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

Abstract

Providing non-human animals capable of DH-DH rearrangement in immunoglobulin heavy chain coding sequences. [Solution] A non-human animal, a method for producing and using the same, and a composition are provided, wherein the non-human animal has a genome containing engineered or recombinant diversity clusters within the immunoglobulin heavy chain variable region, and each engineered or recombinant diversity cluster has one or more D operably bound to a 23-mer recombinant signal sequence. H This includes segment insertion. Methods for producing antibodies derived from non-human animals are also provided, the antibodies optionally containing a human variable region and a rodent, for example, a constant region.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits under 35 U.S.C. 119(3) of U.S. Provisional Patent Applications No. 62 / 685,203 (filed June 14, 2018), No. 62 / 702,206 (filed July 23, 2018), and No. 62 / 812,580 (filed March 1, 2019), each of which is incorporated herein by reference in its entirety.

[0002] Sequence List An official copy of the sequence listing has been submitted electronically via EFS-Web as an ASCII format sequence listing with the filename "10347_ST25.txt," created on June 13, 2019, approximately 50 kilobytes in size, and created concurrently with the specification. The sequence listing contained in this ASCII format document is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Monoclonal antibody products have revolutionized the biopharmaceutical industry, bringing about significant advances in the treatment of several diseases. Many of these monoclonal antibody products leverage the innate properties of antibody molecules (i.e., conventional immunoglobulin gene segments), and in some cases, incorporate other properties such as labeling (e.g., pegylation, radiolabeling) or binding to other drugs. At current approval rates, approximately 70 monoclonal antibody products are expected to be on the market by 2020. Despite these advances and the knowledge gained from the use of monoclonal antibodies for therapeutic purposes, diseases associated with targets that are difficult for monoclonal antibodies to bind to and / or access remain, highlighting the need for different approaches to develop effective treatments. [Overview of the Initiative]

[0004] The present invention involves the manipulation of non-human animals (e.g., rodents (e.g., rats, e.g., mice)) that can be used for the treatment of various diseases 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 their fragments). Further, the present invention also includes the recognition that it is desirable to increase the expression of antibodies containing a complementarity determining region 3 (CDR3) characterized by a longer amino acid length and diversity compared to wild-type (or reference) CDR3, and in some embodiments, diversity that directs binding to a specific antigen. H and D H operated to be operably linked to a recombinant signal sequence that allows for the recombination of one or more D H segments, including immunoglobulin heavy chain variable regions (e.g., heterologous immunoglobulin heavy chain variable regions, e.g., human immunoglobulin heavy chain variable regions) having an engineered heavy chain diversity (D H ) cluster (or engineered D H region). In some embodiments, the non-human animals described herein provide an in vivo system for the development of antibodies and / or antibody-based therapies for administration to humans.

[0005] As used herein, nucleotide molecules are described that contain at least one immunoglobulin heavy chain diversity (D H ) gene segment operably linked to a 23mer recombinant signal sequence (RSS), and optionally, the D H gene segment may be located within an engineered D H region of an immunoglobulin heavy chain variable region (e.g., a human or humanized immunoglobulin heavy chain variable region). Thus, in some embodiments, the nucleotide molecules described herein contain an engineered immunoglobulin heavy chain variable diversity (D H ) containing at least one D H gene segment operably linked to a 23mer (RSS). In some embodiments, the engineered D HThe region is (i) at least one D operably coupled to the RSS of 23mer. H (ii) an unreconstructed D with a 12-mer RSS at one end and another 12-mer RSS at the other end adjacent to it. H (i) At least one D comprising a gene segment operably bound to the RSS of 23mer H (ii) a germline D with a 12-mer RSS at one end and another 12-mer RSS at the other end. H The gene segments are (i) and (ii), according to the 12 / 23 rule, D H -D H The molecules are operably bound so that they can participate in recombination. 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)) containing the nucleotide molecules described herein, methods for using the nucleotide molecules described herein, and so on.

