human animal genome. In some embodiments, the immunoglobulin heavy chain variable region is a V in germline configuration. H 3-74 to V H 6-1 Human V H In some embodiments, the immunoglobulin heavy chain variable region comprises a human J gene segment. H In some embodiments, the immunoglobulin heavy chain variable region optionally comprises two human V H inserted into a gene segment (e.g., human V H 1-2 gene segment and human V H In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent genome comprises one or more nucleotide molecules encoding one or more rodent Adam6 polypeptides (e.g., rodent Adam6 genes), which may be inserted in place of a human Adam6 pseudogene (e.g., inserted between the 6-1 gene segment and the 6-2 gene segment), and / or inserted in place of a human Adam6 pseudogene. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent genome comprises one or more nucleotide molecules encoding one or more rodent Adam6 polypeptides (e.g., rodent Adam6 genes), which may be inserted in place of a human Adam6 pseudogene. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent genome comprises one or more engineered D HIn some embodiments, the rodent genome is heterozygous for the engineered D H In some embodiments, the rodent may be a rat. In some embodiments, the rodent may be a mouse. In some embodiments, the rodent, rodent genome, or rodent cell is a rat, mouse, rat genome, mouse genome, rat cell, or mouse cell, for example, a rodent (rat or mouse) embryonic stem cell.

[0035] In some embodiments, the rodent, rodent genome, or rodent cell described herein comprises a rearranged heavy chain VDJ and / or V encoding an immunoglobulin heavy chain variable domain. H (D H AD H B)J H In some embodiments, the gene encoding the 23-mer RSS further comprises a coding sequence. H engineered D containing gene segments H and (2) an immunoglobulin heavy chain locus containing a rearranged heavy chain variable region V in its somatic genome, e.g., in a B cell. H (D H AD H B)J H A non-human animal, e.g., a rodent, e.g., a rat or mouse, is described, comprising a coding sequence, and a first or second D H Gene segments (respectively, D H A or D H B) D operably linked to the 23mer RSS H A gene segment, or a portion thereof (e.g., a D operably linked to a 23-mer RSS) H In some embodiments, the B cell is a naive B cell, and / or the rearranged heavy chain variable region coding sequence is operably linked to an IgM constant region sequence, and the B cell is derived from a DH A and D H At least one of the B gene segments comprises a D gene operably linked to a 23-mer RSS. H containing at least 9 consecutive nucleotides that align with a nucleotide molecule encoded by the gene segment, H A and D H Each B gene segment is a germline D H In some embodiments, the B cell is a plasma cell or a memory B cell, and / or the rearranged heavy chain variable region coding sequence is somatically hypermutated and / or operably linked to a non-IgM constant region sequence (e.g., IgG, IgA, IgE, etc.), and D H A and D H Each of B is different from the first and second germline D by at most one nucleotide variation, respectively. H In some embodiments, all rearranged heavy chain VDJ and / or VDJ gene segments in a rodent exhibit 40% identity. H (D H AD H B)J H At least 95% of the coding sequences have a CDR3 of at least 10 amino acids in length, and optionally all rearranged heavy chain VDJ and / or VDJ sequences in the rodent. H (D H AD H B)J H At least 70% of the coding sequences have a CDR3 of at least 11 amino acids in length, and optionally all rearranged heavy chain VDJ and / or VDJ sequences in the rodent. H (D H AD H B)J H At least 15% of the coding sequences have a CDR3 of at least 14 amino acids in length. H (D H AD H B)J HThe population of coding sequences has a CDR3 that is at least 15 amino acids in length, optionally at least 16 amino acids in length, optionally at least 17 amino acids in length, and optionally at least 18 amino acids in length. H (D H AD H B)J H A rodent or rodent cell expressing a coding sequence, or a nucleic acid or immunoglobulin locus comprising said coding sequence, is described herein and is a second germline D H The gene segment is D H 3-3 and optionally a rearranged heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 that is greater than 20 amino acids in length. In some embodiments, the rearranged heavy chain V H (D H AD H B)J H A rodent or rodent cell expressing a coding sequence, or a nucleic acid or immunoglobulin locus comprising said coding sequence, is described herein and is a first germline D H The gene segment is D H 2-2, D H 2-8 or D H 2-15, and optionally a rearranged heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 that is greater than 20 amino acids in length. In some embodiments, the rearranged heavy chain V encodes a CDR3 that is greater than 20 amino acids in length. H (D H AD H B)J H 6 Described herein are rodents or rodent cells that express the coding sequence.

[0036] In some embodiments, a rearranged human immunoglobulin heavy chain V operably linked to a rodent immunoglobulin heavy chain constant region sequence. H (D HAD H B)J H A rodent genome, nucleic acid, or immunoglobulin locus comprising a coding sequence is provided. H B gene segments are derived from human germline D H 3-3 gene segment. H The A gene segment is a human germline D H 2-2, D H 2-8 or D H 2-15 gene segments. H A and D H Each of B is different from the first and second germline D by at most one nucleotide variation, respectively. H The rearranged heavy chain shows 40% identity with the gene segment V H (D H AD H B)J H In some embodiments, the rearranged heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 that is greater than 20 amino acids in length. H The gene segment is a human germline J H 6 gene segments. H (D H AD H B)J H A rodent or rodent cell comprising the coding sequence is provided. In some embodiments, the rodent is a rat or mouse cell, or the rodent cell is a rat or mouse cell. In some embodiments, the rodent cell is a rodent B cell. In some embodiments, the rearranged heavy chain V fused with a myeloma cell is provided. H (D H AD H B)J H Hybridomas comprising rodent B cells expressing the coding sequence are provided. H (D HAD H B)J H The sequences are D H A and D H The sequences identified as B are identical to the first and second germline D sequences, each with a maximum of one nucleotide variation. H Even when showing 40% identity with the gene segment, D H -D H It has been confirmed to be the result of recombination.

[0037] In some embodiments, the genome is an engineered D H A non-human animal or cell is provided that contains an immunoglobulin heavy chain variable region comprising an engineered D H The region comprises at least one D operably linked to a first and a second recombination signal sequence (RSS). H In some embodiments, the first RSS is a 23-mer RSS and the second RSS is a 12-mer RSS. In some embodiments, the first RSS is a 12-mer RSS and the second RSS is a 23-mer RSS.

[0038] In some embodiments, the genome is an engineered D H A non-human animal is provided, comprising an immunoglobulin heavy chain variable region comprising an engineered D H The region comprises one or more D nucleotides each operably linked to a 23-mer recombination signal sequence (RSS). H Includes segments.

[0039] In some embodiments, the genome is an engineered D H A non-human cell or tissue is provided that contains an immunoglobulin heavy chain variable region comprising an engineered D H The region comprises one or more D nucleotides each operably linked to a 23-mer recombination signal sequence (RSS). HIn some embodiments, the cell comprises a segment. In some embodiments, the cell is of lymphoid or myeloid lineage origin. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is selected from a B cell, a dendritic cell, a macrophage, a monocyte, and a T cell. In some embodiments, the tissue is selected from adipose, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, or any combination thereof.

[0040] In some embodiments, immortalized cells made from non-human cells described herein, e.g., hybridoma cells made from fusing B cells isolated from a non-human animal described herein with myeloma cells, are provided.

[0041] In some embodiments, the non-human cells are non-human embryonic stem (ES) cells. In some embodiments, the non-human embryonic stem cells are rodent embryonic stem cells. In some embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are derived from the 129 strain, the C57BL strain, or a mixture thereof. In some embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are a mixture of the 129 and C57BL strains.

[0042] In some embodiments, there is provided a use of the non-human embryonic stem cells described herein for generating a non-human animal. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells, and are engineered D as described herein. H In some embodiments, the non-human embryonic stem cells are rat embryonic stem cells, and the non-human embryonic stem cells are used to generate mice containing an immunoglobulin heavy chain variable region comprising an engineered D region, as described herein. H In some embodiments, the engineered D H Non-limiting exemplary methods for generating rats comprising immunoglobulin heavy chain variable regions comprising the regions may include those disclosed in U.S. Patent No. 20140309487, which is incorporated herein by reference in its entirety.

[0043] In some embodiments, a non-human embryo is provided that comprises, is produced from, is obtained from, or is generated from the non-human embryonic stem cells described herein. In some embodiments, the non-human embryo is a rodent embryo, in some embodiments, a mouse embryo, and in some embodiments, a rat embryo.

[0044] In some embodiments, there is provided a use of a non-human embryo described herein to generate a non-human animal. In some embodiments, the non-human embryo is a mouse embryo, and is engineered as described herein. H In some embodiments, the non-human embryo is a rat embryo, and the non-human embryo is used to generate a mouse comprising an immunoglobulin heavy chain variable region comprising an engineered D region, as described herein. H The immunoglobulin heavy chain variable region containing the nucleotide sequence is used to generate rats containing the immunoglobulin heavy chain variable region.

[0045] The genome has been engineered H A method for producing a rodent comprising: (a) one or more D domains, each operably linked to a 23-mer RSS; H modifying the genome of rodent embryonic stem cells to include a DNA fragment comprising a segment, e.g., the DNA fragment is a nucleotide molecule, a targeting vector, and / or an engineered DNA fragment described herein; H Provided herein are methods comprising (b) generating a rodent using the modified rodent embryonic stem cells (a) comprising at least one D operably linked to the 23-mer RSS. In some embodiments, the method comprises: H Unrearranged immunoglobulin heavy chain variable region to contain the D segment H A step of modifying the unreconstructed D H The regions consist of one or more unrearranged D regions, each flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. HIn some embodiments, the modifying further comprises one or more unrearranged human D gene segments, thereby generating the rodent. H Replacing a gene segment, optionally with one or more J H The gene segment is comprised of at least one D operably linked to a 23-mer RSS. H In some embodiments, the immunoglobulin heavy chain variable region is a human immunoglobulin heavy chain variable region, e.g., a human immunoglobulin heavy chain variable region comprising at least one V H Unrearranged human immunoglobulin heavy chain V containing gene segments H One or more unrearranged human D gene clusters H Unrearranged human immunoglobulin heavy chain D containing gene segment H region, and one unrearranged human J H Unrearranged human immunoglobulin heavy chain J containing gene segment H Gene cluster, unrearranged human immunoglobulin heavy chain D H The region comprises at least one D operably linked to a 23-mer RSS. H In some embodiments, the modifying step comprises (i) modifying a functional human V H Gene segments, e.g., V H 3-74 to V H 6-1, including all functional V H (ii) a complete repertoire of gene segments; (iii) at least one D operably linked to a 23-mer RSS; H D replaced by a gene segment H Unrearranged human D, excluding 7-27 H (iii) a complete repertoire of gene segments, and (iv) at least unrearranged human J H 6 gene segments, and possibly at least an unrearranged J H 4 gene segments, unrearranged J H5 gene segments, and unrearranged J H In some embodiments, the immunoglobulin heavy chain variable region comprises at least one human D operably linked to a 23-mer RSS. H The gene segment is operably linked to a 5' 23mer RSS. H In some embodiments, the modification step comprises (i) a functional human V H Gene segments, e.g., V H 3-74 to V H 6-1, including all functional V H (ii) the complete repertoire of gene segments, and (iii) unrearranged human D H 2-2, D H 2-8, and D H 2-15 gene segments, each operably linked at its 3' to a 23mer RSS. H 2-2 gene segment, human D operably linked at its 3' to a 23-mer RSS H 2-8 gene segment and a human D operably linked at its 3' to a 23-mer RSS. H Unrearranged human D except for the replacement of the 2-15 gene segment H The complete repertoire of gene segments, and (iii) unrearranged human J H Gene segments, e.g., unrearranged human J H 1 gene segment, unrearranged human J H 2 gene segments, unrearranged human J H 3 gene segments, unrearranged human J H 4 gene segments, unrearranged human J H 5 gene segments, and unrearranged human J HIn some embodiments, the immunoglobulin heavy chain variable region (a) further comprises one or more rodent Adam6 genes, and optionally, the one or more rodent Adam6 genes comprise two unrearranged V H Between gene segments, e.g., unrearranged human V H 1-2 gene segment and unrearranged human V H and / or (b) is operably linked to an immunoglobulin heavy chain constant region, optionally an endogenous rodent immunoglobulin heavy chain constant region, e.g., an endogenous rodent immunoglobulin heavy chain constant region of an endogenous immunoglobulin heavy chain locus. In some embodiments, the rodent is a rat or a mouse.

[0046] In some embodiments, a kit is provided that includes a non-human animal as described herein, a non-human cell or tissue as described herein, an immortalized cell as described herein, a non-human embryonic stem cell as described herein, or a non-human embryo as described herein.

[0047] In some embodiments, kits described herein are provided for use in the manufacture and / or development of therapeutic or diagnostic drugs (e.g., antibodies or antigen-binding fragments thereof). In some embodiments, kits described herein are provided for use in the manufacture and / or development of drugs (e.g., antibodies or antigen-binding fragments thereof) for the treatment, prevention, or amelioration of a disease, disorder, or condition.

[0048] In some embodiments, a transgene, nucleic acid construct, DNA construct, or targeting vector described herein is provided. In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector is an engineered D gene described herein. HIn some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector comprises one or more D nucleotides operably linked to the 23-mer RSS. H In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector further comprises one or more selectable markers. In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector further comprises one or more site-specific recombination sites (e.g., loxP, Frt, or a combination thereof). In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector is depicted in FIG. 2.

[0049] In some embodiments, there is provided a use of a transgene, a nucleic acid construct, a DNA construct, or a targeting vector described herein to generate a non-human animal, a non-human cell, a non-human embryonic stem cell, and / or a non-human embryo.

[0050] In some embodiments, one or more D H The segments are each operably linked to a 3' 23mer RSS. In some embodiments, one or more D H The segments are each operably linked to a 5' 23mer RSS.

[0051] In some embodiments, the engineered D H The region contains one D operably linked to the 5' 23mer RSS. H In some embodiments, the engineered D H The region comprises one D operably linked to the 3' RSS. H segment. One D operably linked to the 5' 23mer RSS H In some embodiments of the segment, one D H The segment is synthetic D H segment, and in some embodiments, a synthetic human D H segment, and in some embodiments, a human DH A synthetic human D having a sequence identical or substantially identical to the 3-3 segment H A single D operably linked to the 3' 23mer RSS. H In some embodiments of the segment, one D H The segment is synthetic D H segment, and in some embodiments, a synthetic human D H segment, and in some embodiments, a human D H A synthetic human D having a sequence identical or substantially identical to the 3-3 segment H It is a segment.

[0052] In some embodiments, the engineered D H The regions each comprise three Ds operably linked to a 5' 23mer RSS. H Each comprises three D segments operably linked to a 5' 23mer RSS. H In some embodiments of the segment, three D H The segment is synthetic D H Each segment contains three Ds operably linked to a 5' 23mer RSS. H In some embodiments of the segment, three D H The segment is human D H 2 family segments. Each has three Ds operably linked to a 5' 23mer RSS. H In some embodiments of the segment, three D H The segment is human D H 2-2, Human D H 2-8, Human D H 2-15, Human D H 2-21 and combinations thereof. Each of the three Ds operably linked to the 5' 23mer RSS H In some embodiments of the segment, three D H The segment is human D H 2-2, Human D H 2-8, and human D H It's 2-15.

[0053] In some embodiments, the engineered D H The regions each comprise three Ds operably linked to a 3' 23mer RSS. H Each comprises three D segments operably linked to a 3' 23mer RSS. H In some embodiments of the segment, three D H The segment is synthetic D H Each segment contains three Ds operably linked to a 3' 23mer RSS. H In some embodiments of the segment, three D H The segment is human D H 2 family segments. Each has three Ds operably linked to a 3' 23mer RSS. H In some embodiments of the segment, three D H The segment is human D H 2-2, Human D H 2-8, Human D H 2-15, Human D H 2-21 and combinations thereof. Three Ds each operably linked to the 3' 23mer RSS H In some embodiments of the segment, three D H The segment is human D H 2-2, Human D H 2-8, and human D H It's 2-15.

[0054] In some embodiments, the engineered D H The region contains multiple human D H Contains multiple human D segments H At least one of the gene segments is operably linked to a 5' or 3' RSS, and in some embodiments, to a 5' 23mer RSS. H The region contains multiple human D H Contains multiple human D segments HAt least three of the gene segments are each operably linked to a 5' or 3' RSS, and in some embodiments, are operably linked to a 3' 23-mer RSS.

[0055] In some embodiments, the genome of the provided non-human animal, non-human cell, or non-human tissue contains one or more wild-type D H In some embodiments, the genome of the provided non-human animal, non-human cell, or non-human tissue lacks all or substantially all wild-type D segments. H In some embodiments, the genome of the non-human animal, non-human cell, or non-human tissue provided lacks the human D H Contains only the segment.

[0056] In some embodiments, the immunoglobulin heavy chain variable region is a human immunoglobulin heavy chain variable region. In some embodiments, the human immunoglobulin heavy chain variable region is operably linked to an immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an endogenous (e.g., non-human) immunoglobulin heavy chain constant region.

[0057] In some embodiments, the human immunoglobulin heavy chain variable region is V H 3-74 to V H Human V up to 6-1 H In some embodiments, the human immunoglobulin heavy chain variable region comprises at least a human J gene segment. H Gene segment J H 6. In some embodiments, the human immunoglobulin heavy chain variable region comprises at least a human J H Gene segment J H 4. J H 5 and J H 6. In some embodiments, the human immunoglobulin heavy chain variable region comprises a human J H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5 and J HIncludes 6.

[0058] In some embodiments of the non-human animal, non-human cell, or non-human tissue, the genome lacks an endogenous Adam6 gene. In some embodiments of the non-human animal, non-human cell, or non-human tissue, the genome further comprises an insertion of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides, and in some embodiments, the one or more nucleotide sequences encode a first and a second human V H In some embodiments, the one or more nucleotide sequences inserted between gene segments are inserted in place of the human Adam6 pseudogene, and in some embodiments, the one or more nucleotide sequences are inserted in place of the human V H Gene segments and human D H In some embodiments, the first human V H The gene segment is human V H 1-2 and the second human V H The gene segment is human V H The score is 6-1.

[0059] In some embodiments, the non-human animals, non-human cells, or non-human tissues provided are engineered D H In some embodiments, the non-human animal, non-human cell, or non-human tissue provided is homozygous, heterozygous, or hemizygous for the engineered D gene described herein. H Transgenic for the region.

[0060] In some embodiments, the genome is an engineered D H A method for producing a non-human animal comprising an immunoglobulin heavy chain variable region comprising the immunoglobulin heavy chain variable region is provided, the method comprising: (a) inserting DNA fragments into a non-human embryonic stem cell, the DNA fragments each comprising one or more Ds operably linked to a 23-mer RSS. H (b) obtaining the non-human embryonic stem cells produced in (a); and (c) using the non-human embryonic stem cells of (b) to produce a non-human animal.

[0061] In some embodiments, one or more D H The DNA fragments comprising the segments are each operably linked to a 3' 23mer RSS. In some embodiments, the DNA fragments comprise one D operably linked to a 3' 23mer RSS. H In some embodiments, the DNA fragment comprises one synthetic human D operably linked to a 3' 23mer RSS. H In some embodiments, the DNA fragment comprises one synthetic human D operably linked to a 3' 23mer RSS. H In some embodiments, the DNA fragment comprises three Ds each operably linked to a 3' 23mer RSS. H In some embodiments, the DNA fragments each comprise three human D segments operably linked to a 3' 23mer RSS. H In some embodiments, the DNA fragments each comprise three human D segments operably linked to a 3' 23mer RSS. H segment, human D H The segment is human D H 2-2, Human D H 2-8 and human D H It's 2-15.

[0062] In some embodiments, the DNA fragment comprises one or more D H In some embodiments, the DNA fragment comprises one D segment operably linked to a 5' 23mer RSS. H In some embodiments, the DNA fragment comprises one synthetic human D operably linked to a 5' 23mer. H In some embodiments, the DNA fragment comprises one synthetic human D operably linked to a 5' 23mer RSS. HIn some embodiments, the DNA fragment comprises one synthetic human D operably linked to a 5' 23mer RSS. H Contains 3-3 segments and is a synthetic human D H The 3-3 segment is human D H Human D instead of the 7-27 segment H Located in the area.

[0063] In some embodiments, the DNA fragment comprises one or more selectable markers. In some embodiments, the DNA fragment comprises one or more site-specific recombination sites.

[0064] In some embodiments, the genome is an engineered D H A method for producing a non-human animal comprising an immunoglobulin heavy chain variable region comprising a 23-mer RSS is provided, the method comprising: translating the genome of the non-human animal or a cell of the non-human animal into one or more Ds operably linked to a 23-mer RSS; H Manipulated D, including segments H The method includes modifying the genome of a non-human animal or a cell of a non-human animal to contain an immunoglobulin heavy chain variable region, thereby producing the non-human animal.

[0065] In some embodiments of generating a non-human animal, the genome of the non-human animal or non-human animal cell each contains one or more Ds operably linked to a 5' 23mer RSS. H In some embodiments of generating a non-human animal, the genome of the non-human animal or non-human animal cell is modified to contain one or more D segments, each operably linked to a 3' 23-mer RSS. H The segment is modified to include:

[0066] In some embodiments, a method for producing an antibody in a non-human animal is provided. In some embodiments, a method for producing an antibody or obtaining a nucleic acid encoding the same comprises immunizing a non-human animal (e.g., a rodent (e.g., a rat or mouse)) with an antigen, the rodent each containing one or more Ds operably linked to a 23-mer RSS. H Manipulated D containing segments H The method includes causing a rodent having a germline genome containing the region to produce an immune response to the antigen comprising an antibody, or a nucleic acid encoding the same, that binds to the antigen. In some embodiments, the method further includes recovering the antibody or nucleic acid encoding the same from the rodent or a rodent cell, e.g., a B cell, or a hybridoma. In some embodiments, one or more D H The segments are each operably linked to a 5' 23mer RSS. In some embodiments, one or more D H The segments are each operably linked to a 3' 23-mer RSS.

[0067] In some embodiments, a method is provided for producing antibodies in a non-human animal, the method comprising: (a) immunizing the non-human animal with an antigen, the non-human animal each comprising one or more Ds operably linked to a 23-mer RSS; H Manipulated D, including segments H (b) a non-human animal has a genome comprising an immunoglobulin heavy chain constant region comprising a region; (b) a non-human animal has a genome to an antigen; (b) maintaining the rodent under conditions sufficient for the non-human animal to generate a response to the antigen; and (c) recovering an antibody that binds to the antigen from the non-human animal or non-human animal cell. In some embodiments, the non-human cell is a B cell. In some embodiments, the non-human cell is a hybridoma.