[0006] In some embodiments, D is operably coupled to the 23mer RSS. H The gene segment is operably linked to the 23-mer RSS of human D H Includes a gene segment. In some embodiments, human D H The gene segment contains at least 19 nucleotides and / or encodes 2 cysteines. In some embodiments, human D H The gene segment contains at least 20 nucleotides and / or encodes 2 cysteines. In some embodiments, human D H The gene segment contains at least 23 nucleotides and / or encodes 2 cysteines. In some embodiments, human D H The gene segment contains at least 28 nucleotides and / or encodes 2 cysteines. In some embodiments, human D HThe gene segment contains at least 31 nucleotides and / or encodes 2 cysteines. In some embodiments, human D H The gene segment contains at least 37 nucleotides and / or encodes 2 cysteine ​​molecules. In some embodiments, a 23-mer human D2 is operably bound to the RSS. H The gene segment is operably linked to the 23-mer RSS of human D H It includes two gene segments. In some embodiments, D H The gene segment contains at least 30 nucleotides and / or encodes 2 cysteine ​​molecules. In some embodiments, a 23-mer human D2 is operably bound to the RSS. 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 Selected from a group consisting of 2-15 gene segments. H Includes a gene segment. In some embodiments, human D2 is operably bound 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 a group consisting of 2-15 gene segments H Includes a gene segment. In some embodiments, human D2 is operably bound 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 a group consisting of 2-15 gene segments H Includes a gene segment. In some embodiments, human D H The gene segment is human D H Includes 3-3 gene segments. In some embodiments, human D H The gene segment is human D H Includes 2-2 gene segments. In some embodiments, human D H The gene segment is human D H Includes 2-8 gene segments. In some embodiments, human D H The gene segment is human D H Includes 2-15 gene segments.

[0007] In some embodiments, D is operably coupled to the 23mer RSS. H The gene segment is (a) Human D operably linked to the RSS of 23mer H 3-3 gene segments, in some cases, 23-mer RSS, Human D H (b) Human D operably bound to the RSS of 23mer, adjacent to the 5' end of the 3-3 gene segment H 2-2 gene segments, and in some cases, 23-mer RSS, are human D H Human D2, operably bound to the RSS of (c)23mer adjacent to the 3' end of the 2-2 gene segment. H 2-8 gene segments, and in some cases, 23-mer RSS, are human D H (d) Human 2-15 gene segment operably ligated to the RSS of a 23mer adjacent to the 3' end of the 2-8 gene segment, and optionally the 23mer RSS is human D H 2-15 include the 3' end of the gene segment, or any combination of (e)(a)~(d).

[0008] In some embodiments, a nucleotide molecule described herein (e.g., D operatively bound to a 23-mer RSS) is used. H Gene segment, manipulated D HThe region (such as the immunoglobulin heavy chain variable region) contains a nucleotide sequence that includes the sequence described as SEQ ID NO: 52.

[0009] In some embodiments, a nucleotide molecule described herein (e.g., D operatively bound to a 23-mer RSS) is used. H Gene segment, manipulated D H The region (such as the immunoglobulin heavy chain variable region) contains a nucleotide sequence that includes the sequence described as SEQ ID NO: 61.

[0010] In some embodiments, a nucleotide molecule described herein (e.g., D operatively bound to a 23-mer RSS) is used. H Gene segment, manipulated D H The region (such as the immunoglobulin heavy chain variable region) contains a nucleotide sequence that includes the sequence described as SEQ ID NO: 70.

[0011] In some embodiments, a nucleotide molecule described herein (e.g., D operatively bound to a 23-mer RSS) is used. H Gene segment, manipulated D H The region (such as the immunoglobulin heavy chain variable region) contains a nucleotide sequence that includes the sequence described as SEQ ID NO: 71.

[0012] In some embodiments, a nucleotide molecule described herein (e.g., D operatively bound to a 23-mer RSS) is used. H Gene segment, manipulated D H The region (such as the immunoglobulin heavy chain variable region) contains a nucleotide sequence that includes the sequence described as SEQ ID NO: 72.

[0013] In some embodiments, D is operably coupled to the 23mer RSS. H The gene segment consists of 23-mer RSS and D from 5' to 3'. H Includes gene segments, for example, RSS of 23mers from 5' to 3', (human) D HA gene segment, and a 12mer RSS, for example, a 23mer RSS is D H Adjacent to the 5' end of the gene segment, for example, D H The gene segment is operably linked to the 23mer RSS at the 5' end. In some embodiments, D operably linked to the 23mer RSS H The gene segment is a human D operably linked to the 23mer RSS at the 5' end H Contains a 3-3 gene segment, for example, the nucleotide molecule is 23mer RSS, human D from 5' to 3' H Contains a 3-3 gene segment, and a 12mer RSS.