[0068] In some embodiments, the genome comprises one or more human V H A human immunoglobulin heavy chain variable region comprising a gene segment, at least one human D operably linked to a 23-mer RSS. HManipulated D containing segments H region, and at least one human J H A non-human animal containing a gene segment is provided, and the rodent, when immunized with an antigen, produces a human D H -D H Recombinant and / or J H In some embodiments, the human immunoglobulin heavy chain variable region is operably linked to one or more endogenous immunoglobulin constant region genes, such that the antibody exhibits specific binding to an antigen, characterized in that the antibody comprises a human heavy chain variable domain comprising a CDR3 region generated by enhanced recombination into a 6 gene segment. In some embodiments, the antibody exhibits specific binding to an antigen, characterized in that the human immunoglobulin heavy chain variable region is operably linked to one or more endogenous immunoglobulin constant region genes, such that the antibody comprises at least one human D operably linked to a 23-mer RSS. H The segment is human D H In some embodiments, the human immunoglobulin heavy chain variable region contains fewer than all six human J segments. H In some embodiments, the human immunoglobulin heavy chain variable region comprises six human J gene segments. H In some embodiments, the human immunoglobulin heavy chain variable region comprises only one of the human J gene segments. H Contains 6 gene segments and functional J H 1 gene segment and functional J H 2 gene segments and functional J H 3 gene segments and functional J H 4 gene segments and functional J H In some embodiments, the human immunoglobulin heavy chain variable region lacks six human J gene segments. H In some embodiments, the human immunoglobulin heavy chain variable region comprises only three of the human J gene segments. H 4 gene segments, human J H 5 gene segments and human J H Contains only 6 gene segments and is functional J H 1 gene segment and functional J H 2 gene segments and functional J HIn some embodiments, the genome lacks less than all six human J gene segments. H A human immunoglobulin heavy chain variable region comprising a gene segment, e.g., a human immunoglobulin heavy chain variable region comprising only one of six human gene segments (e.g., a human J H Contains 6 gene segments and functional J H 1 gene segment and functional J H 2 gene segments and functional J H 3 gene segments and functional J H 4 gene segments and functional J H A non-human animal whose genome contains all six human J gene segments (including human immunoglobulin heavy chain variable regions) lacks all six human J gene segments. H compared to a control non-human animal containing a human immunoglobulin heavy chain variable region containing the J gene segment H 6 gene segments, e.g., non-human animals exhibit enhanced recombination of J H In some embodiments, the genome contains fewer than all six human J gene segment sequences or portions thereof, and / or a higher percentage of rearranged immunoglobulin heavy chain sequences that encode a CDR3 of at least 20 amino acids in length than the control non-human animal. H A human immunoglobulin heavy chain variable region containing gene segments, e.g., six human J H A human immunoglobulin heavy chain variable region (e.g., human J) containing only three of the gene segments H 4 gene segments, human J H 5 gene segments, and human J H Contains 6 gene segments and functional J H 1 gene segment and functional J H 2 gene segments and functional J H Non-human animals whose genomes contain all six human J gene segments (human immunoglobulin heavy chain variable regions) lack all six human J gene segments. H compared to a control non-human animal containing a human immunoglobulin heavy chain variable region containing the J gene segment H6 gene segments, e.g., non-human animals exhibit enhanced recombination of J H 6 gene segment sequences or portions thereof and / or contain a higher percentage of rearranged immunoglobulin heavy chain sequences encoding CDR3s of at least 20 amino acids in length than the control non-human animal.

[0069] In some embodiments, the genome comprises one or more human V H A human immunoglobulin heavy chain variable region containing a gene segment, at least one human D flanked at the 3' by a 23-mer RSS. H Manipulated D containing segments H region, and at least two human J H A non-human animal containing a gene segment is provided, and the rodent, when immunized with an antigen, produces a human D H -D H In some embodiments, the engineered D gene is operably linked to one or more endogenous immunoglobulin constant region genes to produce an antibody comprising a human heavy chain variable domain comprising a recombinantly produced CDR3 region, wherein the antibody exhibits specific binding to an antigen. H The regions each consist of at least three human D-terminal fragments flanked at the 3' by a 23-mer RSS. H In some embodiments, the engineered D H The regions consist of three human D-seq sequences, each flanked at the 3' by a 23-mer RSS. H Contains three human D segments H The segment is human D H 2-2, Human D H 2-8 and human D H 2-15. In some embodiments, the human immunoglobulin heavy chain variable region is V H 3-74 to V H Human V up to 6-1 H Contains gene segments.

[0070] In some embodiments, the antigen is a pathogen, e.g., a bacterial, fungal, or viral pathogen. In some embodiments, immunizing a non-human animal herein comprises infecting the non-human animal with a pathogen, e.g., a bacterial, fungal, or viral pathogen. In some embodiments, immunizing a non-human animal herein comprises administering to the non-human animal genomic or proteinaceous material isolated from a pathogen, e.g., a bacterial, fungal, or viral pathogen. In some embodiments, the antigen is a receptor (e.g., a complement receptor, a chemokine receptor, etc.), or a portion thereof. In some embodiments, the antigen is a nucleic acid encoding a receptor, or a portion thereof. In some embodiments, the antigen is a cell expressing a receptor, or a portion thereof. In some embodiments, the antigen is an ion channel, or a portion thereof. In some embodiments, the antigen is a nucleic acid encoding an ion channel, or a portion thereof. In some embodiments, the antigen is a cell expressing an ion channel, or a portion thereof.

[0071] In some embodiments, the non-human animal, non-human cell, or non-human tissue provided comprises one or more human VV ... L A gene segment and one or more human J L In some embodiments, the genome further comprises an insertion of a human V gene segment. L and J. L The segment is V κ and J. κ In some embodiments, the human V is a gene segment inserted into the endogenous κ light chain locus. κ and J. κ The gene segment is operably linked to a rodent Cκ gene (e.g., a mouse or rat Cκ gene). L and J. L The segment is V λ and J. λ In some embodiments, the human Vλ and Jλ gene segments are operably linked to a rodent Cλ gene (e.g., a mouse or rat Cλ gene) and are inserted into the endogenous λ light chain locus.

[0072] In some embodiments, there is provided the use of a non-human animal, non-human cell, or non-human tissue described herein in the manufacture and / or development of a drug or vaccine for use in medicine, such as for use as a medicament. In some embodiments, there is provided the use of a non-human animal, non-human cell, or non-human tissue described herein in the manufacture and / or development of an antibody for administration to a human. In some embodiments, there is provided the use of a non-human animal, non-human cell, or non-human tissue described herein in the manufacture of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition.

[0073] In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are provided for use in the manufacture and / or development of therapeutic or diagnostic drugs. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are provided for use in the manufacture of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition.

[0074] In some embodiments, the non-human animals provided herein are rodents, in some embodiments, mice, and in some embodiments, rats. In many embodiments, the non-human animal cells provided herein are rodent cells, in some embodiments, mouse cells, and in some embodiments, rat cells. In many embodiments, the non-human animals provided herein are rodent tissues, in some embodiments, mouse tissues, and in some embodiments, rat tissues.

[0075] In some embodiments, the 23-mer RSS comprises a nucleotide sequence comprising the sequence represented as SEQ ID NO:151. [Brief explanation of the drawings]

[0076] The drawings contained herein, consisting of the following figures, are for illustration purposes only and not for limitation:

[0077] [Figure 1] Figure 1 shows a non-scale generalized illustration of an embodiment of the present invention, illustrating the ordered assembly of gene segments in a DJ recombination event for immunoglobulin heavy chain variable region gene segments with unmodified D regions (top panel) and engineered D regions (bottom panel). A 12-mer recombination signal sequence (RSS) is shown as an open triangle. A 23-mer RSS is shown as a triangle with a vertical stripe. Exemplary unmodified VH gene segments (open squares) and DH gene segments (filled squares) are shown as open squares and filled squares, respectively, and are provided with their general designations using alphabetic characters. A D gene segment (e.g., D3-3) and an unmodified JH gene segment operably linked to a 23-mer RSS are shown as horizontally striped filled squares and open squares, respectively, and are provided with their appropriate designations. The μ0 promoter (μ0pro) is also shown. Recombination of the VH gene segments into the recombined DJ gene segments is not shown.

[0078] [Figure 2] Figure 2 shows a non-scale illustration of an exemplary embodiment of a targeting vector prepared according to Example 1. Hashed lines represent DH gene segments included in the targeting vector but not specifically shown.

[0079] [Figure 3] 3 shows a non-scale illustration of a non-limiting exemplary embodiment of inserting the 23:DH3-3:12 / JH6 targeting vector into the humanized immunoglobulin heavy chain variable region locus in the genome of mouse ES cells via electroporation (EP). Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown.

[0080] [Figure 4] 4 shows a non-scale representation of a non-limiting exemplary embodiment of Cre-mediated deletion of a selection cassette in a humanized immunoglobulin heavy chain locus following electroporation and integration of the 23:DH3-3:12 / JH6 targeting vector, as described in Examples 1 and 2. Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown.

[0081] [Figure 5] 5 shows a non-scale illustration of a non-limiting exemplary embodiment of inserting the 23:DH3-3:12 / JH4-6 targeting vector into the humanized immunoglobulin heavy chain variable region locus in the genome of a mouse ES cell via electroporation (EP). Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown.

[0082] [Figure 6] 6 shows a non-scale representation of a non-limiting exemplary embodiment of Cre-mediated deletion of a selection cassette in a humanized immunoglobulin heavy chain locus following electroporation and integration of the 23:DH3-3:12 / JH4-6 targeting vector, as described in Examples 1 and 2. Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown.

[0083] [Figure 7] 7 shows a non-scaled illustration of a non-limiting exemplary embodiment of inserting the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 / JH1-6 targeting vector into the humanized immunoglobulin heavy chain variable region locus in the genome of mouse ES cells via electroporation (EP). Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown. Filled triangles represent pseudogenes.

[0084] [Figure 8] 8 shows a non-scale representation of an exemplary, non-limiting embodiment of Cre-mediated deletion of a selection cassette in a humanized immunoglobulin heavy chain locus following electroporation and integration of the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vector, as described in Examples 1 and 2. Hashed lines represent VH or DH gene segments that are included in the heavy chain variable region locus but are not specifically shown.

[0085] [Figure 9A] Figure 9A shows results related to an embodiment of the invention, graphing the percentage (y-axis) of all functional immunoglobulin (Ig) reads resulting from DH-DH recombination events (bottom panel) with CDR3s of specific amino acid lengths (x-axis) isolated from animals modified with the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vector. "S1," "S2," and "S3" each represent a different experimental mouse.

[0086] [Figure 9B] Figure 9B shows results related to an embodiment of the invention, graphing the percentage (y-axis) of all Ig reads resulting from DH-DH recombination events (bottom panel) with CDR3s of specific amino acid lengths (x-axis) isolated from animals modified with the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vector. "S1," "S2," and "S3" each represent a different experimental mouse.

[0087] [Figure 10A]10A shows results related to an embodiment of the present invention, graphing the percentage (y-axis) of all functional immunoglobulin (Ig) reads resulting from DH-DH recombination events with CDR3s containing a particular number of cysteine ​​residues (x-axis) isolated from animals modified with the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vector. "S1," "S2," and "S3" each represent a different experimental mouse.

[0088] [Figure 10B] Figure 10B shows results related to an embodiment of the invention, graphing the percentage (y-axis) of all immunoglobulin (Ig) reads resulting from DH-DH recombination events with CDR3s containing a particular number of cysteine ​​residues (x-axis) isolated from animals modified with the 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vector. "S1," "S2," and "S3" each represent a different experimental mouse.

[0089] [Figure 11] 11 shows results related to an embodiment of the present invention, graphing typical antibody titers (y-axis) in individual mice with humanized immunoglobulin heavy and kappa light chain variable region loci (VI; see, e.g., U.S. Pat. Nos. 8,697,940 and 8,642,835; each of which is incorporated by reference herein in its entirety), both cohorts immunized with DNA immunogens encoding G protein-coupled receptors (GPCRs), and mice modified with the 23:DH3-3:12 / JH6 targeting vector described herein. Antibody titers were determined by MSD cell binding on 293 cells engineered to express GPCRs (GPCRs; x-axis) and cells that do not express GPCRs (control; x-axis).

[0090] [Figure 12A]Figure 12A shows results related to embodiments of the invention showing the percentage (%; y-axis) of rearranged immunoglobulin heavy chain VHDHJH gene segments estimated to be the result of VHDHA-DHBJH rearrangement according to the strict criteria set forth in Table 7, isolated from the bone marrow (BM) or spleen of mice modified with the 23:DH3-3:12 / JH6 targeting vector and heavy chain CDR3s (HCDR3s) having amino acids (AA) between 5 and 30 amino acids in length (x-axis). BM cell numbers and spleen cell numbers are not normalized to each other. n=1.

[0091] [Figure 12B] Figure 12B provides an enlargement of the graph shown in panel Figure 12A and shows results related to an embodiment of the invention. BM cell numbers and spleen cell numbers are not normalized to each other. n=1.

[0092] [Figure 12C] Figure 12C shows results related to an embodiment of the invention showing the percentage of reads with a CDR3 longer than 21 amino acids in both the bone marrow or spleen of mice modified with the 23:DH3-3:12 / JH6 targeting vector. BM cell numbers and spleen cell numbers were not normalized to each other. n=1.

[0093] [Figure 13] 13 shows results related to embodiments of the invention showing the percentage (%; y-axis) of putative rearranged immunoglobulin heavy chain VHDHA-DHBJH gene sequences isolated from bone marrow (BM) or spleen (according to the strict criteria set forth in Table 7) from mice modified with the 23:DH3-3:12 / JH6 targeting vector and heavy chain CDR3s (HCDR3s) all having an amino acid (AA) length greater than 20 amino acids (x-axis). BM cell numbers and spleen cell numbers are not normalized to each other. n=1. DETAILED DESCRIPTION OF THE INVENTION

[0094] definition The scope of the present invention is defined by the claims appended hereto, and is not limited by the specific embodiments described herein. Those skilled in the art will recognize, upon reading this disclosure, various modifications that may be equivalent to such described embodiments or that may otherwise be within the scope of the claims. Generally, terminology follows its art-understood meaning unless clearly indicated otherwise. Explicit definitions of certain terms are provided herein and below, but the meaning of these and other terms in specific instances throughout the specification will be apparent to those skilled in the art from the context. Further definitions of the following terms, and other terms, are set forth throughout the specification. References cited within this specification, or relevant portions thereof, are incorporated herein by reference in their entirety.

[0095] The use of ordinal terms such as "first," "second," "third," etc. to modify the claims does not, in itself, imply any priority, precedence, or order of one claim element over another, or the chronological order in which the actions of a method are performed, but is used solely as a marker to distinguish one claim element with a particular name from another element with the same name (other than the use of ordinal terms).

[0096] In this specification and the claims, the articles "a" and "an" should be understood to include plural referents unless clearly indicated otherwise. A claim or statement including "or" between one or more elements of a group is satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless otherwise indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which two or more, or the entire group member, is present in, employed in, or otherwise relevant to a given product or process. Furthermore, the invention should be understood to cover all variations, combinations, and permutations of one or more limitations, elements, phrases, descriptive terms, etc. from one or more of the claims described herein that are introduced into another claim (or any other related claim) relying on the same base claim, unless otherwise specified or unless a contradiction or inconsistency would arise apparent to one of ordinary skill in the art. Where elements are present as recited (e.g., in a Markush group or similar format), each subgroup of elements is also disclosed, and any element can be removed from the group. It will be understood that, generally, when the invention, or aspects of the invention, are referred to as including particular elements, features, etc., an embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For the sake of brevity, these embodiments will not in all instances be specifically described in so many words herein. It will also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited in the specification.

[0097] As used herein, the terms "about" and "approximately" are used interchangeably. Any numbers used herein, with or without about / approximately, are intended to cover any normal variation understood by one of ordinary skill in the art, for example, + / - 5%.

[0098] Administration: refers to the administration of a composition to a subject or system (e.g., a cell, organ, tissue, organism, or related component or components thereof). One of skill in the art will recognize that the route of administration can vary depending, for example, on the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc.

[0099] For example, in some embodiments, administration to an animal subject (e.g., a human or rodent) may be bronchial (including bronchial infusion), buccal, enteral, intercutaneous, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal infusion), transdermal, vaginal, and / or intravitreal administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, an antibody produced by a non-human animal disclosed herein may be administered to a subject (e.g., a human subject or a rodent). In some embodiments, a pharmaceutical composition comprises an antibody produced by a non-human animal disclosed herein. In some embodiments, the pharmaceutical composition may include a buffer, a diluent, an excipient, or any combination thereof. In some embodiments, a pharmaceutical composition comprising an antibody produced by a non-human animal disclosed herein may be included in a container for storage or administration, such as, for example, a vial, a syringe (e.g., an IV syringe), or a bag (e.g., an IV bag).

[0100] Biological activity: means the characteristic of any agent that has activity in a biological system, in vitro or in vivo (e.g., in an organism). For example, an agent is considered to be biologically active if it has a biological effect in an organism when it is present in that organism.

[0101] In certain embodiments, if a protein or polypeptide is biologically active, a portion of the protein or polypeptide that confers at least one biological activity of the protein or polypeptide is generally referred to as a "biologically active" portion.

[0102] Equivalent: means two or more agents, entities, situations, groups of conditions, etc. that may not be identical to one another, but that are similar enough to permit a comparison that allows reasonable conclusions to be made based on observed differences or similarities. One of ordinary skill in the art will understand, in context, the degree of identity required in a given situation for two or more such agents, entities, situations, groups of conditions, etc. to be considered equivalent.

[0103] Conservative: A conservative amino acid substitution refers to the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution does not substantially alter the functional properties of the protein of interest, such as the ability of a receptor to bind to a ligand. Examples of amino acids with side chains with similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine ​​and methionine. Conservative amino acids substitution groups include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine.

[0104] In some embodiments, conservative amino acid substitutions can be substitutions of any native residue in a protein, including alanine, such as those used in alanine-scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, the substitution is a moderately conservative substitution, in which the substitution has a non-negative value in the PAM250 log-likelihood matrix.

[0105] Control: Refers to the art-recognized meaning of "control," a standard against which results are compared. Generally, controls are used to increase the integrity of an experiment by isolating a variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a comparator. "Control" may refer to a "control animal." A "control animal" may have a modification described herein, a modification different from those described herein, or be unmodified (i.e., a wild-type animal). In one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, which is the "control," the variable being tested is not administered. A control may be a positive or negative control.

[0106] In some embodiments, the control is a historical control (i.e., of a previously performed test or assay, or of a previously known amount or result). In some embodiments, the control is or includes a printed or otherwise kept record.

[0107] Disruption: means the result of a homologous recombination event with a DNA molecule (e.g., an endogenous homologous sequence such as a gene or locus).

[0108] In some embodiments, the disruption may achieve or exhibit an insertion, deletion, substitution, replacement, missense mutation, or frameshift of a DNA sequence, or any combination thereof. The insertion may include the insertion of an entire gene, a fragment of a gene (e.g., an exon), which may be of a source other than the endogenous sequence (e.g., a heterologous sequence), or a coding sequence derived from or isolated from a particular gene of interest. In some embodiments, the disruption may increase the expression and / or activity of a gene or gene product (e.g., of a protein encoded by the gene). In some embodiments, the disruption may decrease the expression and / or activity of a gene or gene product. In some embodiments, the disruption may alter the sequence of a gene or encoded gene product (e.g., the encoded protein). In some embodiments, the disruption may alter the sequence of a chromosome or chromosomal location within a genome. In some embodiments, the disruption may truncate or fragment a gene or encoded gene product (e.g., the encoded protein). In some embodiments, the disruption may extend a gene or encoded gene product. In some such embodiments, the disruption may enable the assembly of a fusion protein. In some embodiments, the disruption may affect the level of the gene or gene product but not its activity. In some embodiments, the disruption may affect the activity of the gene or gene product but not its level. In some embodiments, the disruption may have no significant effect on the level of the gene or gene product. In some embodiments, the disruption may have no significant effect on the activity of the gene or gene product. In some embodiments, the disruption may have no significant effect on either the level or activity of the gene or gene product. In some embodiments, a significant effect can be measured by, for example, but not limited to, a Student's T-test.

[0109] Endogenous locus or endogenous gene: means the locus present in a parent or reference organism prior to the introduction of an alteration, disruption, deletion, insertion, modification, substitution or replacement as described herein.

[0110] In some embodiments, the endogenous locus comprises, in whole or in part, a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a genetically engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (wild-type or genetically engineered).

[0111] Endogenous promoter: refers to the promoter naturally associated with an endogenous gene or locus, for example, in a wild-type organism.

[0112] Engineered: Generally refers to aspects that have been manipulated by the hand of man. As is common and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell are also generally considered to be "engineered," even if the actual manipulation was performed on a previous entity. Furthermore, as will be recognized by those of skill in the art, a variety of techniques are available through which the "manipulation" described herein can be achieved.

[0113] In some embodiments, two or more sequences that are not linked together in natural order may be considered "engineered" when they are manipulated by the hand of man to be directly linked to each other in an engineered polynucleotide. In some embodiments, an engineered polynucleotide may include regulatory sequences that are naturally operably linked to a first coding sequence but not to a second coding sequence, and that have been operably linked to the second coding sequence by the hand of man. The engineered D herein refers to a sequence that is operably linked to a first coding sequence but not to a second coding sequence. H In an embodiment of the gene segment, D H The gene segment is engineered by hand to be operably linked to the 23mer RSS (e.g., at least one of which is adjacent to, next to, or immediately adjacent to the 23mer RSS). In some embodiments, the D is engineered to be operably linked to the 23mer RSS. H The gene segment is a separate DH A D resulting from a gene segment and operably linked to, for example, a 23-mer RSS. H The gene segment is similar to other D H Contains a nucleotide sequence identical to the nucleotide sequence of the gene segment, but differs due to degeneracy in the genetic code and / or substitution of a 12-mer RSS for a 23-mer RSS. H Gene segments and the engineered D resulting from them H a gene segment (e.g., a D operably linked to a 23-mer RSS) H A gene segment) can be considered as a corresponding gene segment. For example, a human D operably linked to a 23-mer RSS H The 3-3 gene segment is a D gene flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H D, which can be considered to correspond to a 3-3 gene segment and is operably linked to a 23-mer RSS. H A 3-3 gene segment and a D flanked by a 12-mer RSS on one end and another 12-mer RSS on the other end. H 3-3 gene segments share identical nucleotide sequences, except for differences due to the degeneracy of the genetic code and / or the substitution of two 12-mer RSSs with a 23-mer RSS. Alternatively, or in addition, in some embodiments, a first nucleic acid sequence and a second nucleic acid sequence, each encoding polypeptide elements or domains that are not linked to each other in nature, may be linked to each other in a single engineered polynucleotide. Similarly, in some embodiments, a cell or organism may be considered "engineered" if it has been engineered to thereby alter its genetic information (e.g., by introducing new genetic material not previously present or by altering or removing previously present genetic material).

[0114] For example, in some embodiments, "manipulation" may include selection or design (e.g., of nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) through the use of a computer system programmed to analyze or compare, or otherwise analyze recommended and / or selected sequences. Alternatively, or additionally, in some embodiments, "manipulation" may include the use of in vitro chemical synthesis techniques and / or recombinant nucleic acid techniques, such as nucleic acid amplification hybridization (e.g., via the polymerase chain reaction), mutation, transformation, transfection, and / or the use of any of a variety of controlled mating methods. As will be recognized by those skilled in the art, a variety of such established techniques (e.g., recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and are described in various general and detailed references, which are cited and / or discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; incorporated herein by reference in its entirety).

[0115] Gene: refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). For clarity, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide; the term may optionally include regulatory sequences, which should be clear to one skilled in the art from the context. This definition is not intended to exclude the application of the term "gene" to expression units that encode non-proteins, but is intended to clarify that as used herein, the term often refers to nucleic acids that encode polypeptides.

[0116] In some embodiments, a gene includes coding sequences (i.e., sequences that encode a particular product). In some embodiments, a gene includes non-coding sequences. In some embodiments, a gene includes both coding sequences (e.g., exon sequences) and non-coding sequences (e.g., intron sequences). In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that, for example, can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.).

[0117] Heterologous: refers to an agent or entity from a different source. For example, when used in reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the related polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof (1) has been genetically manipulated by the hand of man, (2) has been introduced into the cell or organism (or a precursor thereof) by the hand of man (e.g., by genetic manipulation), and / or (3) is not naturally produced by or is not present in the related cell or organism (e.g., related cell type or organism type). Another example includes a polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof that is normally present in a particular native cell or organism under the control of a regulatory element (e.g., a promoter) that is not naturally associated with it, and in some embodiments is non-endogenous, but that has been altered, for example, by mutation or substitution.

[0118] Host cell: refers to a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Those skilled in the art will understand, upon reading this disclosure, that such terms are used to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell."

[0119] In some embodiments, the host cell is or comprises a prokaryotic or eukaryotic cell. In some embodiments, the host cell is or comprises a mammalian cell. Generally, a host cell is any cell suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the species to which the cell is designated. Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., Escherichia coli), and the like. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusion products such as hybridomas or quadromas.

[0120] In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) expressing a viral gene. In some embodiments, the host cell is or comprises an isolated cell. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.

[0121] "Humanized": refers to a molecule (e.g., nucleic acid, protein, etc.) that is non-human in origin and in which portions thereof have been replaced with corresponding portions of a corresponding human molecule such that the modified (e.g., humanized) molecule retains its biological function and / or maintains a structure that performs the retained biological function. In contrast, "human" and the like encompasses molecules of exclusively human origin, e.g., having human nucleotides or proteins that contain exclusively human nucleotide and amino acid sequences, respectively. The term "human (humanized)" is used to reflect that the human (humanized) molecule can be (a) a human molecule or (b) a humanized molecule.