[0014] At least one D H An engineered D containing a gene segment together with a 23mer RSS at the 5' end H The region is an unrearranged D with a 12mer RSS adjacent at one end and another 12mer RSS adjacent at the other end H May further contain a gene segment (for example, an unrearranged D with a 12mer RSS adjacent at one end and another 12mer RSS adjacent at the other end H The gene segment is germline D H The gene segment, for example, D in its germline configuration H Includes a gene segment, etc.), an unrearranged D H The gene segment is at least one D operably linked to a 23mer at the 5' end H Upstream of the gene segment and operably linked. In some embodiments, an unrearranged D with a 12mer RSS adjacent at one end and another 12mer RSS adjacent at the other end H The gene segment is an unrearranged human D with a 12mer RSS adjacent at one end and another 12mer RSS adjacent at the other end H Contains a gene segment.

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

[0016] At least one D operably coupled to the RSS of the 23mer at the 3' end H Manipulated D containing gene segment H The region is an unreconstructed D where a 12-mer RSS is adjacent to another 12-mer RSS at one end. H It may further include gene segments (for example, an unreconstructed D with a 12-mer RSS adjacent to another 12-mer RSS at one end and another 12-mer RSS at the other end). H The gene segment is germline D H Gene segment, for example, its germline arrangement D H An unrearranged D (which may include gene segments, etc.) where a 12-mer RSS is adjacent to another 12-mer RSS at one end. H The gene segment has at least one D operably ligated to the 23 mer at the 3' end. H It is upstream of the gene segment and is operablely coupled. In some embodiments, an unreconstructed D has a 12-mer RSS at one end and another 12-mer RSS at the other end. H The gene segment is an unreconstructed human D2 gene segment with a 12-mer RSS adjacent to one end and another 12-mer RSS adjacent to the other end. H Includes gene segments.

[0017] In some embodiments, the operated D described herein H The region is (i) one or more unreconstructed human D H Gene segment, Human D H (ii) Each of one or more gene segments is operably linked to a 23-mer RSS at its 5' end, with (ii) a 12-mer RSS flanking its 5' and 3' ends, and (ii) a human D2 gene segment operably linked to a 23-mer RSS at its 5' end. H Gene segment (e.g., human D H At least one D operably bound to a 23-mer RSS, including a 3-3 gene segment HIncludes a gene segment. In some embodiments, the manipulated DH region described herein is operably ligated from 5' to 3' to (i)23mer RSS of at least one D H A gene segment, for example, at least one human D2 gene segment operably linked to a 23-mer RSS at its 3' end. H Two gene segments, optionally operably linked to a 23-mer RSS at their 3' ends, of at least one human D2 gene segment. H The two gene segments are operably linked to a 23-mer RSS at their 3-terminus in human D H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segment, operably linked to a 23-mer RSS at its 3' end. H (ii) including 2-15 gene segments, or any combination thereof, and one or more human D H Gene segment, one or more human D H Each gene segment contains 12-mer RSSs adjacent to its 5' and 3' ends.

[0018] In some embodiments, the operated D described herein H The domain is Human D H Includes only gene segments.

[0019] In some embodiments, the nucleotide molecules described herein (e.g., manipulated D) H The region (including the variable region of the immunoglobulin heavy chain) is operably bound to the 23-mer RSS. H An unreconstructed D gene segment with a 12-mer RSS at one end and another 12-mer RSS at the other end. H A gene segment containing a 23-mer RSS operably bound to D H An unreconstructed D gene segment with a 12-mer RSS at one end and another 12-mer RSS at the other end. H The gene segment is (i) a different D HGene segment, (ii)V H Gene segment, (iii) J H (iv) Not subjected to recombination with gene segments, or any combination thereof. In some embodiments, the nucleotide molecules described herein are (i) another D H Gene segment, (ii)V H Gene segment, (iii) J H One or more D13s are recombinant with a gene segment, or any combination thereof. H Manipulated D including gene segment H The molecule includes a gene region, for example, a nucleotide molecule containing a reconstituted VDJ or VDDJ coding sequence that encodes a variable region of an immunoglobulin heavy chain.