[0122] Identity: In relation to the comparison of sequences, means identity as determined by a variety of algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity.

[0123] In some embodiments, identity as described herein is determined using ClustalW v.1.83 (slow) alignment using a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008) with a gap opening penalty of 10.0, a gap extension penalty of 0.1.

[0124] In vitro: refers to events that occur not within a multicellular organism but in an artificial environment, such as a test tube or reaction vessel, cell culture, etc.

[0125] In vivo: Refers to events that occur within a multicellular organism, such as a human and / or non-human animal. In the context of cell-based systems, the term may also be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0126] Isolated: (1) refers to a substance and / or entity that has been separated from at least some of the components with which it was associated when originally created (either in natural and / or experimental environments) and / or (2) a substance and / or entity that has been designed, created, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10 or more other components with which they were originally associated. In some embodiments, an isolated agent is at least about 80% or more pure. A substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may still be considered "isolated" or even "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients.

[0127] For example, in some embodiments, a naturally occurring biological polymer, such as a polypeptide or polynucleotide, is considered "isolated" when (a) its origin or source is free from association with some or all of the components that accompany it in its natural state in nature, (b) it is substantially free from other polypeptides or nucleic acids of the same species that produces it in nature, or (c) it is expressed by or is otherwise associated with components from a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered an "isolated" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques can be considered an "isolated" polypeptide so long as it is separated from a) other components with which it is associated in nature and / or b) other components with which it was associated when originally produced.

[0128] Non-human animal: means any vertebrate organism that is not a human.

[0129] In some embodiments, the non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, or a bird. In some embodiments, the non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, the non-human animal is a rodent such as a rat or a mouse.

[0130] Nucleic Acid: In its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain, and is generally interchangeable with nucleic acid molecule, nucleic acid sequence, nucleotide molecule, and nucleotide molecule, and these terms are interchangeable.

[0131] In some embodiments, a "nucleic acid" is an oligonucleotide chain or a compound and / or substance that can be incorporated into an oligonucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), as is clear from the context. In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, "nucleic acid" comprises or consists of one or more naturally occurring nucleic acid residues. In some embodiments, "nucleic acid" comprises or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from "nucleic acids" in that they do not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a "nucleic acid" has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a "nucleic acid" is or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, a "nucleic acid" is or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, a "nucleic acid" comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, a "nucleic acid" has a nucleotide sequence that encodes a polypeptide fragment (e.g., a peptide). In some embodiments, a "nucleic acid" comprises one or more introns. In some embodiments, a "nucleic acid" comprises one or more exons. In some embodiments, a "nucleic acid" comprises one or more coding sequences. In some embodiments, a "nucleic acid" is prepared by one or more of: isolation from a natural source; enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template; replication in a recombinant cell or system; and chemical synthesis. In some embodiments, a "nucleic acid" is at least three or more residues in length. In some embodiments, a "nucleic acid" is single-stranded; in some embodiments, a "nucleic acid" is double-stranded. In some embodiments, a "nucleic acid" has a nucleotide sequence that includes at least one element that encodes, or is the complement of, a sequence that encodes a polypeptide or a fragment thereof. In some embodiments, a "nucleic acid" has enzymatic activity.

[0132] Operably linked: refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

[0133] In some embodiments, the operably linked nucleotide sequences are contiguous with one another, e.g., a nucleic acid sequence comprising an immunoglobulin gene segment operably linked to an RSS comprises an immunoglobulin gene segment nucleotide sequence that is flanked by the RSS nucleotide sequence, e.g., in a contiguous manner such that the immunoglobulin gene segment is immediately adjacent to the RSS nucleotide sequence, and the immunoglobulin gene segment is flanked on at least one side (e.g., contiguous) by the RSS nucleotide sequence.

[0134] In other embodiments, operably linked does not require contiguousity. For example, unrearranged variable region gene segments "operably linked" to each other are capable of rearranging to form a rearranged variable region gene, where the unrearranged variable region gene segments are not necessarily contiguous with each other. Unrearranged variable region gene segments operably linked to each other and to contiguous constant region genes are capable of rearranging to form a rearranged variable region gene that is expressed in conjunction with the constant region gene as a polypeptide chain of an antigen-binding protein. A control sequence "operably linked" to a coding sequence is linked such that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences adjacent to a gene of interest and expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0135] The term "expression control sequence" refers to polynucleotide sequences necessary to effect expression and processing of coding sequences to which they are ligated. "Expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak sequences), sequences that enhance protein stability, and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences generally include a promoter, a ribosomal binding site, and a transcription termination sequence, while in eukaryotes, such control sequences typically include a promoter and a transcription termination sequence. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and can also include additional elements whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0136] Generally, each unrearranged immunoglobulin V, D, or J gene segment is operably linked (e.g., associated with, flanked at one or both ends, flanked, etc.) to a recombination signal sequence (RSS), which may be a 12-mer RSS or a 23-mer RSS. H Any gene segment flanked on both sides by a 23-mer RSS (including a gene segment) has not undergone recombination and thus can be considered an "unrearranged" gene segment. In some embodiments, an unrearranged gene segment herein is a gene segment in its germline (e.g., wild-type) configuration, e.g., a germline V H Gene segments and germline J H In contrast, germline D H Gene segments, e.g., unrearranged D HThe gene segment is flanked on each side by a 12-mer RSS. H Gene segments, e.g., engineered D H The gene segment is also unrecombined and therefore contains (i) a 23-mer RSS and (ii) a 12-mer RSS. H a gene segment (e.g., a D operably linked to a 23-mer RSS) H The gene segment is a 12-mer RSS and another D H Gene segments can be rearranged according to the 12 / 23 rule of recombination.

[0137] Physiological conditions: Includes the art-known meaning referring to the conditions under which a cell or organism lives and / or reproduces. In some embodiments, the term refers to the external or internal environmental conditions that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are conditions found within the body of a human or non-human animal, particularly at and / or within a surgical site. Physiological conditions typically include, for example, temperatures ranging from 20-40°C, 1 atmosphere of pressure, pH 6-8, glucose concentrations of 1-20 mM, atmospheric levels of oxygen, and gravity as occurs on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained at physiological conditions. In some embodiments, physiological conditions are those that occur within an organism (e.g., a non-human animal).

[0138] Polypeptide: Refers to any polymeric chain of amino acids.

[0139] In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that includes portions that occur naturally separately from one another (i.e., from two or more different organisms, e.g., human and non-human portions). In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it is designed and / or produced through the action of man. In some embodiments, a polypeptide may contain or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement from one another than found in the subject polypeptide (e.g., fragments that are directly linked in the parent polypeptide may be spatially separated in the subject polypeptide, or vice versa, and / or fragments may be in a different order in the subject polypeptide than in the parent polypeptide), thereby making the subject polypeptide a derivative of its parent polypeptide.

[0140] Recombinant: refers to a polypeptide that is designed, engineered, prepared, expressed, produced, or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, or a polypeptide isolated from a recombinant combinatorial human polypeptide library (Hoogenboom HR, 1997 TIB Tech. 15:62-70; Hoogenboom H., and Chames P., 2000, Immunology Today 21:371-378; Azzazy H., and Highsmith WE, 2002, Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW, 2002, BioTechniques 29:128-145), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al., 1992, Nucl. Acids Res. 20:6287-6295; Little M. et al., 2000, Immunology Today 21:364-370; Kellermann SA and Green LL, 2002, Current Opinion in Biotechnology 13:593-597; Murphy, AJ, et al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5153-5158, each of which is incorporated herein by reference in its entirety), or any other means involving intersplicing of selected sequence elements.

[0141] In some embodiments, one or more of such selected sequence elements occur in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements arise, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements of natural or synthetic origin. For example, in some embodiments, the recombinant polypeptide comprises a sequence found in the genome (or polypeptide) of a source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide comprises a sequence that occurs naturally separately from one another in two different organisms (e.g., a human and a non-human organism) (i.e., derived from two or more different organisms, e.g., a human and a non-human portion). In some embodiments, the recombinant polypeptide has an amino acid sequence that arises from mutagenesis (e.g., in vitro or in vivo in a non-human animal), and thus the amino acid sequence of the recombinant polypeptide is a sequence that is derived from and related to the polypeptide sequence, but may not naturally occur in the genome of the non-human animal in vivo.

[0142] Reference: Refers to a standard or control agent, animal, cohort, individual, population, sample, sequence, or value to which an agent, animal, cohort, individual, population, sample, sequence, or value of interest is compared. "Reference" can also refer to a "reference animal." A "reference animal" can have a modification described herein, a modification different from those described herein, or be unmodified (i.e., a wild-type animal). Generally, as will be understood by one of skill in the art, the reference agent, animal, cohort, individual, population, sample, sequence, or value will be determined or characterized under conditions comparable to those used to determine or characterize the agent, animal (e.g., mammal), cohort, individual, population, sample, sequence, or value of interest.

[0143] In some embodiments, the reference agent, animal, cohort, individual, population, sample, sequence, or value is tested and / or calculated substantially simultaneously with the testing or calculation of the agent, animal, cohort, individual, population, sample, sequence, or value of interest. In some embodiments, the reference agent, animal, cohort, individual, population, sample, sequence, or value is a known reference, optionally embodied in a tangible medium. In some embodiments, the reference may refer to a control.

[0144] Substantially: refers to the qualitative condition of exhibiting the complete or nearly complete extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or to a perfect state or achieve or avoid an absolute result. Thus, the term "substantially" is used to capture the possible lack of completeness inherent in many biological and chemical phenomena.

[0145] Substantial homology: refers to similarity between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with reasonably similar structural and / or functional characteristics. For example, as will be known to those skilled in the art, certain amino acids are commonly classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid with another amino acid of the same type is often considered a "homologous" substitution. Common amino acid classifications are summarized below. [Table 8] [Table 9]

[0146] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol., 215(3):403-410; Altschul et al., 1996, Methods Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, A. D. and B. F. F. Huellette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1998. In addition to identifying homologous sequences, the above-mentioned programs generally provide an indication of the degree of homology.

[0147] In some embodiments, two sequences are considered to be substantially homologous if at least 95% or more of the corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 9 or more residues. In some embodiments, the relevant stretch includes adjacent residues along the complete sequence. In some embodiments, the relevant stretch includes non-contiguous residues along the complete sequence, e.g., non-adjacent residues brought together by the folded structure of the polypeptide or portion thereof. In some embodiments, the relevant stretch is at least 10 or more residues.

[0148] "Substantial identity" refers to similarity between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN and BLASTP for nucleotide sequences, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are described in Altschul, S. F. et al., 1990, J. Mol. Biol., 215(3):403-410; Altschul et al., 1996, Methods Enzymol. 266:460-80; Altschul, S. F. et al., 1997, Nucleic Acids Res., 25:3389-3402; Baxevanis, A. D. and B. F. F. Feuillette (eds.) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.) Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1998. In addition to identifying identical sequences, the above-mentioned programs often also provide an indication of the degree of identity.

[0149] In some embodiments, two sequences are considered to be substantially identical if at least 95% or more of the corresponding residues over the relevant stretch of residues are identical. In some embodiments, the relevant stretch is the complete sequence. In some embodiments, the relevant stretch is at least 10 or more residues.

[0150] Targeting vector or targeting construct: refers to a polynucleotide molecule that contains a targeting region. The targeting region contains a sequence that is identical or substantially identical to the sequence of a target cell, tissue, or animal, and integrates the targeting construct into a location in the genome of the cell, tissue, or animal by homologous recombination. Also included are targeting regions that use site-specific recombinase recognition sites (e.g., loxP sites or Frt sites) for targeting.

[0151] In some embodiments, the targeting constructs described herein further comprise a nucleic acid sequence or gene of particular interest, a selectable marker, control and / or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated by the exogenous addition of proteins that assist or promote recombination involving such sequences. In some embodiments, the targeting constructs described herein further comprise all or a portion of a gene of interest, where the gene of interest is a heterologous gene that encodes all or a portion of a polypeptide with a similar function to the protein encoded by the endogenous sequence. In some embodiments, the targeting constructs described herein further comprise all or a portion of a humanized gene of interest, where the humanized gene of interest encodes all or a portion of a polypeptide with a similar function to the polypeptide encoded by the endogenous sequence. In some embodiments, the targeting construct (or targeting vector) may comprise a nucleic acid sequence that has been engineered by human hands. For example, in some embodiments, a targeting construct (or targeting vector) can be constructed to contain an engineered or recombinant polynucleotide that includes two or more sequences that are not linked to each other in nature but have been manipulated by the hand of man to be directly linked to each other in the engineered or recombinant polynucleotide.

[0152] Transgene or transgene construct: refers to a nucleic acid sequence (e.g., a sequence encoding all or part of a polypeptide of interest) that has been introduced into a cell by the hand of man (e.g., using the methods described herein). A transgene may be partially or wholly heterologous, i.e., foreign to the transgenic animal or cell into which it is introduced. A transgene may include one or more transcriptional control sequences, e.g., introns or promoters, and any other nucleic acids that may be required for expression of a selected nucleic acid sequence. A transgene may include one or more selectable markers to allow subsequent selection of progeny (e.g., cells) that have incorporated the transgene.

[0153] Transgenic Animals, Transgenic Non-Human Animals, or Tg + : Used interchangeably herein to refer to any non-naturally occurring non-human animal, in which one or more of the cells of the non-human animal contains a heterologous nucleic acid and / or gene encoding all or part of a polypeptide of interest.

[0154] In some embodiments, heterologous nucleic acid sequences and / or genes are introduced directly or indirectly into cells by introduction into progenitor cells via deliberate genetic manipulation, such as by microinjection or infection with a recombinant virus. The term genetic manipulation does not include traditional breeding techniques, but rather covers the introduction of recombinant DNA molecules. This molecule may be integrated into a chromosome or may be extrachromosomally replicating DNA. The term "Tg+" includes animals that are heterozygous or homozygous for the heterologous nucleic acid and / or gene, and / or animals that have a single copy or multiple copies of the heterologous nucleic acid and / or gene.

[0155] Vector: refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been associated.

[0156] In some embodiments, vectors are capable of extrachromosomal replication and / or expression of nucleic acids to which they are linked in host cells, such as eukaryotic and / or prokaryotic cells. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors."

[0157] Wild-type: has the meaning known in the art to refer to an entity having a structure and / or activity that occurs in nature in a "normal" state or situation (as opposed to a mutated, diseased, altered state or situation, etc.). Those of skill in the art will understand that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0158] Other features, objects, and advantages of the present invention will be apparent from the following detailed description of several embodiments. However, it should be understood that, while the detailed description indicates several embodiments of the present invention, they are presented by way of illustration and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0159] Detailed Description of Specific Embodiments The present invention provides, inter alia, transgenic or engineered non-human animals having heterologous genetic material encoding one or more portions (functional fragments, binding portions, etc.) of a human immunoglobulin, wherein the heterologous genetic material is a heavy chain constant (C H) gene. It is intended that such non-human animals will exhibit the ability to produce antibodies encoded by the rearranged V(DD)J sequence. It is also intended that such non-human animals will exhibit an antibody population characterized by heavy chain variable regions with increased CDR3 diversity compared to antibody populations with immunoglobulin heavy chain variable CDR3 diversity produced from wild-type immunoglobulin heavy chain variable region loci (or naturally occurring immunoglobulin heavy chain variable region loci). Thus, the provided non-human animals are particularly useful for the development of antibody-based therapeutics that bind to specific antigens, particularly low and / or poorly immunogenic or antigens characterized by one or more epitopes that are not preferably bound by conventional (or wild-type) antibodies. In particular, the present invention provides a method for the development of antibody-based therapeutics that utilizes a D(DD)J sequence in an immunoglobulin heavy chain variable region. H D in one or more areas H Introduction of a 23-mer recombination signal sequence adjacent to the segment (e.g., 5' or 3'), which allows for D recombination during VDJ recombination. H and D H It provides the ability to reconstruct segments, and a single D H The transgenic non-human animals encompass expression of antibodies having heavy chain variable regions, particularly CDR3 regions, that may be characterized by longer amino acid lengths compared to antibodies generated from VDJ recombination involving these segments. These transgenic non-human animals provide an in vivo system for identifying and developing antibodies and / or antibody-based therapeutics that bind disease targets beyond the targeting capabilities of established drug discovery technologies. Furthermore, these transgenic non-human animals provide a useful animal model system for the development of antibodies and / or antibody-based therapeutics that focus on or are designed to disrupt protein-protein interactions central to various diseases and / or disease conditions that affect humans.

[0160] As shown in the top panel of Figure 1, recombination between immunoglobulin gene segments follows what is commonly referred to as the 12 / 23 rule, in which gene segments flanked by recombination signal sequences (RSSs) are joined by an ordered process. Each RSS is connected to a coding sequence (e.g., V H , D H or J H Each RSS consists of a conserved block of seven consecutive nucleotides (heptamer; 5'-CACAGTG-3'; SEQ ID NO: 144) followed by a non-conserved region known as the spacer, which can be either 12 or 23 bp in length, and a second conserved block of nine nucleotides (nonamer; 5'-ACAAAAACC-3'; SEQ ID NO: 145). The spacer can vary in sequence, but its conserved length corresponds to one or two turns of the DNA double helix. This allows the heptamer and nonameric sequences to migrate to the same side of the DNA helix and bind to the protein complex that catalyzes recombination. An RSS containing a 23-bp spacer is a 23-mer RSS, and an RSS containing a 12-bp spacer is a 12-mer RSS. Although exceptions have been reported, the 12 / 23 rule of recombination generally promotes recombination between a 12-mer RSS and a 23-mer RSS and discourages recombination between a 23-mer RSS and another 23-mer RSS, or between a 12-mer RSS and another 12-mer RSS, e.g., direct germline V H and germline J H Recombinant (i.e., 23mer-to-23mer combination) or germline D H and germline D H Prevents recombination (i.e., 12mer-12mer joining).

[0161] In some embodiments, the non-human animals described herein each comprise a 23-mer RSS operably linked thereto, and thus a D H -D H Recombination-competent, one or more D H The engineered diversity cluster (i.e., engineered D HIn some embodiments, antibodies containing CDR3s generated from such recombination may be characterized by having increased diversity resulting from the longer amino acid length, which directs direct binding to specific antigens (e.g., viruses, membrane channels, etc.). In some embodiments, the non-human animals described herein comprise an immunoglobulin heavy chain variable region containing a VDJ recombination site that is associated with the VDJ recombination site. H , J H and one more D H engineered D to occur between segments to create a heavy chain variable region that binds to the antigen of interest. H a human heavy chain variable (V H ) and bond (J H ) gene segments. In some embodiments, the engineered D H The region is characterized by increased frequency of D compared to a reference immunoglobulin heavy chain variable region locus. H and D H one or more (e.g., 1, 2, 3, 4, 5, 10, or more) human Ds engineered to allow (or promote) recombination H In some embodiments, the non-human animals described herein contain one or more D segments operably linked to a 23-mer recombination signal sequence (RSS) in an immunoglobulin heavy chain variable region within the genome of the non-human animal. H a plurality of V's operably coupled to the segment; H and J H In many embodiments, the V H and J. H The segment is human V H and human J H It is a gene segment.

[0162] In some embodiments, the non-human animals described herein further comprise a human or humanized immunoglobulin light chain locus (e.g., κ and / or λ), such that the non-human animal produces antibodies comprising a human variable region (i.e., heavy and light chain) and a non-human constant region. In some embodiments, the human or humanized immunoglobulin light chain locus comprises a human Vκ or Cλ operably linked to a rodent light chain constant region (e.g., rodent Cκ or Cλ). L and J. L In some embodiments, the non-human animals described herein comprise gene segments. In some embodiments, the non-human animals described herein are those described in U.S. Patent Nos. 9,796,788; 9,969,814; U.S. Patent Publication Nos. 2011 / 0195454A1, 2012 / 0021409A1, 2012 / 0192300A1, 2013 / 0045492A1, 2013 / 0185821A1, 2013 / 0302836A1, 2018 / 01 25043; International Patent Application Publication Nos. 2011 / 097603, 2012 / 148873, 2013 / 134263, 2013 / 184761, 2014 / 160179, 2014 / 160202, and 2019 / 113065 (each of which is incorporated by reference in its entirety).

[0163] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the embodiments described herein. Each section may be applicable to any aspect or embodiment described herein. In this application, the use of "or" means "and / or" unless otherwise specified.

[0164] VDJ recombination Genes involved in immunoglobulin synthesis are found in all cells of an animal and are arranged in gene segments located sequentially along chromosomes. The organization of inherited human gene segments, e.g., the germline configuration of human gene segments, e.g., the order of human gene segments in a human germline genome (e.g., the genome passed on to the next generation), is found in Lefranc, M.-P., Exp. Clin. immunogenet., 18, 100-116 (2001), which is incorporated herein by reference in its entirety, and which also shows the functional gene segments and pseudogenes found within the human immunoglobulin heavy chain locus in their germline configuration. A series of recombination events involving several genetic components helps assemble immunoglobulins from an ordered sequence of gene segments (e.g., V, D, and J). This assembly of gene segments is known as imprecise; therefore, immunoglobulin diversity is achieved both by the combination of different gene segments and by the formation of unique combinations through imprecise combinations. Further diversity is generated through a process known as somatic hypermutation, in which immunoglobulin variable region sequences are modified to increase affinity and specificity for antigens. Immunoglobulin molecules are Y-shaped polypeptides composed of two identical heavy chains and two identical light chains, each of which has two structural components: one variable domain and one constant domain. It is the heavy and light chain variable domains that are formed by the assembly of gene segments, while the constant domain is fused to the variable domain through RNA splicing. The mechanism for assembling (or joining) the gene segments is similar for heavy and light chains, but only one joining event is required for the light chain (i.e., V to J), while two events are required for the heavy chain (i.e., D to J and V to DJ).

[0165] The assembly of gene segments for heavy and light chain variable regions (referred to as VDJ recombination and VJ recombination, respectively) is guided by conserved non-coding DNA sequences flanking each gene segment, called recombination signal sequences (RSSs), which ensure DNA rearrangement at precise locations relative to the V, D, and J coding sequences (see, e.g., Ramsden, DA et al., 1994, Nuc. Acids Res. 22(10):1785-96; incorporated herein by reference in its entirety). A representative schematic of the sequences involved in VDJ recombination of heavy chain gene segments as understood by those skilled in the art is set forth in FIG. 1. Each RSS consists of a coding sequence (e.g., a V, D, or J segment), followed by a conserved block of seven nucleotides (heptamer) contiguous with a spacer (either 12 bp or 23 bp), and a second conserved block of nine nucleotides (nonamer). Although considerable sequence variance is tolerated in the 12-bp or 23-bp spacers between individuals, the lengths of these sequences typically do not vary. Recombination between immunoglobulin gene segments follows what is commonly referred to as the 12 / 23 rule, in which a gene segment flanked by RSSs having a 12-bp spacer (or 12-mer) typically combines with a gene segment flanked by a 23-bp spacer (or 23-mer; see, e.g., Hiom, K. and M. Gellert, 1998, Mol. Cell., 1(7):1011-9, which is incorporated herein by reference in its entirety).The sequence of the RSS has been reported to affect the efficiency and / or frequency of recombination with a particular gene segment (see, e.g., Ramsden, DA and GEWu, 1991, Proc. Natl. Acad. Sci. USA, 88:10721-5; Boubnov, NV et al., 1995, Nuc. Acids Res., 23:1060-7; Ezekiel, UR et al., 1995, Immunity 2:381-9; Sadofsky, M. et al., 1995, Genes Dev. 9:2193-9; Cuomo, CA et al., 1996, Mol. Cell Biol., 16:5683-90; Ramsden, DA et al., 1996, EMBO J 15:3197-3206; each of which is incorporated herein by reference in its entirety). Indeed, numerous reports have shown that gene segments, particularly D. H It has been pointed out that segment usage is highly biased and variable between individuals. Unless otherwise indicated, or unless it is clear to one skilled in the art that a contradiction or inconsistency occurs, without reference to an RSS, an unrearranged gene segment is presumed to comprise two RSSs that the gene segment is naturally associated with, e.g., adjacent, and in some cases operably linked, etc. In some embodiments, an unrearranged gene segment herein refers to a gene segment in its germline (e.g., wild-type) configuration, e.g., a germline V, each flanked by 23-mer RSSs on both ends. H Gene segments and germline J H In contrast, germline D H Gene segments, e.g., unrearranged D H The gene segment is flanked on each side by a 12-mer RSS.