[0020] Therefore, in some embodiments, the operated D described herein H The nucleotide molecules described herein, including the region, are (a) manipulated D by an operable bond. H Located upstream of the region and operably bound, at least one unreconstituted, variable (V) immunoglobulin heavy chain H ) Gene segment (e.g., unreconstructed human V H 6-1 gene segment), (b) manipulated D H Located upstream of the region and operably bound, at least one unreconstituted immunoglobulin heavy chain bind (J H ) Gene segment (e.g., unreconstructed human J H 6) A gene segment, or a combination of (a) and (b).

[0021] In some embodiments, at least one unreconfigured V H The gene segment is from unreconstructed human V H Human V 3-74 and unreconstructed H Across the 1-6 gene segments, including those present, in a functionally unreconstructed human V H Includes a complete repertoire of gene segments. In some embodiments, at least one unreconstructed VH The gene segment is in germline arrangement and is not reconstructed in human V. H Human V 3-74 and unreconstructed H Across the 1-6 gene segments, including those present, in a functionally unreconstructed human V H Includes a complete repertoire of gene segments. In some embodiments, at least one unreconstructed J H The gene segment is from an unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H It includes 6 gene segments. In some embodiments, at least one unreconstructed J H The gene segment is from an unreconstructed human J H 1 gene segment, unreconstructed human J H Two gene segments, unreconstructed human J H 3 gene segments, unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H It includes 6 gene segments. In some embodiments, at least one unreconstructed J H The gene segment is in germline arrangement and is not rearranged in human J. H 1 gene segment, unreconstructed human J H Two gene segments, unreconstructed human J H 3 gene segments, unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H It contains 6 gene segments.

[0022] In some embodiments, the nucleotide molecules described herein are modified D as described herein. H Variable immunoglobulin heavy chains including the region (V H) including the region, for example, from 5' to 3' in a movable joint: (a) At least one unreconstituted immunoglobulin heavy chain variable (V H ) gene segment, (b) At least one D operably coupled to the RSS of 23mer H Manipulated D containing gene segment H region 、 (c) At least one unreconstituted immunoglobulin heavy chain binding (J H ) Includes gene segments.

[0023] In some embodiments, (a) At least one unreconstructed V H The gene segment is, (i) Unreconstructed human V H 6-1 gene segment, (ii) Unreconstructed human V H 2-1 gene segment and unreconstructed human V H 6-1 gene segment, and / or (iii) Unreconstructed human V H Human V not reconstructed from 3-74 H Across 6-1 gene segments, all functionally unreconstituted human V genes, including those containing them. H A gene segment, for example, a human V that has not undergone all functional reconfiguration of germline arrangement. H In some cases, the gene segment, the rodent Adam6 gene, is not rearranged in human V H 2-1 and V H 6-1 Includes substituting a pseudogene between gene segments; (b) Manipulated D H The region is from 5' to 3': (i) One or more, for example, multiple unreconstructed human D H Gene segment, multiple unreconstructed human D HEach gene segment has 12-mer RSSs flanked at its 5' and 3' ends, and is operably linked to a 23-mer RSS at its 5' end, human D H Gene segments, and in some cases, multiple unrearranged human D H The gene segment is in germline arrangement and is not rearranged from human D. H 1-1 gene segment and unreconstructed human D H Between 1 and 26, unreconstructed human D including those mentioned above. H A human gene segment containing a gene segment and / or operably ligated to a 23-mer RSS at its 5' end is a human D H 3-3 gene segments, for example, manipulated D H D is operably coupled to the RSS of the 23 mer at the 5' end. H Unreconstituted human D 3-3. H Human D19, with germline configuration unreconstructed, excluding the 7-27 gene segment. H Includes a complete repertoire of gene segments; (ii) At least one human D operably bound to a 23-mer RSS at its 3' end H Gene segments, and one or more, for example, multiple human D H Gene segment, one or more human D H Each gene segment is operably linked to at least one human D2 gene segment, flanked by a 12-mer RSS at its 5' and 3' ends, and optionally operably linked to a 23-mer RSS at its 3' end. H The gene segment is operably linked to a 23-mer RSS at its 3' end. H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segment, operably linked to a 23-mer RSS at its 3' end. H Includes 2-15 gene segments and / or one or more human D H The gene segment is from unreconstructed human D H1-1 gene segment and unreconstructed human D H Across the 7-27 gene segments, including D H Includes gene segments, and in some cases, manipulated D H This is unreconstructed human D H 2-2, D H 2-8, and D H The 2-15 gene segment is operably linked to a 23-mer RSS at its 3' end. H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segments and their 3' ends operably linked to a 23-mer RSS. H Excluding the 2-15 gene segment, the germline arrangement is unreconstructed human D2. H Includes 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) Unreconstructed human J H 6 gene segments, (ii) Unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H 6 gene segments, and / or (iii) Unreconstructed human J H A complete repertoire of gene segments, e.g., unreconstructed human J H 1 gene segment, unreconstructed human J H Two gene segments, unreconstructed human J H 3 gene segments, unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H 6 gene segments, in some cases, unreconstructed human JH 1, J H 2, J H 3, J H 4, J H 5, and J H The 6 gene segments are germline arrangement, including immunoglobulin V. H Domain (at least one functional V) H Gene segment, manipulated V H Domain, and at least one functional J H (Including gene segments) is human immunoglobulin V H This is a region, for example, D operably coupled to the RSS of 23mer. H Each gene segment (for example, each V within it) H , D H , and J H The gene segment is human (V H , D H , or J H ) This is a gene segment.