[0166] Thus, an unrearranged gene segment also refers to a gene segment in its germline configuration, including any RSSs associated with such germline configuration. Furthermore, multiple gene segments in their germline configuration generally refer not only to each individual gene segment in its germline (e.g., unrearranged) configuration, but also to the order and / or position of functional gene segments. See, for example, Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), which is incorporated herein by reference in its entirety, for a discussion of the germline configuration of human V, D, and J gene segments.

[0167] The assembly of gene segments to form heavy and light chain variable regions results in the formation of an immunoglobulin antigen-binding region (or site). Such antigen-binding regions are characterized, in part, by the presence of hypervariable regions commonly referred to as complementarity-determining regions (CDRs). There are three CDRs for both heavy and light chains (i.e., six CDRs total), with both CDR1 and CDR2 being fully encoded by the V gene segment. However, CDR3 is encoded by the sequence resulting from the joining of the V and J segments for the light chain and the V, D, and J segments for the heavy chain. Thus, during recombination, the additional gene segments utilized to form the heavy chain variable region coding sequence significantly increase the diversity of the heavy chain antigen-binding site. Thus, the engineered D CDRs described herein H The provided non-human animal containing the region is H From D H This results in CDR3 diversity characterized by recombination into a CDR3 region having an increased amino acid length compared to the CDR3 region of a heavy chain variable region generated by conventional VDJ recombination.

[0168] Without being bound by theory, it is believed that further diversity in the heavy chain CDR3 repertoire may be possible through increasing the number of bonds forming the rearranged heavy chain variable region gene sequence and / or increasing the length of the CDR3 region. One mechanism for increasing the number of bonds and / or increasing the length of the CDR3 region is to increase the number of bonds. H (D H AD H B)J H Derive the gene sequence, followed by D H -J H and V.D. H J H Prior to recombination, D H -D H In the recombination phenomenon, the first D H Segment (D H Another D (which may be commonly referred to as "A") H Segment (D H In nature, D H -D H Recombination events have long been thought to be prevented by the 12 / 23 rule (Alt, FW et al., 1984, EMBO J. 3(6):1209-19, incorporated herein by reference in its entirety). However, D H -D HRecombination events occur in a 12 / 23 rule (1 in 800 or approximately 0.125% of naive B cells; see e.g., Ollier, PJ et al., 1985, EMBO J. 4(13B):3681-88; Milner, ECB et al., 1986, Immunol. Today 7:36-40; Liu, Z. et al., 1987, Nucleic Acids Res., 15(11):4688; Liu, Z. et al., 1987, Nucleic Acids Res., 15(15):6296; Meek, KD et al., 1989, J. Exp. Med. 169(2):519-33; Meek, KD et al., 1989, J. Exp. Med. 170:39-57; Baskin, B. et al., 1998, Clin. Exp. Immunol., 112:44-7; Briney, B. S. et al., 2012, Immunol. 137:56-64; each of which is incorporated by reference in its entirety) and is thought to be the primary mechanism generating the abnormally long CDR3s observed in some heavy chains (Janeway's Immunobiology., 9th ed., Kenneth Murphy, Casey Weaver., Chapter 5, 2017; incorporated by reference in its entirety). However, some potential therapeutic targets (e.g., but not limited to, viruses, cell surface receptors, type IV transmembrane proteins such as GPCRs, ion channels) mask or conceal epitopes that are inaccessible to normal antibodies but can be recognized by antibodies with long HCDR3 sequences. For example, antibodies with very long HCDR3s are often found in patients with chronic viral infections and, in some cases, have broad neutralizing activity (e.g., broadly neutralizing antibodies against HIV-1 or influenza). To select antibodies capable of reaching these hidden epitopes, it is useful to increase the frequency of heavy chains with very long HCDR3s. Without wishing to be bound by theory, one way to achieve this is to use heavy chains containing a 23-mer RSS and a D H Longer J gene segments H 6 to increase the frequency of recombination into gene segments.H By engineering the heavy chain V segment H (D H AD H B)J H In mice, D H -J H Binding occurs in two ordered steps: (1) the proximal DQ52 segment (D in humans) H 7-27) H Segment (J H 1 or J H 2) is thought to arise from a primary rearrangement into one of the two. Then, the strong μ promoter upstream of DQ52 drives the remaining J H Segment (J H 3 and J H 4) becomes more accessible to recombination activating genes (RAGs); (2) distal D H The remaining J of the segment H Segment (J H 3 or J H 4). See the top panel of Figure 1. This is a second reconstruction into one of the mouse (J H 3-J H 4) and humans (J H 4-J H 6) Downstream J observed in both H This is consistent with the more frequent use of the D segment (Nitschke et al., 2001, J. Immunol., 166:2540-52, incorporated herein by reference in its entirety). Without wishing to be bound by theory, H 7-27 and J H 1-J H 3 (or J H 1-J H 5) Synthetic D having a 5' 23mer RSS and a 3' 12mer RSS H Genes (e.g., synthetic D H 3-3 segment) substitutions are common in V H (D H -D H )J H It is hypothesized that this can occur by a three-step mechanism: (1) 23(D H)J H To produce J H 4. J H 5, or J H 23-D to 6 H -12 reconstruction, (2)12(D H -D H )J H Distal D to produce H (D H 1-1~D H 1-26) 23(D H )J H Reconstruction to (23(D H )J H V to H (3) V to generate the V V coding sequence encoding the immunoglobulin heavy chain variable domain. H 12(D H -D H )J H See, e.g., the bottom panel of Figure 1. Also, long Ds that can form disulfide bonds that are thought to stabilize long HCDR3 regions (see, e.g., Wang et al., 2013, Cell 153:1379-93, incorporated herein by reference in its entirety) are reconstituted into H A gene segment (longer than 31 nucleotides that encodes two cysteines) called D H 2-2, D H 2-8, and D H Replacement of gene segments 2-15 with those gene segments operably linked to a 12-mer RSS at the 5' end and a 23-mer RSS at the 3' end, respectively, also occurs via a three-step mechanism: (1) 12(D H )J H To produce J H 4. J H 5, or J H 6 to 12:D H -12 reconstruction, (2)12(D H -D H )J H Distal 12:D to produce H 2-2:23, 12:D H 2-8:23 or 12:DH 2-15-23 12(D H )J H and (3) a V to generate a V coding sequence encoding an immunoglobulin heavy chain variable domain. H 12(D H -D H )J H High frequency V that can be generated by rearrangement to H (D H -D H )J H It is also hypothesized that recombination occurs. As described herein, engineered D H A region consists of one or more D H segment, and placed a 23-mer spacer at either the 5' or 3' flanking position, thereby creating a D in the humanized immunoglobulin heavy chain variable region locus. H From D H It will be possible to convert it into

[0169] In some embodiments, the non-human animals described herein comprise immunoglobulin heavy chain variable region loci that exhibit VDJ recombination that does not follow the 12 / 23 rule compared to a reference non-human animal. In some embodiments, the non-human animals described herein comprise wild-type D H Compared to the segment, one or more modified D H In some embodiments, the non-human animal immunoglobulin heavy chain variable region comprises one or more RSSs adjacent to or adjacent to the D segment. H The gene segment is D H -D HEach is operably linked to either a 5' or 3' 23-mer RSS such that the frequency of recombination is increased in the non-human animal compared to a reference non-human animal. Recombination efficiency and / or frequency can, in some embodiments, be determined by the frequency of gene segment usage in a population of antibody sequences (e.g., from an individual or group of individuals) (see, e.g., Arnaout, R. et al., 2011, PLoS One 6(8):e22365; Glanville, J. et al., 2011, Proc. Natl. Acad. Sci. USA, 108(50):20066-71, the entire contents of which are incorporated herein by reference). Thus, the non-human animals described herein, in some embodiments, can be D H -D H Recombination in the reference non-human animal H -D H one or more Ds each operably linked to or adjacent to the 23mer RSS so as to occur at an increased frequency compared to recombination. H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least three-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least four-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least 5-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -DH At least 10-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least 20-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least 30-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least 40-fold increased frequency of D compared to recombinant H -D H In some embodiments, the engineered D H The rodent containing region is the reference D in non-human animals. H -D H At least 50-fold increased frequency of D compared to recombinant H -D H Recombination is shown.

[0170] Provided in vivo systems The present invention is based on the recognition that certain antigens are associated with low and / or poor immunogenicity and are therefore poor targets for antibody-based therapies. Indeed, many disease targets (e.g., viruses, channel proteins) have been characterized as intractable or not conducive to the development of new drugs. Thus, the present invention is based on the creation of an in vivo system for developing antibodies and antibody-based therapies that overcomes the challenges associated with established drug development technologies and / or approaches. In some embodiments, the present invention provides a method for the development of antibodies and antibody-based therapies that utilize one or more D HRSS is a 12-mer D H -12mer format to 12mer D H -23mer or 23mer D H -12mer format, modified, or engineered to achieve the D observed in the reference in vivo system. H From D H increased frequency of recombination compared to H From D H engineered D that allows recombination into H The present disclosure provides an in vivo system characterized by the presence of an immunoglobulin locus, particularly a humanized immunoglobulin heavy chain variable region locus, comprising an engineered D H We demonstrate the construction of transgenic rodents containing immunoglobulin heavy chain variable regions containing engineered D H The area is D with increased frequency H -D H One or more Ds that allow recombination H One or more Ds each operably linked to a 23-mer RSS located relative to each of the segments H The methods described herein include the engineered D H To generate the region, any number of Ds each operably linked to a 5' or 3' 23mer RSS H Segments (e.g., conventional or synthetic) can be tailored to achieve engineered D H Once the region is assembled into an immunoglobulin heavy chain variable region (i.e., V H and J. H and / or one or more constant regions) and heavy chains with added diversity to direct binding to specific antigens (i.e., long CDR3s, e.g., at least 95% of the heavy chain CDR3 sequences are at least 14 amino acids in length). H and J. H More than one D in a gene segment HIn some embodiments, such heavy chain variable regions have the ability to access hard-to-reach epitopes of viruses, channel proteins, GPCRs, and the like.

[0171] While not wishing to be bound by any particular theory, the inventors believe that the data provided herein suggest that, in some embodiments, the genome is composed of a D H The engineered D characterized by the inclusion of H The inventors have noted that rodents containing immunoglobulin heavy chain variable loci containing the V(DD)J region are shown to efficiently generate antibodies produced by V(DD)J recombination. The inventors have noted that the data provided herein demonstrate that, in some embodiments, the genome contains three D(DD)J regions operably linked to a 3' 23-mer RSS, each of which is a nucleotide sequence. H The engineered D characterized by the inclusion of H We also noted that rodents containing immunoglobulin heavy chain variable loci containing the J region exhibit efficient production of antibodies produced by V(DD)J recombination. H Some or all five J genes upstream of the six gene segments H Deletion of a gene segment results in J H It is also shown herein that the J H The six gene segments contain 63 nucleotides, e.g., 52, 53, 50, 40, and 51 nucleotides, respectively. H 1. J H 2. J H 3. J H 4, and J H Thus, at least in some embodiments, the present disclosure provides a method for identifying a rearranged immunoglobulin heavy chain variable region gene sequence, e.g., a V gene encoding a heavy chain variable domain having a CDR3 of at least 20 amino acids in length, e.g., a CDR3 of 20 to 30 amino acids in length. H D H J H and V H (DH AD H B)J H , e.g., V H (D H AD H B)J H In some embodiments, the present invention encompasses the development of in vivo systems for generating antibodies and / or antibody-based therapies against intractable disease targets by providing rodents that produce the rearranged immunoglobulin heavy chain variable region gene sequences, e.g., V6 sequences, of the non-human animals described herein. H D H J H and V H (D H AD H B)J H At least 8-10% of the gene sequence encodes a CDR3 region that is at least 21 amino acids in length.

[0172] In some embodiments, (1) a D operably linked to a 23-mer RSS in its germline genome, e.g., a germline cell. H engineered D containing gene segments H (2) the immunoglobulin heavy chain locus containing the region, and (3) the somatic genome, e.g., in B cells, the rearranged heavy chain V H (D H AD H B)J H A non-human animal, e.g., a rodent, e.g., a rat or mouse, comprising a coding sequence is described herein, and the first or second D H gene segments (i.e., V H (D H AD H B)J H D in the coding sequence H A or D H B) D operably linked to the 23mer RSS H a gene segment, or a portion thereof, e.g., a first or second D H The gene segment is operably linked to a 23-mer RSS. Hhaving at least 9 consecutive nucleotides aligned with the gene segment, and H Each gene segment has an unrelated corresponding germline D H It comprises at least 5 consecutive nucleotides aligned with the gene segment.

[0173] In some embodiments, the non-human animal provided comprises a plurality of human Vs configured in germline configuration. H , D H and J. H In some embodiments, the non-human animals provided comprise an immunoglobulin heavy chain locus operably linked to a non-human immunoglobulin heavy chain constant region, enhancer, and regulatory region, characterized by the presence of one or more human V gene segments. H gene segment, one or more human D H gene segments, and one or more human J gene segments operably linked to a non-human immunoglobulin heavy chain constant region. H Contains gene segments.

[0174] In some embodiments, the non-human animal provided has at least one human V H Gene segment V H 3-74, V H 3-73, V H 3-72, V H 2-70, V H 1-69, V H 3-66, V H 3-64, V H 4-61, V H 4-59, V H 1-58, V H 3-53, V H 5-51, V H 3-49, V H 3-48, V H 1-46, V H 1-45, V H 3-43, V H 4-39, V H 4-34, V H 3-33, V H 4-31, V H3-30, V H 4-28, V H 2-26, V H 1-24, V H 3-23, V H 3-21, V H 3-20, V H 1-18, V H 3-15, V H 3-13, V H 3-11, V H 3-9, V H 1-8, V H 3-7, V H 2-5, V H 7-4-1, V H 4-4, V H 1-3, V H 1-2 and V H Including 6-1.

[0175] In some embodiments, the provided non-human animal is a human D H Gene segment D H 1-1, D H 2-2, D H 3-3, D H 4-4, D H 5-5, D H 6-6, D H 1-7, D H 2-8, D H 3-9, D H 3-10, D H 5-12, D H 6-13, D H 2-15, D H 3-16, D H 4-17, D H 5-18, D H 6-19, D H 1-20, D H 2-21, D H 3-22, D H 6-25 and D H 1-26. In some embodiments, the non-human animal provided is D H 1-14, D H 4-11, D H 4-23, D H5-24, or a combination thereof. In some embodiments, the non-human animal provided further comprises a human D H Also includes 7-27.

[0176] In some embodiments, (1) a D operably linked to a 23-mer RSS in its germline genome, e.g., a germline cell. H engineered D containing gene segments H (2) the immunoglobulin heavy chain locus containing the region, and (3) the somatic genome, e.g., in B cells, the rearranged heavy chain V H (D H AD H B)J H A non-human animal, e.g., a rodent, e.g., a rat or mouse, comprising a coding sequence is described herein, and the first or second D H gene segments (i.e., V H (D H AD H B)J H D in the coding sequence H A or D H B) D operably linked to the 23mer RSS H a gene segment, or a portion thereof, e.g., a first or second D H The gene segment is operably linked to a 23-mer RSS. H having at least 9 consecutive nucleotides aligned with the gene segment, and H Each gene segment has a corresponding germline D, irrespective of any overlap. H In some embodiments, the non-human animal comprises a human D operably linked to a 23-mer RSS. H In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H In some embodiments, the gene segment comprises two cysteine ​​codons. HThe gene segment contains at least 37 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment contains at least 19 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment contains at least 20 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment contains at least 23 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment contains at least 28 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H The gene segment contains at least 31 nucleotides. In some embodiments, the gene segment comprises a human D operably linked to a 23-mer RSS. H A gene segment contains at least 37 nucleotides.

[0177] In some embodiments, a human D operably linked to a 23mer RSS H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D3 gene segment operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D5 gene segment operably linked to a 23-mer RSS. HThe gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H It contains 7 gene segments.

[0178] In some embodiments, a human D operably linked to a 23mer RSS H The gene segment is human D H 1-1 gene segment. In some embodiments, the human D H The gene segment is human D H 2-2 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a 4-4 gene segment. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the 6-6 gene segment comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H 1-7 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H 2-8 gene segments. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. HThe gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23-mer RSS. H The gene segment is human D H 2-15 gene segments. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H 4-17 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H 1-20 gene segments. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H 2-21 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D HIn some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H 1-26 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H 1-14 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H 4-11 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H 4-23 gene segment. In some embodiments, a human D operably linked to a 23mer RSS. H The gene segment is human D H In some embodiments, the RSS comprises a human D operably linked to a 23mer RSS. H The gene segment is human D H Contains 7-27 gene segments.

[0179] In some embodiments, the non-human animals provided comprise human D loci typically arranged in the order found in unrearranged human genomic variable loci. H A complete or substantially complete repertoire of human D gene segments. H One of the gene segments was engineered to be operably linked to a 23mer RSS. H In some embodiments, a complete or substantially complete repertoire of human D gene segments is replaced. H One of the gene segments is a corresponding D engineered to be operably linked to a 23-mer RSS. H The gene segment is replaced with, for example, the wild-type D H The 2-2 gene segment is engineered to be operably linked to a 23mer RSS. HIn some embodiments, a complete or substantially complete repertoire of human D H One of the gene segments is engineered to be operably linked to a 23-mer RSS. H The gene segment is replaced with, for example, the wild-type D H The 7-27 gene segment was engineered to be operably linked to a 23-mer RSS. H In some embodiments, the 3-3 gene segments are replaced with human D sequences typically arranged in the order found in unrearranged human genomic variable loci. H D of a complete or substantially complete repertoire of gene segments H The 1-1 gene segment may be a corresponding or another D gene segment engineered to be operably linked to a 23-mer RSS, e.g., a 3' 23-mer RSS. H In some embodiments, the gene segments are replaced with human D gene segments generally arranged in the order found in unrearranged human genomic variable loci. H D of a complete or substantially complete repertoire of gene segments H 1-1, D H 2-2, D H 2-8, D H 2-15, D H The 2-21 gene segment or any combination thereof can be a corresponding or another D gene segment engineered to be operably linked to a 23-mer RSS, e.g., a 3' 23-mer RSS. H In some embodiments, the gene segments are replaced with human D gene segments generally arranged in the order found in unrearranged human genomic variable loci. H D of a complete or substantially complete repertoire of gene segments H 2-2, D H 2-8, and D H Each of the 2-15 gene segments may be a corresponding or another D gene segment engineered to be operably linked to a 23-mer RSS, e.g., a 3' 23-mer RSS. H It is replaced by a gene segment.

[0180] In some embodiments, the non-human animal provided comprises at least one human J H Gene segment J H In some embodiments, the non-human animal provided comprises at least one human J H Gene segment J H 4. J H 5 and J H In some embodiments, the non-human animal provided comprises a human J H Gene segment J H 1. J H 2. J H 3. J H 4. J H 5 and J H Includes 6.

[0181] In some embodiments, the non-human immunoglobulin heavy chain constant region comprises one or more non-human immunoglobulin heavy chain constant region genes, such as, for example, immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin A (IgA). In some embodiments, the non-human immunoglobulin heavy chain constant region comprises rodent IgM, rodent IgD, rodent IgG3, rodent IgG1, rodent IgG2b, rodent IgG2a, rodent IgE, and rodent IgA constant region genes. In some embodiments, the human V H , D H and J. H The gene segment is operably linked to one or more non-human immunoglobulin heavy chain enhancers (i.e., enhancer sequences or enhancer regions). H , D H and J. H The gene segment is operably linked to one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences). H , D H and J. H The gene segment is operably linked to one or more non-human immunoglobulin heavy chain enhancers (or enhancer sequences) and one or more non-human immunoglobulin regulatory regions (or regulatory sequences).

[0182] In some embodiments, the provided non-human animals do not contain (or lack) an endogenous Adam6 gene. In some embodiments, the provided non-human animals do not contain or lack an endogenous Adam6 gene (or Adam6 coding sequence) in the same germline genomic location as found in the germline genome of a wild-type non-human animal of the same species. In some embodiments, the provided non-human animals do not contain or lack a human Adam6 pseudogene. In some embodiments, the provided non-human animals comprise an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion may be within an engineered immunoglobulin heavy chain locus or elsewhere (e.g., a randomly introduced non-human Adam6 coding sequence) in the germline genome of the non-human animal, cell, or tissue, outside of the engineered immunoglobulin heavy chain locus described herein (e.g., the 5'-most V H In some embodiments, the non-human animal provided does not contain or lacks a functional endogenous Adam6 pseudogene.

[0183] In some embodiments, the provided non-human animals, non-human cells, or non-human tissues described herein contain endogenous non-human V in antibody molecules. H In some embodiments, the non-human animals, cells, or tissues provided herein do not detectably express, in whole or in part, the endogenous non-human V region in the antibody molecule. H Region (e.g., V H , D H and / or J H In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not contain (or lack or contain a deletion of) one or more nucleotide sequences encoding, in whole or in part, an endogenous non-human V. H , D H and J. H and having a germline genome that contains, in whole or in part, a deletion of a gene segment. In some embodiments, the non-human animals provided are capable of reproduction.

[0184] In some embodiments, the provided non-human animals further comprise an immunoglobulin κ light chain locus characterized by the presence of multiple human Vκ and Jκ gene segments arranged in a germline configuration and inserted upstream of and operably linked to non-human Cκ gene segments, hi some embodiments, the engineered immunoglobulin κ light chain locus comprises at least a human Vκ gene segment that appears in the proximal variable cluster (or proximal arm, or proximal duplication) of the human immunoglobulin κ light chain locus. In some embodiments, the engineered immunoglobulin κ light chain locus comprises at least human Vκ gene segments Vκ2-40, Vκ1-39, Vκ1-33, Vκ2-30, Vκ2-8, Vκ1-27, Vκ2-24, Vκ6-21, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, and Vκ4-1. In some embodiments, the engineered immunoglobulin κ light chain locus comprises at least 1, 2, 3, 4, 5, 10, or 15 human Vκ gene segments selected from Vκ2-40, Vκ1-39, Vκ1-33, Vκ2-30, Vκ2-8, Vκ1-27, Vκ2-24, Vκ6-21, Vκ3-20, Vκ1-17, Vκ1-16, Vκ3-15, Vκ1-12, Vκ3-11, Vκ1-9, Vκ1-8, Vκ1-6, Vκ1-5, Vκ5-2, and Vκ4- 1. In some embodiments, the engineered immunoglobulin κ light chain locus comprises human Jκ gene segments Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In some embodiments, the engineered immunoglobulin κ light chain locus comprises at least one, two, three, or four human Jκ gene segments selected from Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5.

[0185] In many embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or regions). In some embodiments, the human Vκ and Jκ gene segments are operably linked to a mouse Igκ light chain intronic enhancer region (Igκ Ei or Eiκ). In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences). In some embodiments, the human Vκ and Jκ gene segments are operably linked to a mouse Igκ light chain 3' enhancer region (Igκ3'Ei or 3'Eiκ). In some embodiments, the human Vκ and Jκ gene segments are operably linked to a mouse Eiκ and operably linked to a mouse 3'Eκ. In some embodiments, the human Vκ and Jκ gene segments are operably linked to one or more non-human immunoglobulin κ light chain enhancers (or enhancer sequences or enhancer regions) and one or more non-human immunoglobulin κ light chain control regions (or control sequences). In some embodiments, the engineered immunoglobulin κ light chain locus contains the same non-human immunoglobulin κ light chain enhancer region (or enhancer sequence) that appears in a wild-type immunoglobulin κ light chain locus. In some embodiments, the engineered immunoglobulin κ light chain locus contains a non-human Igκ light chain enhancer region (or enhancer sequence) that appears in a wild-type immunoglobulin κ light chain locus of a different species (e.g., a different rodent species).

[0186] In some embodiments, the engineered immunoglobulin κ light chain loci described herein do not contain (ie, lack) the human VpreB gene (or human VpreB gene coding sequence).

[0187] In some embodiments, the non-human Cκ gene of the engineered immunoglobulin κ light chain locus comprises a rodent Cκ gene, such as, for example, a mouse Cκ gene or a rat Cκ gene. In some embodiments, the non-human Cκ gene of the engineered immunoglobulin κ light chain locus is or comprises a mouse Cκ gene derived from a genetic background comprising a 129 strain, a BALB / c strain, a C57BL / 6 strain, a mixed 129xC57BL / 6 strain, or a combination thereof.

[0188] In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not detectably express an endogenous non-human Vκ region, in whole or in part, in their antibody molecules. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not contain (or lack, or contain deletions of) one or more nucleotide sequences encoding, in whole or in part, an endogenous non-human Vκ region in their antibody molecules. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that comprises deletions of endogenous non-human Vκ and Jκ gene segments, in whole or in part.

[0189] In some embodiments, the provided non-human animals further comprise a wild-type or inactivated (eg, by gene targeting) immunoglobulin κ light chain locus.

[0190] In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not detectably express, in whole or in part, an endogenous non-human Vλ region in their antibody molecules. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not contain (or lack, or contain deletions of) one or more nucleotide sequences encoding, in whole or in part, an endogenous non-human Vλ region in their antibody molecules. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that comprises, in whole or in part, a deletion of endogenous non-human Vλ and Jλ gene segments. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that comprises, in whole or in part, a deletion of endogenous non-human Vλ, Jλ, and Cλ gene segments.

[0191] Guidance for the generation of targeting vectors, non-human cells, and animals with such engineered immunoglobulin loci can be found in U.S. Patent Nos. 8,642,835, 8,697,940, 9,006,511, 9,012,717, 9,029,628, 9,035,128, 9,066,502, 9,150,662, and 9,163,092, which are incorporated herein by reference in their entireties. Those skilled in the art will recognize a variety of technologies known in the art for accomplishing such genetic engineering and / or manipulation of non-human (e.g., mammalian) genomes or for engineering such sequences for introduction into the germline genome of non-human animals.

[0192] DNA constructs In many cases, polynucleotide molecules containing the immunoglobulin gene segments described herein, particularly those containing one or more D-terminal fragments, each operably linked to a 5' or 3' 23-mer RSS, are also provided. H The segment is inserted into a vector, preferably a DNA vector, for replicating the polynucleotide molecule in a suitable host cell.

[0193] Depending on the size, D H Segments can be cloned directly from commercially available genomic sources or designed in silico based on published sequences available from GenBank. Alternatively, bacterial artificial chromosome (BAC) libraries can provide immunoglobulin sequences. BAC libraries contain an average insert size of 100-150 kb and can carry inserts as large as 300 kb (Shizuya et al., 1992, Proc. Natl. Acad. Sci., USA 89:8794-8797; Swiatek et al., 1993, Genes and Development 7:2071-2084; Kim et al., 1996, Genomics 34 213-218, the entire contents of which are incorporated herein by reference). For example, human and mouse genomic BAC libraries have been constructed and are commercially available (e.g., Invitrogen, Carlsbad, CA). Genomic BAC libraries can also be used as a source of immunoglobulin sequences as well as transcriptional regulatory regions.

[0194] Alternatively, immunoglobulin sequences may be isolated, cloned, and / or transferred from yeast artificial chromosomes (YACs). The entire immunoglobulin locus, or a substantial portion thereof, can be cloned and contained within one or several YACs. When multiple YACs are used and contain overlapping regions of homology, they can be recombined in a yeast host strain to produce a single construct representing the entire locus. YAC arms can be further modified with a mammalian selection cassette by introducing the construct into embryonic stem cells or embryos by methods known in the art and / or described herein.

[0195] The DNA construct can be prepared using methods known in the art. For example, the DNA construct can be prepared as part of a larger plasmid. Such preparation allows for cloning and selection of the correct construct using efficient methods known in the art. One or more Ds each operably linked to a 5' or 3' 23-mer RSS as described herein can be used.H The DNA fragments containing the segments can be located between convenient restriction enzyme sites on the plasmid so that they can be easily isolated from the remaining plasmid sequences for incorporation into the desired animal.

[0196] In some embodiments, the methods used in preparing the plasmids and transforming the host organisms are known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombinant methods, see Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989; the entire contents of which are incorporated herein by reference.

[0197] Non-human animal production D in the immunoglobulin heavy chain variable region of the genome of a non-human animal H and D H One or more Ds that allow recombination H Non-human animals are provided that express antibodies with heavy chain CDR3 diversity characterized by long amino acid lengths resulting from the integration of 23-mer RSSs flanking the segments. Suitable examples described herein include rodents, particularly mice. One or more D H The segment may, in many embodiments, be a heterologous D H segment (e.g., human D H segment) at increased frequency compared to wild-type or reference non-human animals. H and D H Recombinable D H Non-human animals, embryos, cells and targeting constructs for generating non-human animals, non-human embryos and cells containing the segments are also provided.

[0198] In some embodiments, one or more D H The segments are located in the diversity clusters (i.e., D) of immunoglobulin heavy chain variable regions in the genomes of non-human animals. HIn some embodiments, the D of an immunoglobulin heavy chain variable region is modified to be adjacent to (or operably linked to) a 5' or 3' 23-mer RSS within the D of an immunoglobulin heavy chain variable region. H In some embodiments, the D region (or portion thereof) of the immunoglobulin heavy chain variable region is not deleted (i.e., is intact). H The region (or a portion thereof) may each comprise one or more D nucleotides operably linked to a 5' or 3' 23-mer RSS. H In some embodiments, the D H One or more synthetic Ds in which all or substantially all of the regions are operably linked to a 5' or 3' 23-mer RSS, respectively. H segment, and in some embodiments, one or more conventional D H The gene segment is the D of the immunoglobulin heavy chain variable region. H In some embodiments, the D H The area is the conventional D H segments (i.e., one or more D operably linked to the 5' 12-mer RSS and the 3' 12-mer RSS, respectively). H segment) and manipulated D H A gene segment (i.e., one or more Ds operably linked to a 5' or 3' 23-mer RSS, respectively) H In some embodiments, the D H The area is composite D H In some embodiments, D H The region is human D H In some embodiments, D H The region is mouse D H In some embodiments, the engineered D H The region (or part thereof) is the manipulated D H A region (or part of it) contains one or more V H gene segment and / or one or more J HIn some embodiments, the engineered D is inserted into an immunoglobulin heavy chain variable region so as to be operably linked to the gene segment. H The region is inserted into one of the two copies of the immunoglobulin heavy chain variable region, and the engineered D H In some embodiments, the engineered D H Non-human animals homozygous for the region are provided. In some embodiments, the engineered D H A non-human animal heterozygous for the region is provided.

[0199] In some embodiments, the non-human animals described herein each contain one or more Ds operably linked to a 5' or 3' 23mer RSS in their genome. H D containing segment H Such non-human animals contain randomly integrated human immunoglobulin heavy chain variable regions, including engineered D H The engineered D gene may be described as having a human immunoglobulin heavy chain transgene containing the D region. H The region can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or gain of allele (GOA) or loss of allele (LOA) assays. In some such embodiments, the non-human animals described herein contain the engineered D H In some embodiments, the non-human animals described herein are heterozygous for the engineered D H In some embodiments, the non-human animals described herein are homozygous for the engineered D H In some embodiments, the non-human animals described herein are hemizygous for the engineered D H Contains one or more copies of the region.

[0200] In some embodiments, the engineered D of a non-human animal described herein HThe region comprises at least one D associated with (or operably linked to) the 5' 23mer RSS. H In some embodiments, the engineered D of a non-human animal described herein comprises a D segment. H The region comprises at least one D associated with (or operably linked to) the 3' 23mer RSS. H In some embodiments, the engineered D of a non-human animal described herein comprises a D segment. H The region comprises at least one human D H In some embodiments, the engineered D of a non-human animal described herein comprises a 3-3 segment. H The region comprises at least one human D H Contains two segments, human D H 2 segments are human D H 2-2, Human D H 2-8, Human D H 2-15 and human D H 2-21.

[0201] In some embodiments, the engineered D of a non-human animal described herein H The regions each have one or more Ds associated with (or operably linked to) a 5' 23mer RSS. H In some embodiments, the engineered D of a non-human animal described herein comprises a D segment. H The regions each have one or more Ds associated with (or operably linked to) a 3' 23mer RSS. H In some embodiments, the engineered D of a non-human animal described herein comprises a D segment. H The regions each comprise a human D-like protein associated with (or operably linked to) a 3' 23-mer RSS. H 2-2, Human D H 2-8, and human D H Includes 2-15 segments.

[0202] a human V operably linked to one or more non-human heavy chain constant region genes H and J. H More than one D from the immunoglobulin heavy chain locus containing gene segments H The present invention relates to a method for producing a non-human animal having an engineered D gene, comprising producing an antibody comprising a heavy chain variable region having a CDR3 region having an amino acid sequence encoded by the D gene. H Compositions and methods for producing a non-human animal comprising an immunoglobulin heavy chain variable region comprising an engineered D H The regions each comprise one or more Ds associated with (or operably linked to) a 5' or 3' 23-mer RSS. H In some embodiments, compositions and methods are provided for generating non-human animals that express such antibodies under the control of endogenous enhancers and / or endogenous regulatory sequences. In some embodiments, compositions and methods are provided for generating non-human animals that express such antibodies under the control of heterologous enhancers and / or heterologous regulatory sequences. The methods involve inserting one or more D(DD)J segments into the genome of the non-human animal such that an antibody is expressed that comprises an immunoglobulin heavy chain resulting from V(DD)J recombination. H segment, and wild-type D H D at increased frequency compared to segment H and D H This includes inserting other sequences that allow for recombination.

[0203] In some embodiments, the method further comprises: H D having a 5' 23mer RSS and a 3' 12mer RSS operably linked to the gene segment H As described herein, the method includes inserting DNA containing a gene segment. H In some embodiments, the method further comprises: HThe gene segment has a 5' 23-mer RSS and a 3' 12-mer RSS so that it is accessible to the RAG genes (e.g., RAG-1 and / or RAG-2) during recombination. H The method includes inserting the segment into a position relative to the μ promoter sequence. H The genetic material containing the segment and the flanking RSS is inserted into the genome of a non-human animal, thereby forming the flanking D H Gene segments and V H and J. H to allow recombination with the gene segment H The engineered D containing the segment and the required RSS H Non-human animals can be generated that have the region.

[0204] In some embodiments, the method comprises: H Three Ds with a 5' 12-mer RSS and a 3' 23-mer RSS operably linked to the gene segment H As described herein, the method includes inserting DNA containing a gene segment. H The gene segments each contain multiple Ds associated with 5' and 3' 12-mer RSSs. H In some embodiments, the method comprises: determining a D associated with a 5' 12-mer RSS and a 3' 23-mer RSS, respectively, located within a gene segment; H 2-2, D H 2-8 and D H 2-15 gene segments into a diversity cluster having a plurality of other DH gene segments, each associated with a conventional or wild-type RSS. H The genetic material, including the gene segment and flanking RSS, is inserted into the genome of a non-human animal, thereby forming the flanking D H Gene segments and V H and J. H to allow recombination with the gene segment H The engineered D containing the segment and the required RSS HNon-human animals can be generated that have the region.

[0205] Where appropriate, D H The sequence corresponding to (or encoding) the segment may be modified to contain codons that are optimized for expression in a non-human animal (see, e.g., U.S. Pat. Nos. 5,670,356 and 5,874,304, each of which is incorporated by reference in its entirety). A codon-optimized sequence is a synthetic sequence that preferably encodes the same polypeptide (or a biologically active fragment of the full-length polypeptide that has substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide. In some embodiments, D H The sequence corresponding to (or encoding) the segment may include sequences that have been altered to optimize codon usage for a particular cell type (e.g., rodent cells). For example, a D H The codons of the sequences corresponding to the segments may be optimized for expression in cells of a non-human animal, and such sequences may be described as codon-optimized sequences.

[0206] D operably linked to the 5' or 3' 23mer RSS H Segment D H Insertion into the area is H The segment is V H and J. H Gene segments (e.g., multiple V H and J. H The present invention utilizes relatively few genomic modifications to operably link the CDR3 fragments (gene segments) and results in the expression of antibodies comprising heavy chains characterized by CDR3s with longer amino acid lengths.

[0207] Methods for generating transgenic non-human animals, including knockouts and knockins, are well known in the art (see, e.g., Gene Targeting: A Practical Approach, Joyner, ed., Oxford University Press, Inc., (2000); incorporated herein by reference in its entirety). For example, the generation of transgenic rodents optionally involves disrupting the locus of one or more endogenous rodent genes (or gene segments) and, in some embodiments, inserting one or more Ds into the rodent genome, each operably linked to a 23-mer RSS, at the same location as the endogenous rodent gene (or gene segment). H In some embodiments, the RSS may include the introduction of one or more Ds each operably linked to a 23-mer RSS. H The segment is a randomly inserted fragment of the immunoglobulin heavy chain locus in the rodent genome. H In some embodiments, one or more Ds each operably linked to a 23mer RSS are introduced into the region. H The segment is the D of the endogenous immunoglobulin heavy chain locus in the rodent genome. H In some embodiments, an endogenous immunoglobulin heavy chain locus is altered, modified, or engineered to contain human gene segments (e.g., V and / or J) operably linked to one or more constant region genes (e.g., human or mouse).

[0208] The genome comprises one or more diversity clusters each operably linked to a 5' or 3' 23-mer RSS. H The engineered diversity clusters (i.e., D H and (iii) generating a rodent containing an immunoglobulin heavy chain variable region comprising an engineered D HA schematic diagram (not to scale) of a representative targeting vector for constructing the region and its integration into rodent embryonic stem (ES) cells is provided in Figure 2. Exemplary strategies and methods for inserting such vectors into an immunoglobulin heavy chain variable region in the genome of rodent ES cells are provided in Figures 3-8. In each of Figures 2-8, the NotI restriction enzyme recognition site shown is appropriate, the names and approximate locations (dotted lines) of various primer / probe sets (see Table 4) are shown for the various alleles indicated (not to scale), and unless otherwise noted, open symbols and lines represent human sequences, and filled symbols and black lines represent mouse sequences. The following abbreviations are used for each of the figures: spec: spectinomycin resistance gene; neo: neomycin resistance gene; hyg: hygromycin resistance gene; lp: loxP site-specific recombination recognition site; Ei: mouse heavy chain intron enhancer; IgM: mouse immunoglobulin M constant region gene; L: loxP site sequence; Frt: flippase recognition target sequence; μ0pro: μ0 promoter sequence.

[0209] As illustrated in Figure 2, one D operably linked to the 5' 23mer RSS. H segment (Figure 2, top and middle) and three segments D each operably linked to a 3' 23mer RSS H (Fig. 2, bottom) H DNA fragments containing the segments are generated using VELOCIGENE® technology (e.g., U.S. Patent No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659; incorporated herein by reference in their entireties) and molecular biology techniques known in the art. In Figure 2, unless otherwise indicated, open symbols and lines represent human sequences, while filled symbols and black lines represent mouse sequences. Human V H 6-1, D H 2-2, D H 2-8, D H2-15, D H 3-3, and J H 1. J H 2. J H 3. J H 4. J H 5 and J H The non-scaled relative positions of the six gene segments are shown in Figure 1. Any remaining gene segments that are present and unnamed are not included in the D H When a gene segment is present, it is indicated. Generally, the D is indicated by a dotted line. H The region is unrearranged human D H It contains a complete repertoire of gene segments (see, e.g., www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=locus&species=human&group=IGH; incorporated herein by reference in its entirety), with the following exceptions: the top two targeting vectors are D-seq vectors flanked by a 23-mer RSS at the 5' end and a 12-mer RSS at the 3' end; H The last D has been replaced by a sequence containing 3-3 gene segments. H The bottom targeting vector lacks the 7-27 gene (shown as open arrows); the bottom targeting vector is a D flanked by a 12-mer RSS at the 5' end and a 23-mer RSS at the 3' end. H 2-2 gene segment, flanked by a 12-mer RSS at the 5' end and a 23-mer RSS at the 3' end. H 2-8 gene segments and flanked by a 12-mer RSS at the 5' end and a 23-mer RSS at the 3' end. H Unrearranged human D replaced by the 2-15 gene segment H 2-2 gene segment, unrearranged human D H 2-8 gene segments, and D H 2-15 gene segment (engineered D H 2-2, D H 2-8, and D HEach of the 2-15 gene segments is shown as an open arrow. The open bars are 12:D to help ensure correct modification of the targeting vector / ES cells. H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 represent the positions of unique artificial 40mers homologous to primer and probe sequences located in the targeting vector. The sequences of the unique artificial 40mers designated as "1", "2", "10", "16", "8", and "18" are set forth as SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively.

[0210] The DNA fragments are assembled with homology arms for precise targeted insertion into the humanized immunoglobulin heavy chain variable region locus (Figures 3, 5, 7). A selection cassette (e.g., neomycin) flanked by site-specific recombination recognition sites (e.g., loxP) is included in the targeting vector to facilitate screening of ES cell clones for proper insertion and can be removed by transient expression of a recombinase (e.g., Cre) in positive ES cell clones (Figures 4, 6, 8). Once integrated into the immunoglobulin heavy chain locus, the DNA fragments contain sequences essential for the correct assembly (i.e., recombination), transcription, and expression of the heavy chain variable region. The targeting vector contains engineered D H The region is 5' human V H Genomic DNA contains a human J H The final targeting vector for integration into the genome of a non-human cell (e.g., rodent embryonic stem cell) is designed to be flanked by genomic DNA and non-human (e.g., rodent) genomic heavy chain constant region DNA (e.g., an intronic enhancer and an IgM constant region gene). H Genomic DNA (e.g., one or more V H containing gene segments), engineered D H area, 3'J Hgenomic DNA, and non-human (e.g., rodent) genomic heavy chain constant region DNA, all of which, once integrated into the genome of the non-human animal, form a V H Gene segment, engineered D H Area and J H The gene segments are operably linked to allow recombination between them. Once assembled, the targeting vector is linearized and electroporated into rodent ES cells.

[0211] The targeting vector is a vector containing a sequence (i.e., an engineered D H area) has more than one D H The segment is introduced into rodent (e.g., mouse) embryonic stem cells to render the non-human cell or animal (e.g., mouse) capable of expressing antibodies containing a CDR3 having the amino acids encoded by the segment.

[0212] As described herein, engineered D H A transgenic rodent is generated in which the region has been introduced into an immunoglobulin heavy chain locus of the rodent genome (e.g., an immunoglobulin heavy chain locus engineered to contain human variable region gene segments, which may be an engineered endogenous immunoglobulin heavy chain locus).

[0213] Immunoglobulin loci comprising human variable region gene segments are known in the art and are described in, e.g., U.S. Patent Nos. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,2 No. 32,449, No. 8,502,018, No. 8,697,940, No. 8,703,485, No. 8,754,287, No. 8,791,323, No. 8,809,051, No. No. 8,907,157, No. 9,035,128, No. 9,145,588, No. 9,206,263, No. 9,447,177, No. 9,551,124, No. 9,580,491 and 9,475,559, each of which is incorporated herein by reference in its entirety, and U.S. Patent Application Publication Nos. 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287, and 2015 / 0113668, each of which is incorporated herein by reference in its entirety. The entireties of which are incorporated herein by reference and can be found in PCT Application Publication Nos. 2007117410, 2008151081, 2009157771, 2010039900, 2011004192, 2011123708, and 2014093908, each of which is incorporated herein by reference in its entirety.

[0214] In some embodiments, the non-human animals disclosed herein contain engineered D2B1 cells that can be reconstituted in precursor B cells in mice. H These embodiments include endogenous complete human immunoglobulin transgenes containing the engineered D region (Alt et al., 1985, Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236, incorporated herein by reference in its entirety). HCompletely human immunoglobulin transgenes containing the IgG1 gene may be inserted (randomly), or endogenous immunoglobulin genes may be knocked out (Green et al., 1994, Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs, Nat Genet 7:13-21; Lonberg et al., 1994, Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 368:856-859; Jakobovits et al., 2007, From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice, Nat Biotechnol 25:1134-1143; each of which is incorporated by reference in its entirety), for example, endogenous immunoglobulin heavy and kappa light chain loci are inactivated, for example, by targeted deletion of a small but significant portion of each endogenous locus, followed by introduction of human immunoglobulin gene loci as randomly integrated large transgenes or minichromosomes (Tomizuka et al., 2000; Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies, PNAS USA 97:722-727; incorporated herein by reference in its entirety).

[0215] In some embodiments, the engineered D HHuman or humanized immunoglobulin heavy and light chain loci, including these regions, are located at endogenous immunoglobulin heavy and light chain loci, respectively. Large-scale in situ gene replacement methods for mouse germline immunoglobulin variable loci with human germline immunoglobulin variable loci while maintaining the reproductive ability of the mouse have been reported. See, for example, U.S. Patent Nos. 6,596,541 and 8,697,940, each of which is incorporated herein by reference in its entirety. Specifically, precise replacement of six megabases of both the mouse heavy and kappa light chain immunoglobulin variable loci with their human counterparts while leaving the mouse constant regions intact has been described. As a result, mice have been generated that have precise replacement of their entire germline immunoglobulin variable repertoire with equivalent human germline immunoglobulin variable sequences while maintaining the mouse constant regions. The human variable regions are then ligated to the mouse constant regions to form chimeric human-mouse immunoglobulin loci that are expressed at physiologically relevant levels. The antibodies expressed are "reverse chimeric," i.e., they contain human variable region sequences and mouse constant region sequences. These mice with humanized immunoglobulin variable regions that express antibodies with human or humanized variable regions and mouse constant regions are called VELOCIMMUNE® mice.

[0216] VELOCIMMUNE® humanized mice exhibit a fully functional humoral immune system essentially indistinguishable from that of wild-type mice. They exhibit normal cell populations at all stages of B cell development. They exhibit normal lymphoid organ morphology. Antibody sequences in VELOCIMMUNE® mice exhibit normal V(D)J rearrangements and normal somatic hypermutation frequencies. Antibody populations in these mice reflect the isotype distribution resulting from normal class switching (e.g., normal isotype switching). Immunization of VELOCIMMUNE® mice generates a stable humoral immune response that generates a large and diverse antibody repertoire with human immunoglobulin variable domains suitable for use as therapeutic candidates. This platform provides a rich source of native affinity-matured human immunoglobulin variable region sequences for generating pharmaceutically acceptable antibodies and other antigen-binding proteins. A single endogenous V H Gene segment, human V H Substitution with gene segments has also been shown to be capable of generating immune responses containing humanized immunoglobulin variable domains. See, e.g., Tien et al. (2016) Cell 166:1471-84, incorporated herein by reference in its entirety. By precisely substituting mouse immunoglobulin variable sequences for human immunoglobulin variable sequences, the human immunoglobulin variable sequences are operably linked to endogenous non-human constant region gene sequences in a reverse chimeric manner to generate VELOCIMMUNE® mice.