[0024] In some embodiments, immunoglobulin V described herein H Nucleotide molecules containing the region are linked by a operable bond from 5' to 3'. (a) At least, unreconstructed human V H 6-1 gene segment, e.g., unreconstructed human V H Human V 3-74 and unreconstructed H 6-1 across gene segments, including all unreconstructed human V H All or part of a gene segment, for example, two unreconstructed human V H Between gene segments, the rodent Adam6 gene, for example (for example, the rodent Adam6 gene is human V H 1-2 gene segments and human V H Functionally unreconstructed human V (including those located between the 6-1 gene segments) H Complete repertoire of gene segments (b) Unreconstructed human D in germline configurationH 1-1 gene segment and unreconstructed human D H Across the 1-26 gene segments, including those, unreconstructed human D2 from 5' to 3'. H Human-engineered D, including gene segments H The region and the RSS of the 23-mer at the 5' end are operably bound to unreconstituted human D2. H 3-3 gene segments, e.g., unreconstituted human D operably linked to the 23-mer RSS at the 5' end. H Unreconstructed human D with 3-3 gene segment substitutions. H Human D2, not reconfigured in germline configuration, excluding the 7-27 gene segment. H The complete repertoire of gene segments, and (c) Human J that has not been reconstructed at least H It includes 6 gene segments.

[0025] In some embodiments, immunoglobulin V described herein H Nucleotide molecules containing the region are connected by a movable bond from 5' to 3': (a) At least, unreconstructed human V H 6-1 gene segment, e.g., unreconstructed human V H Human V 3-74 and unreconstructed H 6-1 across gene segments, including all unreconstructed human V H All or part of a gene segment, for example, two unreconstructed human V H Between gene segments, the rodent Adam6 gene, for example (for example, the rodent Adam6 gene is human V H 1-2 gene segments and human V H Functionally unreconstructed human V (including those located between the 6-1 gene segments) H Complete repertoire of gene segments (b) Unreconstructed human D in germline configuration H1-1 gene segment and unreconstructed human D H Across the 1-26 gene segments, including those, unreconstructed human D2 from 5' to 3'. H Human-engineered D, including gene segments H The region and the RSS of the 23-mer at the 5' end are operably bound to unreconstituted human D2. H 3-3 gene segments, e.g., unreconstituted human D operably linked to the 23-mer RSS at the 5' end. H Unreconstructed human D with 3-3 gene segment substitutions. H Human D2, not reconfigured in germline configuration, excluding the 7-27 gene segment. H The complete repertoire of gene segments, and (c) Unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J H It includes 6 gene segments.