[0217] Mice modified in a reverse chimeric manner contain human (humanized) variable regions (e.g., comprising a D, J, and one or more human V gene segments) operably linked to endogenous constant regions at endogenous immunoglobulin loci, e.g., (a) At the endogenous heavy chain locus: (i) an unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region, the unrearranged human (humanized) immunoglobulin heavy chain variable region comprising a plurality of unrearranged human heavy chain variable region Vs; Hgene segments (e.g., all functional human unrearranged human V H gene segment), one or more unrearranged immunoglobulin heavy chains D H gene segment, and one or more unrearranged immunoglobulin heavy chain J H including gene segments, optionally, one or more unrearranged immunoglobulin heavy chains D H a gene segment and one or more unrearranged immunoglobulin heavy chain J H The gene segment may comprise one or more unrearranged human immunoglobulin heavy chain D H gene segments (e.g., all functional human D H gene segments) and / or one or more unrearranged human immunoglobulin heavy chain J H gene segments (e.g., all functional human J H gene segment), (ii) a restricted unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the restricted unrearranged human (humanized) heavy chain variable region being operably linked to one or more unrearranged immunoglobulin heavy chain D H a gene segment and one or more unrearranged immunoglobulin heavy chain J H A single unrearranged human heavy chain variable region V operably linked to a gene segment H Optionally, one or more unrearranged immunoglobulin heavy chain D gene segments H a gene segment and one or more unrearranged immunoglobulin heavy chain J H Each gene segment encodes one or more unrearranged human immunoglobulin heavy chain D H gene segments and / or one or more unrearranged human immunoglobulin heavy chain J H A gene segment, (iii) a histidine-modified unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the histidine-modified unrearranged human (humanized) heavy chain variable region comprising an unrearranged immunoglobulin heavy chain variable gene sequence comprising a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon in the complementarity-determining region 3 (CDR3) coding sequence; (iv) a heavy chain-only immunoglobulin coding sequence comprising an unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, the endogenous heavy chain constant region comprising (1) an intact endogenous IgM gene encoding an IgM isotype associated with a light chain, and (2) a non-IgM gene lacking a sequence encoding a functional CH1 domain, e.g., an IgG gene, the non-IgM gene encoding a non-IgM isotype lacking a CH1 domain capable of covalently linking to a light chain constant domain; and / or (b) At the endogenous light chain locus: (i) an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region, the unrearranged human (humanized) immunoglobulin light chain variable region comprising a plurality of unrearranged human light chain variable region Vs; L gene segments (e.g., all functional human unrearranged human V L gene segment) and one or more unrearranged immunoglobulin light chain J L including gene segments, optionally, one or more unrearranged immunoglobulin light chains J L The gene segment comprises one or more unrearranged human immunoglobulin light chain J L gene segments (e.g., all functional human J H L gene segment), Optionally, the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain kappa (κ) locus, and the unrearranged human (humanized) immunoglobulin light chain variable region comprises human variable κ (Vκ) and joining κ (Jκ) gene segments, and the endogenous light chain constant region is an endogenous κ chain constant region sequence, and / or the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain lambda (λ), and the unrearranged human (humanized) immunoglobulin light chain variable region comprises human variable λ (Vλ) and joining λ (Jλ) gene segments, and the endogenous light chain constant region is an endogenous κ chain constant region sequence. is an endogenous λ chain constant region sequence, optionally wherein the endogenous immunoglobulin light chain λ locus comprises (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) and a rodent immunoglobulin light chain constant (Cλ) gene segment, and wherein the endogenous immunoglobulin λ light chain locus comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ), and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally comprising three human Eλs; (ii) a consensus light chain coding sequence comprising a rearranged human (humanized) light chain variable region sequence operably linked to an endogenous light chain constant region, the rearranged human (humanized) light chain variable region sequence being an immunoglobulin light chain J L Gene segments and rearranged human light chain variable region V L including gene segments, (iii) a restricted unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, the restricted unrearranged human (humanized) light chain variable region comprising one or more unrearranged human immunoglobulin light chain binding (J) constant regions; L and up to two unrearranged human immunoglobulin light chain variable (V) gene segments operably linked to the L ) gene segments, (iv) a histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, the histidine-modified unrearranged human (humanized) light chain variable region comprising an unrearranged human (humanized) immunoglobulin light chain variable gene sequence comprising a substitution of at least one non-histidine codon with a histidine codon, or an insertion of at least one histidine codon, in the complementarity-determining region 3 (CDR3) coding sequence; or (v) a mouse modified to comprise a histidine-modified reshaped human (humanized) light chain variable region operably linked to an endogenous light chain constant region, the histidine-modified reshaped human (humanized) light chain variable region comprising a reshaped human (humanized) immunoglobulin light chain variable gene sequence comprising a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon in the complementarity-determining region 3 (CDR3) coding sequence; Optionally, the mouse further comprises: (i) a human (humanized) immunoglobulin heavy chain locus containing a functional ADAM6 gene, such that the mouse exhibits wild-type fertility of a non-human animal; and / or (ii) optionally, comprising an exogenous terminal deoxynucleotidyl transferase (TdT) gene to increase antigen receptor diversity, such that at least 10% of the rearranged variable region genes contain non-templated additions; The mouse has been previously described, e.g., in U.S. Patent Nos. 8,697,940, 8,754,287, 9,204,624, 9,334,334, 9,801,362, 9,332,742, and 9,516,868; U.S. Patent Application Publication Nos. 20110195454, 20120021409, 201 See PCT Patent Application Publication Nos. 20192300, 20130045492, 20150289489, 20180125043, 20180244804, PCT Patent Application Publication Nos. 2019 / 113065, 2017210586 and 2011163314, Lee et al. (2014) Nature Biotechnology 32:356, which are incorporated herein by reference in their entireties.

[0218] In some embodiments, the present invention provides a method for detecting a genome, e.g., a live lineage genome, comprising: Human V H Gene segments, human engineered D H Gene region, and human J H an endogenous immunoglobulin locus comprising an immunoglobulin heavy chain variable region comprising a gene segment, the immunoglobulin heavy chain variable region operably linked to a constant region; and / or The present invention also includes a genetically modified non-human animal comprising an endogenous chain locus comprising an immunoglobulin light chain variable region comprising a human VL gene segment, and a human JL gene segment, the immunoglobulin light chain variable region being operably linked to a constant region.

[0219] In some embodiments, the non-human animal, e.g., a rodent, e.g., a rat or mouse, contains one or more human VVs in its genome. H , D H , and J. H One or more endogenous V segments at the endogenous immunoglobulin heavy chain locus H , D H , and J. H containing a substitution of one or more human V segments H , D H , and J. H The segment is a human D operably linked to a 23mer RSS. H and optionally, an unrearranged or rearranged human V gene segment operably linked to a non-human, e.g., rodent, e.g., mouse or rat, endogenous immunoglobulin heavy chain gene at an endogenous non-human light chain locus. L and human J L segment, or human immunoglobulin light chain constant (C L ) region gene is operably linked to.

[0220] In certain embodiments, the genetically modified non-human animal has engineered D Hand an immunoglobulin locus (endogenous or foreign) containing an immunoglobulin variable region comprising one or more unrearranged human immunoglobulin variable region gene segments comprising a region, wherein the one or more unrearranged human immunoglobulin variable region gene segments are operably linked to the immunoglobulin constant region gene.

[0221] Generally, a genetically modified immunoglobulin locus comprises an immunoglobulin variable region (comprising an immunoglobulin variable region gene segment) operably linked to an immunoglobulin constant region. In some embodiments, the genetically modified immunoglobulin locus comprises an engineered D heavy chain constant region gene operably linked to an immunoglobulin variable region gene. H In some embodiments, the genetically modified immunoglobulin locus comprises one or more human unrearranged immunoglobulin heavy chain variable region gene segments comprising a κ chain constant region. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region κ gene segment operably linked to a κ chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region λ gene segment operably linked to a κ chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region λ gene segment operably linked to a λ chain constant region gene.

[0222] In certain embodiments, the non-human animal comprises an engineered D 10 nucleotide sequence operably linked to an endogenous heavy chain constant region at the endogenous heavy chain locus. H In some embodiments, the immunoglobulin variable region comprises an unrearranged human (humanized) immunoglobulin heavy chain variable region comprising one or more unrearranged human Ig heavy chain variable region gene segments. In some embodiments, the one or more unrearranged human Ig variable region gene segments comprise at least one human immunoglobulin heavy chain variable (V H ) segment, one or more immunoglobulin heavy chain diversity (D H) segment (e.g., one or more unrearranged human D operably linked to a 23-mer RSS) H segment), and one or more immunoglobulin heavy chain joining (J H ) segments (optionally one or more unrearranged human J H In some embodiments, the unrearranged human Ig variable region gene segments comprise a plurality of unrearranged human V H Segment, one or more unrearranged (human) D H segment (e.g., one or more unrearranged (human) D operably linked to a 23-mer RSS) H segment) and one or more unrearranged (human) J H In some embodiments, the unrearranged human Ig variable region gene segment comprises at least three V segments. H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H gene segments, or at least 80 V H In some embodiments, the unrearranged human Ig gene segments comprise functional human D H Contains all gene segments and is functionally human D H At least one of the gene segments is modified to be operably linked to a 23-mer RSS. In some embodiments, the unrearranged human Ig gene segment comprises a functional human J HExemplary variable region, including Ig heavy chain gene segments, are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementary information, which are incorporated herein by reference in their entirety.

[0223] In some embodiments, the non-human animals provided herein comprise, at an endogenous heavy chain locus, a restricted unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprising at least a non-human IgM gene, wherein the restricted unrearranged human (humanized) heavy chain variable region is a single human V H Gene segments, multiple D H a gene segment (e.g., one or more unrearranged (human) D nucleotides operably linked to a 23-mer RSS) H Human D, including gene segments H gene segments) and multiple J H Gene segments (e.g., human J H The restricted immunoglobulin heavy chain locus, characterized by a single V gene segment, can rearrange and form multiple distal rearrangements, each of which is a single human V H Gene segment, D H One of the segments, and J H In some embodiments, a single human VV is derived from one of the VV segments, with each rearrangement encoding a different heavy chain variable domain (e.g., as described in U.S. Patent Application Publication No. 20130096287, which is incorporated herein by reference in its entirety). H The gene segment is V H 1-2 or V H It is 1-69.

[0224] In certain embodiments, the non-human animal comprises an unrearranged human (humanized) immunoglobulin light chain variable region at an endogenous light chain locus operably linked to an endogenous light chain constant region. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region comprises an unrearranged human Igκ variable region gene segment. In some embodiments, the unrearranged human (humanized) immunoglobulin variable region comprises a plurality of unrearranged human Vκ segments and one or more unrearranged human Jκ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human Jκ segments. In some embodiments, the immunoglobulin variable region gene segment comprises four functional Vκ segments and all of the human Jκ segments. In some embodiments, the immunoglobulin variable region gene segment comprises 16 functional Vκ segments and all of the human Jκ segments (e.g., all of the functional human Vκ segments and Jκ segments). In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human JVκ segments and all of the human Jκ segments. Exemplary variable regions comprising Igκ gene segments are provided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA 1 11:5147-52 and supplementary information, which are incorporated herein by reference in their entirety.

[0225] In some embodiments, the restricted unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region comprises no more than two human V L Gene segments and multiple J L In some embodiments, the V gene segment is a dual light chain mouse, or DLC, as described in U.S. Pat. No. 9,796,788, which is incorporated herein by reference in its entirety. L The gene segment is a Vκ gene segment. LThe gene segments are Vλ gene segments. In some embodiments, the Vκ gene segments are IGKV3-20 and IGKV1-39. In some embodiments, the non-human animal comprises exactly two unrearranged human Vκ gene segments and five unrearranged human Jκ gene segments operably linked to a mouse light chain constant region at an endogenous κ light chain locus of the mouse, optionally wherein the exactly two unrearranged human Vκ gene segments are a human Vκ1-39 gene segment and a human Vκ3-20 gene segment, and the five unrearranged human Jκ gene segments are a human Jκ1 gene segment, a human Jκ2 gene segment, a human Jκ3 gene segment, a human Jκ4 gene segment, and a human Jκ5 gene segment, and wherein the unrearranged human kappa light chain gene segment is capable of rearranging and encoding a human variable domain of an antibody, and optionally wherein the non-human animal does not comprise endogenous Vκ gene segments capable of rearranging to form an immunoglobulin light chain variable region.

[0226] In certain embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to the endogenous light chain constant region contains an unrearranged human Igλ variable region gene segment. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises a plurality of human Vλ segments and one or more human Jλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises one or more human Vλ segments, one or more human Jλ segments, and one or more human Cλ constant region sequences. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human Vλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segment comprises all of the human Jλ segments. Exemplary variable regions comprising Igλ gene segments are provided, for example, in U.S. Patent Nos. 9,035,128 and 6,998,514, which are incorporated herein by reference in their entireties. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to the endogenous light chain constant region comprises (a) one or more human Vλ gene segments, (b) one or more human Jλ gene segments, and (c) one or more human Cλ gene segments, wherein (a) and (b) are operably linked to (c) and an endogenous (e.g., rodent) Cλ gene segment, and the endogenous immunoglobulin λ light chain locus further comprises one or more rodent immunoglobulin λ light chain enhancers (Eλ), and one or more human immunoglobulin λ light chain enhancers (Eλ), optionally comprising three human Eλ.

[0227] In certain embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises an unrearranged human Igλ variable region gene segment operably linked to an endogenous (e.g., rodent, e.g., rat or mouse) Cκ gene such that the non-human animal expresses an immunoglobulin light chain comprising a human λ variable domain sequence derived from a Vλ and a Jλ gene segment fused to an endogenous κ constant domain; see, e.g., U.S. Pat. No. 9,226,484, herein incorporated by reference in its entirety.

[0228] In some embodiments, the immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments also comprises a human immunoglobulin variable region intergenic sequence. In some embodiments, the immunoglobulin variable region comprises a non-human (e.g., rodent, rat, mouse) Ig variable region intergenic sequence. In some embodiments, the intergenic sequence is an intergenic sequence of endogenous species origin.

[0229] In some embodiments, the immunoglobulin variable region is a rearranged light chain variable region (a "common light chain variable region"). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. Exemplary rearranged Ig light chain variable regions are provided in, for example, U.S. Patent Nos. 9,969,814, 10,130,181, and 10,143,186, and U.S. Patent Application Publication Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, which are incorporated herein by reference in their entireties. In some embodiments, bispecific antibodies are produced using non-human organisms that contain a common light chain variable region (a "common light chain" organism). In some embodiments, the common light chain coding sequence comprises a single rearranged human immunoglobulin light chain Vκ / Jκ sequence operably linked to an endogenous light chain constant region, and the single rearranged human immunoglobulin light chain Vκ / Jκ sequence is either (i) a human Vκ1-39 / Jκ5 sequence comprising a human Vκ1-39 gene segment fused to a human Jκ5 gene segment, or (ii) a human Vκ3-20 / Jκ1 sequence comprising a human Vκ3-20 gene segment fused to a human Jκ1 gene segment.

[0230] In some embodiments, the immunoglobulin variable regions are light and / or heavy chain immunoglobulin variable regions that contain histidine codon insertions and / or substitutions designed to introduce pH-dependent binding properties into antibodies produced in such non-human organisms. In some such embodiments, the histidine codons are inserted into and / or substituted into the nucleic acid sequences encoding CDR3. Various such light and / or heavy chain immunoglobulin loci are provided in U.S. Patent Nos. 9,301,510, 9,334,334, and 9,801,362, and U.S. Patent Application Publication No. 20140013456, which are incorporated herein by reference in their entireties. In some embodiments, the histidine-modified rearranged human (humanized) light chain variable region operably linked to the endogenous light chain constant region comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human Vκ and Jκ segment sequences, optionally wherein the Vκ segment sequence is derived from a human Vκ1-39 or Vκ3-20 gene segment, and the single rearranged human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon in the Vκ segment sequence with a histidine codon expressed at a position selected from the group consisting of 105, 106, 107, 108, 109, 111, and combinations thereof (according to IMGT numbering). In some embodiments, the histidine-modified unrearranged human (humanized) heavy chain variable region operably linked to the endogenous heavy chain constant region comprises a (human) Dκ modified to be operably linked to a complementarity-determining region 3 (CDR3) coding sequence (e.g., a 23-mer RSS). H In some embodiments, the unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence comprises an unrearranged human V (V gene segment) containing a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon. In some embodiments, the unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence comprises an unrearranged human V (V gene segment). H , D operably linked to the 23mer RSS H Unrearranged human D containing segment H or synthetic D H, and unrearranged human J H an unrearranged human D gene segment, optionally operably linked to a 23-mer RSS; H or synthetic D H Gene segment or D H The gene segment comprises a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon. In some embodiments, a histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous heavy chain constant region comprises an unrearranged V L and unreconstructed J L In some embodiments, the histidine-modified unrearranged human (humanized) light chain variable region comprises no more than two unrearranged human V gene segments. L (e.g., two or fewer Vκ gene segments) and one or more unrearranged human J L (e.g., Jκ) gene segments, and up to two human V L Each of the gene segments comprises a substitution of at least one non-histidine codon with a histidine codon or an insertion of at least one histidine codon in the CDR3-encoding sequence. In some embodiments, the no more than two unrearranged human Vκ gene segments are human Vκ1-39 and human Vκ3-20 gene segments, each comprising one or more substitutions of non-histidine codons with histidine codons, and the human Vκ and Jκ gene segments are capable of rearrangement, and the human Vκ and human Jκ gene segments encode human light chain variable domains comprising one or more histidines at positions selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering), and combinations thereof, wherein the one or more histidines result from one or more substitutions.

[0231] In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region gene. In some embodiments, the heavy chain constant region is a human heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a heavy chain constant region gene of an endogenous species origin. In some embodiments, the heavy chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the constant region gene is a mixture of human and non-human sequences. For example, in some embodiments, the constant region gene encodes a human CH1 region and a non-human (e.g., endogenous species origin, mouse, rat) CH2 and / or CH3 region. In some embodiments, the heavy chain constant region gene is a Cμ, Cδ, Cγ (Cγ1, Cγ2, Cγ3, Cγ4), Cα, or Cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region genes encode mutated CH1 regions so that the non-human animal expresses heavy chain-only antibodies (see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Application Publication No. 2015 / 0289489, the entire contents of which are incorporated herein by reference). For example, in some embodiments where the goal is to generate heavy chains to create bispecific antibodies (in general or dual-light chain organisms), the Fc domain of the heavy chain contains modifications to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, and 8,679,785, and U.S. Patent Application Publication No. 2013 / 0195849, the entire contents of which are incorporated herein by reference.

[0232] In some embodiments, the immunoglobulin constant region comprises a light chain constant region gene. In some embodiments, the light chain constant region gene is a kappa constant region gene. In some embodiments, the light chain constant region gene is a lambda constant region gene. In some embodiments, the light chain constant region gene is a light chain constant region gene of endogenous species origin. In some embodiments, the light chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the light chain constant region gene is a mixture of human and non-human sequences.

[0233] In some embodiments, the immunoglobulin variable region comprising the human variable region gene segment and the immunoglobulin constant region gene to which the variable region gene segment is operably linked are located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous κ locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous λ locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably linked is an endogenous constant region gene.

[0234] In some embodiments, one or more endogenous immunoglobulin loci, or portions of one or more endogenous loci (e.g., variable and / or constant regions) in the genome of a non-human animal provided herein, are inactivated. Endogenous immunoglobulin variable region gene agents and portions thereof can be inactivated using any method known in the art, including, but not limited to, deleting the locus or portions thereof from the genome of the organism, replacing the locus or portions thereof with a different nucleic acid sequence, inverting a portion of the locus, and / or moving a portion of the locus to another location in the genome of the non-human organism. In some embodiments, the inactivation of the locus is only partial. In some embodiments, the variable region of the locus is inactivated, but the constant region remains functional (e.g., because it is operably linked to a non-endogenous variable region gene segment).

[0235] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus, or portion thereof, is inactivated by deletion, replacement, transfer, and / or inversion of at least a portion of the endogenous variable region of the endogenous heavy chain locus. In some embodiments, at least a portion of the variable region of the endogenous heavy chain locus that is deleted, replaced, transferred, and / or inverted comprises a variable region J segment. In some embodiments, the endogenous immunoglobulin heavy chain locus, or portion thereof, is inactivated by deletion, replacement, transfer, and / or inversion of at least a portion of the endogenous constant region of the endogenous heavy chain locus. In some embodiments, at least a portion of the constant region of the endogenous heavy chain locus that is deleted, replaced, transferred, and / or inverted comprises the Oμ gene of the endogenous constant region.

[0236] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous immunoglobulin κ chain locus, or portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of an endogenous variable region of the endogenous κ chain locus. In some embodiments, at least a portion of the variable region of the endogenous κ chain locus that is deleted, replaced, moved, and / or inverted comprises a variable region J segment. In some embodiments, the endogenous immunoglobulin κ chain locus, or portion thereof, is inactivated by deleting, replacing, moving, and / or inverting at least a portion of the endogenous constant region of the endogenous κ chain locus. In some embodiments, at least a portion of the constant region of the endogenous κ chain locus that is deleted, replaced, moved, and / or inverted comprises an endogenous constant region CK gene.

[0237] In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin λ chain locus. In some embodiments, the endogenous immunoglobulin λ chain locus, or a portion thereof, is inactivated by deletion, replacement, relocation, and / or inversion of at least a portion of an endogenous variable region of the endogenous λ chain locus. In some embodiments, at least a portion of at least one VJC gene cluster in the endogenous λ chain locus is deleted, replaced, relocated, and / or inverted. In some embodiments, the endogenous immunoglobulin λ chain locus, or a portion thereof, is inactivated by deletion, replacement, relocation, and / or inversion of at least a portion of an endogenous constant region of the endogenous λ chain locus. In some embodiments, at least the portion of the constant region of the endogenous λ chain locus that is deleted, replaced, relocated, and / or inverted comprises an endogenous constant region C gene.

[0238] In various embodiments, the immunoglobulin locus modification does not affect the reproductive capacity of the non-human animal. In some embodiments, the heavy chain locus comprises a functional, e.g., endogenous ADAM6a gene, ADAM6b gene, or both, and the genetic modification does not affect the expression and / or function of the endogenous ADAM6a gene, ADAM6b gene, or both. In some embodiments, the genome of the genetically modified non-human animal further comprises an ectopically located functional, e.g., endogenous ADAM6a gene, ADAM6b gene, or both. Exemplary non-human animals expressing exogenous ADAM6a and / or ADAM6b are described in U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety.

[0239] In some embodiments, the genetically modified non-human animal further contains and expresses an exogenous terminal deoxynucleotidyl transferase (TdT) gene for increased antigen receptor diversity. Exemplary non-human animals expressing exogenous TdT are described in PCT Patent Application Publication No. 2017210586, which is incorporated herein by reference in its entirety.

[0240] In some embodiments, the genome of the provided non-human animal further comprises one or more human immunoglobulin heavy and / or light chain genes (see, e.g., U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, and U.S. Patent Application Publication Nos. 2013 / 0096287A1 and 2018 / 0125043A1, and PCT Application Publication No. 2019 / 113065, each of which is incorporated by reference in its entirety). Alternatively, engineered D H The regions can be introduced into embryonic stem cells of different modified strains, such as the VELOCIMMUNE® line (see, e.g., U.S. Pat. No. 8,502,018 or U.S. Pat. No. 8,642,835, incorporated herein by reference in their entireties). In some embodiments, the non-human animals described herein can be generated by introducing the targeting vectors described herein into cells from the modified lineages. By way of example, the targeting vectors described herein can be introduced into the non-human animals described in U.S. Pat. Nos. 8,642,835 and 8,697,940, incorporated herein by reference in their entireties, and the non-human animals express antibodies with fully human variable regions and mouse constant regions. In some embodiments, the non-human animals described herein are generated to further comprise human immunoglobulin genes (variable region genes and / or constant region genes). In some embodiments, the non-human animals described herein are generated by introducing the engineered D vectors described herein. H and genetic material of heterologous (e.g., human) origin, wherein the genetic material encodes, in whole or in part, one or more human heavy chain variable regions and / or light chain variable regions.

[0241] The non-human animals described herein can often be prepared, as described above or using methods known in the art, to contain additional human or humanized genes, depending on the intended use of the non-human animal. Such additional human or humanized genetic material can be introduced through further modification of the genome of a cell (e.g., an embryonic stem cell) bearing the genetic modification, as described above or through breeding techniques known in the art with other genetically modified parts as desired.

[0242] For example, engineered D H Non-human animals containing regions are described in U.S. Patent Application Publication No. 2011-0195454, which is incorporated herein by reference in its entirety. A1, 2012-0021409 A1, 2012-0192300 A1, 2013-0045492 A1, 2013-0185821 A1, 2013-0198880 A1, 2013-0302836 A1, 2015-0059009 A1; International Patent Application Publication Nos. 2011 / 097603, 2012 / 148873, 2013 / 134263, 2013 / 184761, 2014 / 160179, 2014 / 160202.

[0243] Transgenic founder non-human animals contain engineered D H The presence of regions and / or D in tissues or cells of non-human animals H -D H The engineered D gene can then be identified based on the expression of an antibody containing a CDR3 region containing the resulting amino acid. H and mating the non-human animals with additional non-human animals carrying the engineered D region, each of which contains one or more copies of the engineered D region. H A range of non-human animals can be generated that contain engineered D HTransgenic non-human animals carrying the region can further be bred to other transgenic non-human animals carrying other desired transgenes (eg, human immunoglobulin genes).

[0244] Transgenic non-human animals may also be generated that contain selected systems that allow for the control or direction of transgene expression. Exemplary systems include the Cre / loxP recombinase system of bacteriophage P1 (see, e.g., Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236, incorporated herein by reference in its entirety) and the FLP / Frt recombinase system of Saccharomyces cerevisiae (O'Gorman, S. et al., 1991, Science 251:1351-1355, incorporated herein by reference in its entirety). Such animals may be modified, for example, by the addition of a selected modification (e.g., an engineered D H This can be achieved by constructing a "double" transgenic animal by mating two transgenic animals, one containing a transgene including a recombinase (e.g., a Cre recombinase) and the other containing a transgene encoding a recombinase (e.g., a Cre recombinase).

[0245] Mouse (i.e., human V H and J. H engineered D gene segments, all of which are operably linked to one or more mouse heavy chain constant region genes. H In mice with the engineered D H Although embodiments utilizing regions are discussed extensively herein, engineered D H Other non-human animals that contain the endogenous V region are also provided. In some embodiments, such non-human animals contain the endogenous V region. H and J. H an engineered D operably linked to a gene segment H In some embodiments, such non-human animals comprise a humanized V region. H and J. H an engineered D operably linked to a gene segmentH Such non-human animals include, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), and the like, which contain D regions at increased frequency in combination with the wild-type immunoglobulin heavy chain loci disclosed herein. H -D H This includes any that can be genetically modified to express an antibody having a CDR3 containing an amino acid resulting from recombination. For example, for non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are used to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) and using somatic cell nuclear transfer (SCNT) to introduce the genetically modified genome into a suitable cell, such as an enucleated oocyte, and gestation of the modified cell (e.g., the modified oocyte) into a non-human animal under conditions suitable for embryo formation.

[0246] Methods for modifying non-human animal genomes (e.g., pigs, cows, rodents, chickens, etc.) include, for example, utilizing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., CRISPR / Cas systems) to engineer Ds described herein. H Guidance on methods for modifying the germline genome of non-human animals can be found, for example, in U.S. Patent Application Publication Nos. 2015-0376628A1, 2016-0145646A1, and 2016-0177339A1, each of which is incorporated by reference in its entirety.

[0247] In some embodiments, the non-human animals described herein are mammals. In some embodiments, the non-human animals described herein are small mammals, such as small mammals of the Jerboidea or Murine superfamily. In some embodiments, the genetically modified animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice, rats, and hamsters. In some embodiments, the rodents described herein are selected from the Murine superfamily. In some embodiments, the genetically modified animals described herein are from a family selected from the family Odontoidea (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (pure-breed mice and rats, gerbils, spiny mice, maned mice), Tetragnathidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Dormiceidae (e.g., spiny dormice), and Odontoidea (e.g., mole rats, bamboo mice, and plateau mole rats). In some embodiments, the genetically modified rodent described herein is selected from a pure breed of mouse or rat (Muridae), a gerbil, a spiny mouse, and a maned mouse. In some embodiments, the genetically modified mouse described herein is from a member of the Muridae family. In some embodiments, the non-human animal described herein is a rodent. In some embodiments, the rodent described herein is selected from a mouse and a rat. In some embodiments, the non-human animal described herein is a mouse.

[0248] In some embodiments, the non-human animals described herein are rodents that are mice of the C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse of the present invention is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032; the entire contents of which are incorporated herein by reference). In some embodiments, the genetically modified mice described herein are a mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In some embodiments, the mice described herein are a mix of the aforementioned 129 strains, or a mix of the aforementioned BL / 6 strains. In some embodiments, the 129 strain in the mix described herein is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mice described herein are a BALB strain, e.g., a BALB / c strain. In some embodiments, the mice described herein are a mix of a BALB strain and another aforementioned strain.

[0249] In some embodiments, the non-human animal described herein is a rat. In some embodiments, the rat described herein is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain described herein is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0250] method A variety of in vitro and in vivo techniques have been developed to generate antibody-based therapeutics. In particular, in vivo techniques have been characterized by the generation of transgenic animals (i.e., rodents) with human immunoglobulin genes, where the genes have been either randomly integrated into the animal's genome (see, e.g., U.S. Pat. No. 5,569,825, incorporated herein by reference in its entirety) or precisely located at the animal's endogenous immunoglobulin loci operably linked to the animal's endogenous immunoglobulin constant regions (see, e.g., U.S. Pat. Nos. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, incorporated herein by reference in their entireties). Both approaches have been successful in generating promising antibody therapeutic candidates for human use. Furthermore, both approaches have an advantage over in vitro approaches in that antibody candidates are selected from an in vivo-generated antibody repertoire, which involves selection for affinity and specificity for the antigen within the internal environment of the host immune system. In this way, antibodies bind to naturally presented antigens (within the relevant biological epitopes and surfaces) rather than to artificial environments or in silico predictions that can accompany in vitro techniques. Despite the robust antibody repertoires obtained from in vivo techniques, antibodies against complex (e.g., viruses, channel polypeptides) or cytoplasmic antigens remain challenging. Furthermore, generating antibodies against polypeptides that share a high degree of sequence identity between species (e.g., humans and mice) also remains a challenge due to immune tolerance.

[0251] Thus, the present invention provides, inter alia, unconventional gene segment rearrangements (i.e., D H From D H The construction of an in vivo system characterized by the production of antibodies with added CDR, particularly CDR3, diversity, generated from the recombination of one or more Ds operably linked to the 5' or 3' 23mer RSS, respectively, can be achieved by the construction of an in vivo system characterized by the production of antibodies with added CDR, particularly CDR3, diversity, generated from the recombination of one or more Ds operably linked to the 5' or 3' 23mer RSS, respectively, HIt is recognized that such engineered Ds can be created using a 5' or 3' 23-mer RSS. H Compared to the immunoglobulin heavy chain locus lacking the gene segment, D H Recombination between segments increases. This added diversity can direct binding to specific antigens (e.g., viruses, channel polypeptides). H Gene segments, at least two D H gene segments, and J H During recombination of gene segments, D H From D H A heavy chain variable region coding sequence is formed that contains a CDR3 region having an amino acid sequence resulting from recombination into D H From D H The resulting CDR3 from recombination contains additional diversity due to its increased amino acid length. Furthermore, such CDR3 regions have the ability to direct binding to specific antigens (or epitopes) that cannot be bound by antibodies generated by conventional VDJ recombination.

[0252] The provided non-human animals can be used to generate human antibodies, which comprise variable domains derived from one or more variable region nucleic acid sequences encoded by the genetic material of the cells of the non-human animals described herein. For example, the provided non-human animals are immunized with an antigen of interest (e.g., a virus or channel polypeptide, in whole or in part) under conditions and for a time sufficient to cause the non-human animal to generate an immune response to the antigen of interest. Antibodies are isolated from the non-human animals (or one or more cells, e.g., one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, epitope mapping, ability to inhibit ligand-receptor interaction, inhibitory receptor activity, etc. In some embodiments, the antibodies produced by the provided non-human animals comprise one or more human variable domains derived from one or more human variable region nucleotide sequences isolated from the non-human animal. In some embodiments, anti-drug antibodies (e.g., anti-idiotypic antibodies) can be generated in the provided non-human animals.

[0253] The non-human animals described herein provide improved in vivo systems and sources of biological material (e.g., cells) for producing human antibodies useful in various assays. In some embodiments, the provided non-human animals are used to develop therapeutics that target one or more viruses and / or modulate viral activity and / or modulate viral interactions with other binding partners (e.g., cell surface receptors). In some embodiments, the provided non-human animals are used to develop therapeutics that target one or more channel proteins and / or modulate channel protein activity and / or modulate channel protein interactions with other binding partners. In some embodiments, the provided non-human animals are used to identify, screen, and / or develop candidate therapeutics (e.g., antibodies, siRNAs, etc.) that bind to one or more viral, channel, or G protein-coupled receptor (GPCR) polypeptides. In some embodiments, the provided non-human animals are used to screen and develop candidate therapeutics (e.g., antibodies, siRNAs, etc.) that inhibit the activity of one or more viral polypeptides, one or more human channel polypeptides, or one or more human GPCR polypeptides. In some embodiments, the provided non-human animals are used to determine the binding characteristics of antagonists of one or more GPCR polypeptides or one or more human channel polypeptides. In some embodiments, the non-human animals provided are used to determine the epitope or epitopes of one or more candidate therapeutic antibodies that bind one or more human GPCR polypeptides or that bind one or more human channel polypeptides.

[0254] In some embodiments, the provided non-human animals are used to determine the pharmacokinetic profile of the antibody. In some embodiments, one or more provided non-human animals and one or more control or reference non-human animals are each exposed to one or more candidate therapeutic antibodies at various doses (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more). The candidate therapeutic antibodies can be administered via any desired route of administration, including parenteral and oral routes. Parenteral routes include, for example, intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intrathecal, intracerebroventricular, intracranial, intrathoracic, or other routes of injection. Oral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, and ocular. Administration may be by continuous infusion, topical administration, sustained release from an implant (gel, membrane, etc.), and / or intravenous injection, e.g., using an intravenous fluid bag. Blood may be isolated from the non-human animals (humanized and control) at various time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). Various assays may be used to determine the pharmacokinetic properties of the administered candidate therapeutic antibody using feed obtained from the non-human animals described herein, including, but not limited to, total IgG, anti-therapeutic antibody response, agglutination, etc.

[0255] In some embodiments, the provided non-human animals are used to measure the therapeutic effect of inhibiting or modulating the activity of a target antigen, and the effect on gene expression or cell surface density of the target antigen (in the case of a cell surface receptor) as a result of cellular changes in the cells of the non-human animals described herein. In some embodiments, the provided non-human animals or cells isolated therefrom are exposed to a candidate therapeutic agent that binds to the antigen of interest, and after a subsequent period of time, analyzed for its effect on a target antigen-dependent process (or interaction), e.g., ligand-receptor interaction or antigen-associated signaling.

[0256] In some embodiments, the provided non-human animals are used to measure the effect of inhibiting or modulating channel activity (or channel signaling, or channel-mediated interactions, or channel action potentials), and the effect of treatments on the resulting cellular changes in gene expression or channel density of the non-human animals described herein. In some embodiments, the provided non-human animals or cells isolated therefrom are exposed to candidate therapeutic agents that bind to the human channel (or portion thereof), and after a subsequent period of time, are analyzed for effects on channel-dependent processes (or interactions), e.g., ligand-receptor interactions or channel action potentials.

[0257] In some embodiments, the provided non-human animals express antibodies, and thus cells, cell lines, and cell cultures can be generated to serve as a source of antibodies for use in binding and functional assays, e.g., to assay for antagonist or agonist binding or function, where the antagonist or agonist is specific for a human polypeptide sequence or epitope, or is specific for a human polypeptide sequence or epitope that functions in ligand-receptor interaction (binding). In some embodiments, the epitope bound by a candidate therapeutic antibody or siRNA can be determined using cells isolated from the provided non-human animals.

[0258] In some embodiments, the cells derived from the provided non-human animals can be isolated and used ad hoc or maintained in culture for many generations, hi some embodiments, the cells derived from the provided non-human animals are immortalized (e.g., through the use of a virus) and maintained in culture (e.g., in continuous culture) indefinitely.

[0259] In some embodiments, the non-human animals described herein provide an in vivo system for the generation of antibody variants that bind to human target antigens. Such variants include antibodies with desired functionality, specificity, and low cross-reactivity against a common epitope shared by two or more human target antigens. In some embodiments, the provided non-human animals are utilized to generate panels of antibodies to generate a series of antibody variants that are screened for desired or improved functionality.

[0260] In some embodiments, the non-human animals described herein provide an in vivo system for generating antibody libraries. Such libraries provide a source of heavy and light chain variable region sequences that can be grafted with different Fc regions based on desired effector functions and / or used as a source for affinity maturation of variable region sequences using techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.).

[0261] In some embodiments, the non-human animals described herein provide an in vivo system for the analysis and testing of drugs or vaccines. In some embodiments, a candidate drug or vaccine is delivered to one or more provided non-human animals, and the non-human animals can then be monitored to determine one or more of the immune response to the drug or vaccine, the safety profile of the drug or vaccine, or its effect on a disease or condition and / or one or more symptoms of the disease or condition. Typical methods used to determine safety profiles include measuring toxicity, optimal dose concentrations, antibody (i.e., anti-drug) responses, drug or vaccine efficacy, and potential risk factors. Such drugs or vaccines may be improved and / or developed in such non-human animals.

[0262] The effectiveness of a vaccine may be determined in a number of ways. Briefly, the non-human animals described herein are vaccinated using methods known in the art, and then challenged with the vaccine, or the vaccine is administered to an already infected non-human animal. The response of the non-human animal to the vaccine may be measured by monitoring the non-human animal (or cells isolated therefrom) and / or performing one or more assays on them to determine the effectiveness of the vaccine. The response of the non-human animal to the vaccine is then compared with that of a control animal using one or more means known in the art and / or described herein.

[0263] The efficacy of the vaccine may be further determined by a virus neutralization assay. Briefly, the non-human animals described herein are immunized, and serum is collected at various days after immunization. Serial dilutions of the serum are pre-incubated with the virus, during which time the antibodies in the serum specific for the virus bind to the virus. The virus / serum mixture is then added to permissive cells, and infectivity is determined by a plaque assay or microneutralization assay. If the antibodies in the serum neutralize the virus, the number of plaques or relative luciferase units will be lower compared to the control group.

[0264] The non-human animals described herein provide improved in vivo systems for the development and characterization of antibody-based therapeutics for use in cancer and / or inflammatory diseases. Inflammation has been implicated in cancer (e.g., Grivennikov, S.I. et al., 2010, Cell 140:883-99; Rakoff-Nahoum, S., 2006, Yale J. Biol. Med. 79:123-30, the entire contents of which are incorporated herein by reference). Indeed, the developing tumor environment is characterized, in part, by the infiltration of various inflammatory mediators. Furthermore, persistent inflammation may lead to a higher likelihood of developing cancer. Thus, in some embodiments, the non-human animals described herein provide an in vivo system for the development and / or identification of anti-cancer and / or anti-inflammatory therapeutics. In some embodiments, a provided non-human animal or a control non-human animal (e.g., with a genetic modification different from those described herein or with no genetic modification, i.e., wild-type) may be implanted with a tumor (or tumor cells), followed by administration of one or more candidate therapies. In some embodiments, the candidate therapy may comprise a multispecific antibody (e.g., a bispecific antibody) or an antibody cocktail. In some embodiments, the candidate therapy comprises a combination therapy, such as, for example, administration of two or more monospecific antibodies administered sequentially or simultaneously. The tumor may be left undisturbed for a sufficient time to establish at one or more locations within the non-human animal, after which one or more candidate therapeutic agents may be administered. Tumor cell proliferation, growth, survival, etc. may be measured both before and after administration of the candidate therapeutic agent. The cytotoxicity of the candidate therapeutic agent may also be measured in the non-human animal, if desired.

[0265] kit The present invention further provides packs or kits comprising one or more containers filled with at least one non-human animal, non-human cell, DNA fragment, and / or targeting vector described herein. The kits may be used in any applicable method (e.g., research method). Optionally, a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical and biological products may be associated with such containers, reflecting (a) regulatory approval for manufacture, use, or sale for human administration, (b) instructions for use, or both, or a contract governing the transfer of materials and / or biological products (e.g., non-human animals or non-human cells described herein) between two or more entities.

[0266] Other features of the present invention will become apparent in the course of the following description of exemplary embodiments, which are given by way of illustration and are not intended to be limiting thereof. [Example]

[0267] The following examples are provided to illustrate to those of ordinary skill in the art how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are given in degrees Celsius and pressures are at or near atmospheric.

[0268] Example 1. Design and construction of targeting vector This example describes the construction of a targeting vector for insertion into the genome of a non-human animal, such as a rodent (e.g., a mouse). In particular, the method described in this example is useful for constructing ...), whose genome contains an engineered D H The regions each comprise one or more D operably linked to a 5' or 3' 23-mer RSS. H Engineered heavy chain diversity (D HThis example demonstrates the generation of targeting vectors for insertion into the genome of rodents (e.g., mice) containing immunoglobulin heavy chain variable regions containing the 5' 23-mer RSS and one human J region. H The proximal (or 3') D segment operably linked to H Segment containing manipulated D H The first targeting vector contains a region operably linked to the 5' 23-mer RSS and three human J H The proximal (or 3') D segment operably linked to H Segment containing manipulated D H A second targeting vector containing six human J regions, each associated with a 3' 23-mer RSS. H three D operably linked to the segment H Segment containing manipulated D H A third targeting vector containing the humanized immunoglobulin heavy chain variable region (Figure 2) was generated and inserted separately into each of these targeting vectors (Figures 3, 5, 7). The engineered D H The heavy chain variable (V H ) and heavy chain binding (J H ) segment, and upon VDJ recombination, H -D H Antibodies with recombinantly generated CDR3 were expressed.

[0269] one or more human Ds associated with a 5' or 3' 23-mer RSS, respectively, for insertion into an immunoglobulin heavy chain variable region. H Targeting vectors containing the segments were generated using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659, which are incorporated herein by reference in their entireties) and molecular biology techniques known in the art. Utilizing the methods described in this example, any DH Segment, D H Set of segments, or D as needed H A combination of segments can be used.

[0270] A.23:D H 3-3:12 / J H 6. Targeting Vector (Figure 2) Briefly, a first targeting vector is inserted into a plurality of human D H segment, and human D H Synthetic human D located at positions 7-27 H The donor for in vitro Cas9 / GA modification was generated by de novo synthesis (Blue Heron Bio) and was constructed using a genomic DNA fragment containing the 3-3 segment, 5' to 3: (a) D H (b) a 100-bp homology arm starting 350 bp upstream of the 5′ 12-mer RSS of 7-27; (c) AgeI and XhoI sites for insertion of a neomycin resistance cassette; (d) D H (d) a 250-bp region upstream of the 12 RSSs at the 5' end of 7-27; (e) a 23-mer RSS at the 5' end (J H 4) and a 12-mer RSS at the 3' end (D H 3-3) engineered synthetic human D H 3-3 segments, and (3)J H The synthetic D100 gene contains a 100-bp homology box beginning 3 bp downstream of the loxp-UbC-Em7-Neo-loxp cassette, e.g., a neomycin resistance gene flanked by loxP site-specific recombination recognition sites, which is ligated into the AgeI and XhoI sites to allow selection in E. coli and mouse ES cells. H The 23:D segment contains a neomycin cassette located approximately 250 bp upstream and downstream. H 3-3:12 / J HThe 6 donor (SEQ ID NO: 61) was used to modify the BAC by in vitro Cas9 / GA using two Cas9:gRNA complexes. Prior to modification, the BAC was identical in sequence to the chimeric IgH locus of VELOCIMMUNE® mice (e.g., J H See Figures 3 and 4, which show exemplary, non-limiting endogenous immunoglobulin heavy chain loci for VELOCIMMUNE® mice that lack gene segments and are heterozygous for the 6394 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence, and the 1460 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence. Specifically, the BACs are located between the most proximal human V H Gene (V H 6-1), 27 human D H Genes, all six human J H The BAC was identical to the 1460 allele, containing the gene, the mouse IgH intronic enhancer (Eμ), the mouse IgM switch region (Sμ), and the first four exons of the mouse IgM gene. The BAC also contained a spectinomycin (spec) resistance cassette and an approximately 29 kb V H The human J6-1 gene was found to contain a human intergenic sequence upstream of the 6-1 gene. H 6 gene and 490 bp of the 5' end of the mouse Eμ enhancer. H 6. Insertion into the donor's BAC H Replacement of the 7-7 gene segment with the engineered 23:D3-3:12 segment and J H 1. J H 2. J H 3. J H 4, and J H Final 23:D3-3:12 / J, containing a deletion of 5 gene segments H Six targeting vectors were generated (Figure 2). Table 1 shows the 23:D H 3-3:12 / J H 6. Provide the sequences of the gRNAs, primers, and probes used to determine the correct construction of the targeting vector. [Table 1]

[0271] The 23:D3-3:12 / JH6 targeting vector was linearized with NotI and engineered to contain multiple human V sequences heterozygous for humanized immunoglobulin heavy chain variable regions (i.e., operably linked to rodent immunoglobulin heavy chain constant regions containing rodent heavy chain enhancer and regulatory regions). H , D H , and J. H a first heavy chain allele (1460het) containing a segment and containing an inserted nucleotide sequence encoding one or more mouse Adam6 genes (e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference herein in its entirety); and a plurality of human V H and D H Segment, J H A second heavy chain allele (6394het) containing a rodent immunoglobulin heavy chain constant region including the region deletion and rodent heavy chain enhancer and control regions, and containing an inserted nucleotide sequence encoding one or more mouse Adam6 genes (e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety), was electroporated into mouse embryonic stem cells having a genome homozygous (HO) for a humanized endogenous κ locus containing a full repertoire of human immunoglobulin light chain Vκ and Jκ gene segments operably linked to an endogenous immunoglobulin heavy chain Cκ region (1293) (see Figure 3). The 6799 allele was electroporated, and the 1460 allele, 23:D3-3:12 / J, containing a neomycin cassette, was inserted (Figure 3). HThese engineered mouse ES cells, heterozygous for the 6394 and 679 alleles, were used to facilitate efficient screening of positive ES clones (see below) and subsequent cre-mediated removal of the drug resistance cassettes; henceforth, deletions of the 6394 and 6799 alleles are referred to as 6643 and 6800, respectively (Figure 4).