[0026] In some embodiments, immunoglobulin V described herein H Nucleotide molecules containing the region are linked by a operable bond from 5' to 3'. (a) At least, unreconstructed human V H 6-1 gene segment, e.g., unreconstructed human V H Human V 3-74 and unreconstructed H 6-1 across gene segments, including all unreconstructed human V H All or part of a gene segment, for example, two unreconstructed human V H Between gene segments, the rodent Adam6 gene, for example (for example, the rodent Adam6 gene is human V H 1-2 gene segments and human V H Functionally unreconstructed human V (including those located between the 6-1 gene segments) H Complete repertoire of gene segments (b) From 5' to 3', unreconstructed human D H 1-1 gene segment, with a 23-mer RSS operably linked at its 3' end. H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segment, operably linked to a 23-mer RSS at its 3' end. H 2-15 gene segments, and unreconstructed human D H 3-16 gene segments and unreconstructed human D H Across the 7-27 gene segments, including those, unreconstructed human D H Human-engineered D, including gene segments H Region, in some cases, manipulated D H This is unreconstructed human D H 2-2, D H 2-8, and D H Each of the 2-15 gene segments is operably linked to a 23-mer RSS at its 3' end. H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segments and their 3' ends operably linked to a 23-mer RSS. H Except for substitutions in gene segments 2-15, the germline configuration is that of unreconstructed human D12. H (c) Unreconstructed human J, including the complete repertoire of gene segments. H A complete repertoire of gene segments, e.g., unreconstructed human J H 1 gene segment, unrearranged J H 2 human gene segments, unreconstructed J H 3 human gene segments, unreconstructed J H 4 human gene segments, unreconstructed J H 5 human gene segments, and unreconstructed J H 6 human gene segments, in some cases, unreconstructed human JH 1, J H 2, J H 3, J H 4, J H 5, and J H The 6 gene segments include those in germline arrangement.

[0027] In some embodiments, the nucleotide molecules described herein have (a) at least one (human) V nucleotide at the 5' to 3' end. H Variable (V) immunoglobulin heavy chains containing gene segments (human) H ) region, (human) manipulated D as described herein H region, and (b) heavy chain immunoglobulin constant region (C H ) or at least one (human) J operably coupled to a part thereof H Includes gene segments, and in some cases, C H This includes rodent intron enhancer regions, rodent IgM genes, rodent IgD genes, rodent IgG genes, rodent IgA genes, rodent IgE genes, or any combination thereof, rodent C H In some embodiments, the nucleotide molecules described herein have (a) at least one human V nucleotide at the 5' to 3' end. H Gene segment, human-modified D as described herein H (b) a region, and (b) at least a rodent intron enhancer region, optionally including the rodent IgM gene, rodent C H At least one human J operably bound to the region H Human immunoglobulin heavy chain V, including gene segments. H The region includes. In some embodiments, the nucleotide molecules described herein have (a) at least one human V nucleotide from 5' to 3'. H Gene segment, human-modified D as described herein H (b) a region, and (b) at least a rodent intron enhancer region, optionally including the rodent IgM gene, rodent C H At least one human J operably bound to the region HHuman heavy chain V, including gene segments H The region includes. In some embodiments, the nucleotide molecules described herein have (a) at least one human V nucleotide from 5' to 3'. H Gene segment, human-modified D as described herein H region, and (b) endogenous rodents C H The region contains, for example, at least one human J activatably bound at the endogenous rodent immunoglobulin heavy chain locus. H Human V, including gene segments H This includes the region. 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 the rodent Adam6 gene. In some embodiments, the rodent Adam6 gene comprises the human V H 2-1 and V H Located between 6-1 gene segments, for example, in germline arrangement, human V H 2-1 and V H The human Adam6 gene located between the 6-1 gene segments 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 include one or more drug selection cassettes, e.g., drug resistance genes adjacent to one or more site-specific recombination sites, e.g., neomycin drug resistance genes adjacent to a loxP site-specific recombination recognition site, and at least one of the one or more drug resistance cassettes optionally has at least one D nucleotide operably bound to a 23-mer RSS. H It is located immediately upstream of the gene segment. In some embodiments, the nucleotide molecule includes the sequence described in Figure 2.