[0272] B.23:D H 3-3:12 / J H 4-6 Targeting Vector (Figure 2) In a similar manner, the first targeting vector (23:D H 3-3:12 / J H 4-6) to multiple human D H segment, and human D H Synthetic human D located at positions 7-27 H This second targeting vector was constructed using the same genomic DNA fragment containing the 3-3 segment. However, this second targeting vector contained six human J H Three of the segments (i.e., J H 4. J H 5. J H 6) included. Final 23:D H 3-3:12 / J H To generate the 4-6 targeting vector, a donor was used to modify a BAC identical in sequence to the chimeric IgH locus of the VELOCIMMUNE® mouse (e.g., J HSee Figures 3 and 4, which show exemplary, non-limiting endogenous immunoglobulin heavy chain loci for VELOCIMMUNE® mice that lack a gene segment and are heterozygous for the 6394 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence, and the 1460 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence. The donor contains, from 5' to 3: (a) a 100 bp homology arm beginning 350 bp upstream of the 12-mer RSS at the 5' end of D7-27, (b) AgeI and XhoI sites for insertion of a neomycin resistance cassette, (c) a 250 bp region upstream of the 12-mer RSS at the 5' end of D7-27, (d) a 23-mer RSS at the 5' end (J H 4) and a 12-mer RSS at the 3' end (D H 3-3) engineered synthetic human D H 3-3 segments, and (e)J H The synthetic D1 gene was constructed by ligating a loxp-UbC-Em7-Neo-loxp cassette, e.g., a neomycin resistance gene flanked by loxP site-specific recombination recognition sites, into the AgeI and XhoI sites to allow selection in E. coli and mouse ES cells. H The 23:D3-3:12 / J gene contains the nucleotide sequence set forth as SEQ ID NO: 52, which includes the neomycin cassette. H Using 4-6 donors, we modified a BAC identical in sequence to the 1460 allele (Figures 3 and 4). Specifically, the BAC was designed to clone the most proximal human V H Gene (V H 6-1), 27 human D H Genes, all six human J H The BAC was identical to the 1460 allele, containing the gene, the mouse IgH intronic enhancer (Eμ), the mouse IgM switch region (Sμ), and the first four exons of the mouse IgM gene. The BAC also contained a spectinomycin (spec) resistance cassette and an approximately 29 kb V HThe human J6-1 gene was found to contain a human intergenic sequence upstream of the 6-1 gene. H The 3' 222 bp of the 6 gene and the 5' 490 bp of the mouse Eμ enhancer were used to transfect 23:D3-3:12 / J in vitro via Cas9 / GA using two Cas9:gRNA complexes. H 4-6 Insertion into the donor's BAC H Replacement of the 7-7 gene segment with the engineered 23:D3-3:12 segment and J H 1. J H 2, and J H Contains a deletion of 3 gene segments, final 23:D3-3:12 / J H 4-6 targeting vectors (Figure 2). Table 2 shows the 23:D H 3-3:12 / J H 6. Provide the sequences of the gRNAs, primers, and probes used to determine the correct construction of the targeting vector. [Table 2]

[0273] 23:D3-3:12 / J H The 4-6 targeting vector was also linearized with NotI and constructed to contain multiple human V sequences heterozygous for humanized immunoglobulin heavy chain variable regions (i.e., operably linked to rodent immunoglobulin heavy chain constant regions containing rodent heavy chain enhancer and control regions). H , D H , and J. H a first heavy chain allele (1460het) containing a segment and containing an inserted nucleotide sequence encoding one or more mouse Adam6 genes (e.g., U.S. Pat. Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference herein in its entirety); and a plurality of human V H and D H Segment, J HA second heavy chain allele (6394het) containing a rodent immunoglobulin heavy chain constant region including the region deletion and rodent heavy chain enhancer and control regions, and containing an inserted nucleotide sequence encoding one or more mouse Adam6 genes (e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated herein by reference in its entirety), was electroporated into mouse embryonic stem cells having a genome homozygous (HO) for a humanized endogenous κ locus containing a full repertoire of human immunoglobulin light chain Vκ and Jκ gene segments operably linked to an endogenous immunoglobulin heavy chain Cκ region (1293) (see Figure 5). The 6797 allele was electroporated, and the 1460 allele, 23:D3-3:12 / J, containing a neomycin cassette, was inserted (Figure 5). H These engineered mouse ES cells, heterozygous for the 6394 and 6797 alleles, were used to facilitate efficient screening of positive ES clones (see below) and subsequent cre-mediated removal of the drug resistance cassettes; the deletions of the 6394 and 6797 alleles are hereafter referred to as 6643 and 6798, respectively (Figure 6).

[0274] C.12:D H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J H 1-6 targeting vector (Figure 2) Briefly, the third targeting vector was constructed from three D-terminal fragments, each with a 12-mer 5' RSS and a 23-mer 3' RSS. H 2 family gene segments (D H 2-2, D H 2-8 and D H 2-15) including multiple D H First, a BAC (e.g., J) identical in sequence to the chimeric IgH locus of VELOCIMMUNE® mice was constructed using a DNA fragment containing the human segment. HSee Figures 3 and 4, which show exemplary, non-limiting endogenous immunoglobulin heavy chain loci for VELOCIMMUNE® mice that lack gene segments and are heterozygous for the 6394 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence, and the 1460 allele comprising a humanized variable region operably linked to an endogenous mouse immunoglobulin heavy chain constant region sequence. Specifically, the BACs are located between the most proximal human V H Gene (V H 6-1), and then the first human D for selection in E. coli and mouse ES cells. H Segment (D H 1-1), 512 bp upstream of the loxp-UbC-Em7-Neo-loxp cassette flanked by loxP site-specific recombination recognition sites, e.g., modified using bacterial homologous recombination (BHR) to insert the neomycin resistance gene. H Genes, all six human J H The resulting BAC was identical to the 1460 allele, containing the gene, the mouse IgH intronic enhancer (Eμ), the mouse IgM switch region (Sμ), and the first four exons of the mouse IgM gene. The resulting BAC was subsequently modified with three donors using in vitro Cas9 / GA. 1. From 5' to 3', D H A 50-bp homology box starting 1419 bp upstream of the 12 RSS at the 5' end of 2-2, multiple cloning sites (MreI-NsiI-EcoRI-KpnI-MreI), human V H 1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC; T→A, G→C; SEQ ID NO: 151), and contained a 50 bp homology box ending 863 bp downstream of the D2-2 3' 23 mer RSS. H First donor for 2-2 modification. D H The nucleotide sequence of this first donor for the 2-2 modification is set forth as SEQ ID NO: 70.H Because the region consists of four direct repeats of approximately 10 kb, designing unique primers and probes for screening is very challenging. To overcome this problem, two unique 40-bp sequences were inserted; one 74 bp downstream of the 5' homology box and the other 40 bp upstream of the 3' homology box. These unique 40-mers were used as binding sites for PCR / sequencing primers and Taqman probes, and are shown as open rectangles "1" and "2" in Figure 2, containing the nucleotide sequences set forth as SEQ ID NO:73 and SEQ ID NO:74, respectively. 2. From 5' to 3', D H The D2-8 gene contained a 50-bp homology box starting 552 bp upstream of the 5'-terminal 12-mer RSS of D2-8, a multiple cloning site (MreI-NsiI-EcoRI-KpnI-MreI), a modified D2-8 gene with a 3'-terminal 12-mer RSS replaced with a putative 3'-terminal 23-mer RSS derived from human VH1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC; T→A, G→C; SEQ ID NO: 151), and a 50-bp homology box ending 867 bp downstream of the 3'-terminal 23-mer RSS of D2-8. H Second donor for modification of 2-8. D H The nucleotide sequence of this second donor for modification 2-8 is set forth as SEQ ID NO: 71. H Because the region consists of four direct repeats of approximately 10 kb, designing unique primers and probes for screening is very challenging. To overcome this problem, two unique 40-bp sequences were inserted; one 74 bp downstream of the 5' homology box and the other 40 bp upstream of the 3' homology box. These unique 40-mers were used as binding sites for PCR / sequencing primers and Taqman probes, and are shown as open rectangles "10" and "16" in Figure 2, containing the nucleotide sequences set forth as SEQ ID NO:75 and SEQ ID NO:76, respectively. 3. From 5' to 3', D HA 50-bp homology box starting 391 bp upstream of the 12 RSS at the 5' end of 2-15, multiple cloning sites (MreI-NsiI-EcoRI-KpnI-MreI), human V H A modified D2-15 gene was prepared with a 12-mer RSS at its 3' end replaced by a putative 23-mer RSS at its 3' end derived from D2-15 1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC; T→A, G→C; SEQ ID NO: 151), and a 50-bp homology box ending 867 bp downstream of the 23-mer RSS at the 3' end of D2-15. H Third donor for 2-15 qualification. D H The nucleotide sequence of this third donor for the 2-2 modification is set forth as SEQ ID NO: 72. H Because the region consists of four direct repeats of approximately 10 kb, designing unique primers and probes for screening is very challenging. To overcome this problem, two unique 40-bp sequences were inserted; one 74 bp downstream of the 5' homology box and the other 40 bp upstream of the 3' homology box. These unique 40-mers were used as binding sites for PCR / sequencing primers and Taqman probes, and are shown as open rectangles "8" and "18" in Figure 2, containing the nucleotide sequences set forth as SEQ ID NO:77 and SEQ ID NO:78, respectively. After in vitro Cas9 / GA modification in three donors, the final targeting vector contained, from 5' to 3', human variable region DNA, a neomycin selection cassette, and three human D segment-engineered human D fragments, each flanked by a 12-mer RSS at the 5' and a 23-mer RSS at the 3'. H Area, Six Human J H The 12:D homology arm contained a 49 kb 5' homology arm containing the mouse heavy chain intronic enhancer (Ei) and the mouse IgM constant region gene (Figure 2). H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J H1-6 provide the sequences of the gRNAs, primers, and probes used to determine the correct construction of the targeting vector. [Table 3-1] [Table 3-2] [Table 3-3] [Table 10-1] [Table 10-2]

[0275] 12:D H 2-2:23:|12:D H 2-8:23|12:D H 2-15:23x3 / J H The 1-6 targeting vector was also linearized with NotI to express functional human V H Complete repertoire of gene segments, D H D excluding 7-27 H Deletion of the region and functional human J H Mouse embryonic stem cells with a genome homozygous for a humanized immunoglobulin heavy chain variable region containing a full repertoire of gene segments (6011) and homozygous (HO) for a humanized endogenous κ locus containing a full repertoire of human immunoglobulin light chain Vκ and Jκ gene segments operably linked to an endogenous mouse immunoglobulin heavy chain Cκ region (1293) were electroporated (see Figure 7). The 20187 allele was electroporated and the 6011 allele was transfected with the 12:D fusion protein containing the neomycin cassette. H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J HThe 20187 allele is generated by appropriate homologous recombination with the 1-6 targeting vector (Figure 7). These engineered mouse ES cells are used to facilitate efficient screening of positive ES clones (see below) and subsequent cre-mediated removal of the neomycin drug resistance cassette, henceforth referred to as 20188 (Figure 8).

[0276] Example 2. ES cell screening This example demonstrates that the genome contains engineered heavy chain diversity (D H The present invention provides a method for producing a non-human animal (e.g., a rodent) that includes an immunoglobulin heavy chain variable region containing an engineered D H The regions each comprise one or more D operably linked to a 23-mer RSS. H Includes segments.

[0277] The correct assembly of the targeting vectors described in Example 1, which targeted the insertion of DNA fragments into the diversity cluster of the humanized immunoglobulin heavy chain locus contained with BAC DNA, was confirmed by sequencing and polymerase chain reaction during the construction of each targeting vector. The target BAC DNA was confirmed by polymerase chain reaction and then transfected into embryonic stem (ES) cells via electroporation, followed by culturing in selective medium. The genomes of the ES cells used for electroporation of each targeting vector are shown in Figures 3, 5, and 7, respectively. Drug-resistant colonies were picked 10 days after electroporation and manipulated. H Primers / probes detecting correct integration of the gene segment (Table 4; F: forward primer, P: probe, R: reverse primer) were used to screen for correct targeting by TAQMAN™ and karyotyping as previously described (Valenzuela et al., supra; Frendewey, D. et al., 2010, Methods Enzymol. 476:295-307, incorporated herein by reference in its entirety).

[0278] Using the VELOCIMOUSE® method (DeChiara, TM et al., 2010, Methods Enzymol. 476:285-294; DeChiara, TM, 2009, Methods Mol. Biol., 530:311-324; Poueymirou et al., 2007, Nat. Biotechnol., 25:91-99, incorporated herein by reference in their entireties), targeted ES cells were injected into uncompacted 8-cell stage Swiss Webster embryos to produce engineered D H Heterozygous for the region, D H -D H Healthy, whole ES cell-derived F0 mice expressing antibodies containing the recombinantly generated heavy chain variable region containing the CDR3 region were generated. F0 heterozygous males were bred with C57B16 / NTac females to generate F1 heterozygotes, which were then interbred to generate F2 homozygous and wild-type mice.

[0279] For example, to remove any lox-added selection cassette introduced by the unremoved targeting construct at the ES cell stage or in the embryo, the drug selection cassette may be optionally removed by subsequent addition of recombinase (e.g., by Cre treatment) by crossing with a Cre-deficient mouse strain (see, e.g., International Patent Application Publication No. 2009 / 114400, incorporated herein by reference in its entirety). Optionally, the selection cassette is retained in the mouse. The selection cassette engineered into the targeting vector described herein was removed in positive ES cells by transient expression of Cre recombinase (see Figures 4, 6, and 8, respectively). [Table 4-1] [Table 4-2]

[0280] Example 3.23:D H 3-3:12 / J H6,23:D H 3-3:12 / J H 4-6, or 12:D H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J H Characterization of mice modified with the 1-6 targeting vector immunophenotype

[0281] 23:D H 3-3:12 / J H 6 or 23:D H 3-3:12 / J H Immunophenotyping of animals modified with the 4-6 targeting vector was performed on mice heterozygous for the respective 6800 or 6795 modified alleles (see Figures 4 and 6). H 2-2:23|12:D H 2-8:2-23|12:D H 2-15:23 / J H Immunophenotypic analysis of animals modified with the 1-6 targeting vector was performed on mice bred homozygous for the 20188 modified allele. B cell development was analyzed in these animals by fluorescence-activated cell sorting (FACS). Briefly, spleens and leg bones (femurs and tibias) were collected from: VELOCIMMUNE® mice (n=3, 26% C57BL / 6, 23% 129S6 / SvEvTac 51% Balb / cAnNTac; see, e.g., U.S. Pat. Nos. 8,502,018 and 8,642,835), 6643het / 6800het / 1293ho mice (23:D H 3-3:12 / J H modified with six targeting vectors: 25% C57BL / 6NTac, 25% 129S6 / SvEvTac, and 50% Balb / cAnNTac; see Figure 4 ; n = 3); 6643het / 6798het / / 1293ho mouse (23:D H 3-3:12 / J H25% C57BL / 6NTac, 25% 129S6 / SvEvTac, 50% Balb / cAnNTac modified with 4-6 targeting vectors; see Figure 6; n = 3); or 20188ho / 1293ho mice (12:D H 2-2:23|12:D H 2-8:23|12:D H 2.1523 / J H 25% modified with 1-6 targeting vector C57BL / 6NTac, 25% 129S6 / SvFvTac, 50% Balb / cAnNTac;n=3). Bone marrow was collected from the femur by centrifugation. Red blood cells from spleen and bone marrow preparations were lysed with ACK lysis buffer (Gibco) followed by washing with 1x PBS containing 2% FBS. Isolated cells (1x10 6 100 pieces) were incubated with the selected antibody cocktail for 30 minutes at 4°C. Staining 1: Rat anti-mouse CD43-FITC (Biolegend 121206, clone 1B11), rat anti-mouse c-kit-PE (Biolegend 105808, clone 2B8), rat anti-mouse IgM-PeCy (eBiosciences 25-5790-82, clone II / 41), rat anti-mouse IgD-PerCP-Cy5.5 (Biolegend 405710, clone 11-26c.2a), rat anti-mouse CD3-PB (Biolegend 100214, clone 17-A2), rat anti-mouse B220-APC (eBiosciences 17-0452-82, clone RA3-6B2), and rat anti-mouse CD19-APC-H7 (BD 560143, clone 1D3). Stain 2: rat anti-mouse kappa-FITC (BD 550003, clone 187.1), rat anti-mouse lambda-PE (Biolegend 407308, clone RML-42), rat anti-mouse IgM-PeCy (eBiosciences 25-5790-82, clone II / 41), rat anti-mouse IgD-PerCP-Cy5.5 (Biolegend 405710, clone 11-26c.2a), rat anti-mouse CD3-PB (Biolegend 100214, clone 17-A2), rat anti-mouse B220-APC (eBiosciences 17-0452-82, clone RA3-6B2), and rat anti-mouse CD19-APC-H7 (BD 560143, clone 1D3). After staining, cells were washed and fixed in 2% formaldehyde. Data collection was performed on a BD LSRFORTESSA™ flow cytometer (BD Biosciences) and analyzed using FLOWJO™ software (BD Biosciences).

[0282] Mice heterozygous for the 6800 or 6798 allele had reduced total B cell numbers in both the spleen and bone marrow, with an increase in pro-B cells observed in the bone marrow when compared to control VELOCIMMUNE® mice (data not shown). Overall, B cells were similar in kappa and lambda usage (data not shown). Mice homozygous for the 20188 allele had slightly higher levels of lambda in the spleen. +B cells were observed (data not shown). The differences observed in B cell populations in mice expressing the 6800, 6798, or 20188 alleles compared to control VELOLCIMMUNE® mice were not observed to affect B cell development in these animals (data not shown).

[0283] D H -D H Reconstruction and characterization frequencies

[0284] 23:D H 3-3:12 / J H 6 targeting vector, 23:D H 3-3:12 / J H 4-6 targeting vector, or 12:D H 2-2:23|12:D H 2-8:23|12:D H Gene rearrangements in unimmunized, naive mice modified with the 2-15:23 targeting vector were analyzed by IgM repertoire sequencing. Splenic B cell-derived IgM repertoire libraries were prepared using the SMARTer™ RACE (rapid amplification of cDNA ends) cDNA Amplification Kit (Clontech) with primers specific for mouse constant IgM (Table 5, where "nnnnnn" represents a 6-bp index sequence that allows multiplexing of samples for sequencing). The prepared repertoire libraries were then sequenced on a MiSeq sequencer (Illumina).

[0285] Illumina MiSeq paired-end sequences (2 x 300 cycles) were mixed, merged, and selected based on the quality and perfect match of the heavy chain IgM constant region primers. Rearranged heavy chain sequences were aligned to the germline V, D, and J reference database (IMGT). Subsequent analysis included only productive rearrangements, defined as sequences with in-frame VDJ bonds and no stop codons within the open reading frame. [Table 5]

[0286] 23:D H 3-3:12 / J H 6 targeting vector or 23:D H 3-3:12 / J H In unimmunized animals modified with 4-6 targeting vectors, D H 7-27 expression was not detected, and J H The use of J H 6, or J H 4. J H 5, and J H 6 (data not shown). H Recombination with 4 was seen at a higher frequency (approximately 50%) in both modified and control animals (data not shown), whereas 12:D H 2-2:23|12:D H 2-8:23|12:D H In unimmunized animals modified with the 2-15:23 targeting vector, all J H Recombination with the 1-6 gene segment was detected. In all modified animals, the engineered D H Various Vs recombined by gene segments H Gene segments (e.g., but not limited to, V H 4-39, V H 3-23, etc.), and various D H Gene segments (e.g., but not limited to, D H 1-7, D H 3-10, and D H 5-12). Interestingly, D H 2-2, D H 2-8, and D H The use of the 2-15 gene segment resulted in a significantly higher incidence of 12:D compared to control animals (2.57%, 3.44%; 3.58%; n = 3). H 2-2:23|12:D H 2-8:23|12:D H There was a tendency for this to be higher in animals modified with the 2-15:23 targeting vector (2.41%; 6.34%; 6.91%; n = 3) (Table 6).

[0287] The minimum two consecutive Ds identified within the binding region H Functional sequences with segments were identified using the following criteria: H -D H This was further confirmed as arising from a recombination event. H 3-3:12 / J H 6 targeting vector or 23:D H 3-3:12 / J H In the sequence isolated from mice modified with the 4-6 targeting vector, (1)IGHD3-3 D, which is independent of any duplication, H Identical "contiguous" or 3' D sequences aligned with the germline sequence of the gene segment by a minimum of 9 consecutive base pairs H Segmental and (2) germline D H Identified leading 5' and contiguous 3' DNA aligned to the gene segment with a minimum of 5 contiguous base pairs. H For both gene segments, D H -D H Recombination was confirmed. 12:D H 2-2:23|12:D H 2-8:23|12:D H In the sequence isolated from mice modified with the 2-15:23 targeting vector, (1)IGHD2-2 D, independent of any duplications, H Gene segment, IGHD2-8 D H Gene segment, IGHD2-15 D H Identified "leading" or 5' D aligned with the germline sequence of the gene segment for a minimum of 9 consecutive base pairs. H segment, and (2) germline D H Identified leading 5' and contiguous 3' DNA aligned to the gene segment with a minimum of 5 contiguous base pairs. H For both gene segments, D H -D H The recombination phenomenon was confirmed.

[0288] Table 6 shows the functional Ig readings as absolute amounts and as percentages of the total readings, D H 2-2 gene segment, D H 2-8 gene segments, or D H 2-15 Percentage of all functional reads contributed by gene segments, and D H -D H D, which meets the criteria to be identified as the result of a recombination event. H 2-2 gene segment, D H 2-8 gene segments, or D H Th...

Claims

1. A method for obtaining nucleic acid encoding an immunoglobulin heavy chain variable region, said method comprising the step of obtaining nucleic acid comprising a rearranged heavy chain VH(DHA-DHB)JH-encoding nucleotide sequence from a rodent, wherein said rodent is: (i) a D H gene segment immediately adjacent to a 23-mer recombination signal sequence (RSS) (the "engineered D H gene segment"); and (ii) an unrearranged D H gene segment flanked at its 5′ end by a first 12-mer RSS and at its 3′ end by a second 12-mer RSS (“unrearranged D H gene segment”). a germline genome comprising an engineered immunoglobulin heavy chain diversity (DH) region (an "engineered DH region") comprising: (i) the engineered D H gene segment and (ii) the unrearranged D H gene segment are operably linked such that (i) the engineered D H gene segment and (ii) the unrearranged D H gene segment can combine in a D H -D H recombination event according to the 12 / 23 rule; the DHA-DHB nucleotide sequence results from the engineered DH region after the DH-DH recombination event; method.

2. The rodent germline genome comprises: (a) at least one unrearranged V H gene segment operably linked to said engineered D H region; and / or (b) at least one unrearranged JH gene segment operably linked to said engineered DH region; The method of claim 1 further comprising: (a) the at least one unrearranged V H gene segment comprises a complete repertoire of functional unrearranged human V H gene segments; and / or (b)(i) the at least one unrearranged JH gene segment comprises an unrearranged human JH6 gene segment; (ii) the at least one unrearranged JH gene segment comprises an unrearranged human JH4 gene segment, an unrearranged human JH5 ​​gene segment, and an unrearranged human JH6 gene segment; and / or (iii) the at least one unrearranged JH gene segment comprises an unrearranged human JH1 gene segment, an unrearranged human JH2 gene segment, an unrearranged human JH3 gene segment, an unrearranged human JH4 gene segment, an unrearranged human JH5 ​​gene segment, and an unrearranged human JH6 gene segment; The method of claim 2.

4. The method of claim 3(b)(iii), wherein the unrearranged human J H 1 gene segment, the unrearranged human J H 2 gene segment, the unrearranged human J H 3 gene segment, the unrearranged human J H 4 gene segment, the unrearranged human J H 5 gene segment, and the unrearranged human J H 6 gene segment are in a germline configuration.

5. A method according to any one of claims 1 to 4, further comprising the steps of immunising the rodent with an antigen and causing the rodent to generate an immune response to the antigen.

6. The method of any one of claims 1 to 5, wherein DHA or DHB is derived from the DH gene segment or a portion thereof operably linked to a 23mer RSS, and at least 9 base pairs of DHA or DHB are identical to at least 9 base pairs of the DH gene segment or the portion thereof operably linked to the 23mer RSS.

7. The method of any one of claims 1 to 6, wherein the rearranged heavy chain VH(DHA-DHB)JH-encoding nucleotide sequence comprises a rearranged human VH gene segment, a human engineered DH gene segment, a rearranged human DH gene segment, and a rearranged human JH gene segment.

8. The method of claim 7, wherein the D H B gene segment is derived from the engineered D H gene segment comprising a rearranged human germline D H 3-3 gene segment.

9. The method of claim 8, wherein the rearranged human JH gene segment comprises a rearranged human germline JH4 gene segment, a rearranged human germline JH5 gene segment, or a rearranged human germline JH6 gene segment.

10. The method of claim 7, wherein the D H A gene segment is derived from the engineered D H gene segment comprising a rearranged human germline D H 2-2 gene segment, a rearranged human germline D H 2-8 gene segment, or a rearranged human germline D H 2-15 gene segment.

11. The method of claim 10, wherein the rearranged human JH gene segment comprises a rearranged human germline JH1 gene segment, a rearranged human germline JH2 gene segment, a rearranged human germline JH3 gene segment, a rearranged human germline JH4 gene segment, a rearranged human germline JH5 gene segment, or a rearranged human germline JH6 gene segment.

12. The method of any one of claims 1 to 11, wherein the rearranged heavy chain VH(DHA-DHB)JH-encoding nucleotide sequence encodes a complementarity determining region 3 (CDR3) greater than 20 amino acids in length.

13. The method of any one of claims 1 to 12, wherein the rearranged heavy chain VH(DHA-DHB)JH-encoding nucleotide sequence is operably linked to an immunoglobulin heavy chain constant region (CH) nucleotide sequence or a portion thereof.

14. The immunoglobulin C H nucleotide sequence or the portion thereof, (a) is a rodent immunoglobulin C H nucleotide sequence or a portion thereof; and / or (b) comprising a rodent intronic enhancer region and a rodent IgM C H nucleotide sequence; The method of claim 13.

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