[0030] Furthermore, the genome (e.g., germline genome) of a non-human animal (e.g., a rodent such as a rat or mouse) is modified according to the D described herein. H Targeting vectors for modification to include a region are also described herein. Generally, the targeting vectors described herein include one of the nucleotide molecules described herein, optionally including 5' and 3' homologous arms for homologous recombination within the immunoglobulin heavy chain variable region, optionally the immunoglobulin heavy chain variable region being human or humanized immunoglobulin heavy chain variable region. In some embodiments, the targeting vectors described herein include an unreconstituted human gene segment, e.g., V H 6-1 gene segment containing a 5' homologous arm, and / or rodents (e.g., mouse) C H A region or part thereof, for example, rodents (mouse) C H It includes an intron enhancer region and / or a 3' homologous arm containing the rodent (mouse) IgM gene.

[0031] In some embodiments, the targeting vector includes the nucleotide molecules described herein and 5' and 3' homologous arms positioned to allow homologous recombination with the immunoglobulin heavy chain sequence, which may be located at the endogenous rodent immunoglobulin heavy chain locus and / or include a human or humanized immunoglobulin heavy chain variable region. In some embodiments, an unreconstituted human gene segment (e.g., V H 6-1 gene segment), human-engineered D H Region, at least one unreconstructed human J H 5' homologous arm containing gene segment, and rodent (mouse) C HThe targeting vectors described herein, comprising an intron enhancer region and / or a 3' homologous arm including a rodent (mouse), may optionally include a modified endogenous heavy chain constant region gene sequence containing an intact endogenous IgM gene and another endogenous modified constant region gene (e.g., IgG) for the production of reverse chimeric non-IgM antibodies lacking a functional ADAM6 gene, a functional CH1 domain, a reverse chimeric humanized common light chain, a reverse chimeric humanized kappa light chain, a reverse chimeric humanized lambda light chain, or a hybrid kappa / lambda or kappa / lambda light chain. Rodents containing a humanized immunoglobulin heavy chain locus, which may include an unreconstituted germline human heavy chain gene segment and / or a reconstituted (or unreconstituted) germline light chain gene segment, which may contain a sequence encoding a histidine amino acid and be modified with a histidine codon for the expression of a variable domain capable of exhibiting terminal deoxyribonucleotide transferase (TdT) for pH-sensitive antigen binding and / or increased antigen receptor diversity, for example, mice containing a substitution of a mouse immunoglobulin variable sequence with a human immunoglobulin variable sequence, for example, in VELOCIMMUNE® mice. HIt may be useful for manipulating the domain. For example, 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, 8,697,940; 8,7 See Patent Nos. 54,287; Nos. 9,334,334; Nos. 9,801,362; Nos. 9,332,742; Nos. 9,969,814; U.S. Patent Publications 2011 / 0195454, 2012 / 0021409, 2012 / 0192300, 2013 / 0185821, 2013 / 0302836, 2013 / 0045492, and 2018 / 0125043; and International Patent Application Publications 2017210586 and 2019 / 113065, each of which, in whole, is incorporated herein by reference.

[0032] Therefore, for example, D in rodents H -D H D for rearrangement H Methods for manipulating the region are described herein. In some embodiments, the method involves at least one D operably coupled to the RSS of 23mer. H One or more unreconstructed D H D containing gene segment H This includes the step of modifying the region. In some embodiments, D H -D H Methods for modifying the variable region of an immunoglobulin heavy chain to manipulate recombination include one or more unreconstituted D H D containing gene segment H A process to obtain a variable region of immunoglobulin heavy chains that includes the region, and unreconstituted D HEach gene segment has at least one D operatively linked to a 23-mer RSS, with a 12-mer RSS adjacent to one end and another 12-mer RSS adjacent to the other end. H D further includes gene segments H This includes the step of modifying the region. In some embodiments, D H The region is one or more unreconstructed human D H Human D2 including gene segment H This is a region, for example, multiple unreconstructed human D2 H In some cases, the gene segment may be, for example, in germline arrangement, D H 1-1 and D H Across the 7-27 gene segments, all functionally unreconstituted human D2 genes, including those segments, are included. H Includes a gene segment. In some embodiments, the modification is one or more unreconstructed D2s adjacent to a 12-mer RSS at one end and another 12-mer RSS at the other end. H Gene segments, for example, unreconstructed human D19. H One or more gene segments are operably linked to at least one D23mer RSS. H This includes substitution with a gene segment. In some embodiments, D H A 12-mer RSS at one end of the region and another 12-mer RSS at the other end are adjacent to a large portion of the unreconstructed 3' D H The gene segment is operably linked to the 23-mer RSS. H The gene segment is replaced and operably linked to the 23-mer RSS. H The gene segment is a 23-mer RSS from 5' to 3', D H Includes a gene segment and a 12-mer RSS. In some embodiments, an unreconstructed D is found where a 12-mer RSS is adjacent to another 12-mer RSS at one end. H The gene segment was engineered to operably bind to the 23-mer RSS of the corresponding D H It is replaced with a gene segment. In some embodiments, DH The region is unreconstructed human D H 7-27 gene segments (e.g., germline D H Includes 7-27 gene segments) and unreconstituted human D H The 7-27 gene segment is operably linked to the RSS of the 23-mer at the 5' end of human D H Gene segment (e.g., unreconstructed human D H It is replaced by the 3-3 gene segment. In some embodiments, D H The region is unreconstructed D H 2-2 gene segment, unrearranged D H 2-8 gene segments, and / or unrearranged D H Includes 2-15 gene segments (e.g., manipulated D H This is a germline arrangement of unreconstructed human D H (Including the complete repertoire of gene segments), unreconstructed D H 2-2 gene segment, unrearranged D H 2-8 gene segments, and / or unrearranged D H Each of the 2-15 gene segments is operably linked to a 23-mer RSS at its 3' end. H Human D2-2 gene segment, operably linked to a 23-mer RSS at its 3' end. H Human D2-8 gene segments and / or their 3' ends operably linked to a 23-mer RSS. H 2-15 gene segments are substituted. In some embodiments, the modification is not rearranged. H Gene segment (e.g., human germline D H This involves replacing one of two 12-mer RSSs adjacent to a gene segment with a 23-mer RSS. In some embodiments, the immunoglobulin heavy chain variable region to be modified is D H In addition to the region, D H J operably coupled to the region H Region (in some cases, 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 6 gene segments H Includes a complete repertoire of gene segments, and in some cases, human germline J H 1, J H 2, J H 3, J H 4, J H 5, and J H The 6 gene segment contains (in germline arrangement) an unreconstructed D with a 12-mer RSS at one end and another 12-mer RSS adjacent at the other end. H Gene segment substitution is J H At least one unreconstructed J included in the region H This includes deleting a gene segment, for example, D H Unreconstructed human J adjacent to the region H 1, J H 2, J H 3, J H 4, and / or J H This results in the deletion of 5 gene segments. In some embodiments, J H At least one germline J included in the region H Deleting a gene segment is possible in unreconstructed human J H 1, J H 2, and J H Deleting three gene segments, and, if applicable, J H 4 and J H This includes further deletion of 5 gene segments. In some embodiments, all functional D H Substituting one or more gene segments, for example, D H The 7-27 gene segment was operably linked to the 23-mer RSS at the 5' end of D HA gene segment, for example, D operably bound to the 23-mer RSS at the 5' end. H Substitution in the 3-3 gene segment is D H Unreconstructed human J adjacent to the region H 1, J H 2, and J H This results in the deletion of three gene segments. In some embodiments, all functional D H Substituting one or more gene segments, for example, D H The 7-27 gene segment was operably linked to the 23-mer RSS at the 5' end of D H A gene segment, for example, D operably bound to the 23-mer RSS at the 5' end. H Substitution in the 3-3 gene segment is D H Unreconstructed human J adjacent to the region H 1, J H 2, J H 3, J H 4, and J H This results in the deletion of 5 gene segments. In some embodiments, D H The region is operably coupled to the RSS of 23mer, with at least one D H At least one D modified to include a gene segment and operably bound to a 23-mer RSS H The gene segment is (a) Human D operably linked to the RSS of 23mer H 3-3 gene segments, in some cases, 23-mer RSS, D H (b) Human D operably bound to the RSS of 23mer, adjacent to the 5' end of the 3-3 gene segment H 2-2 gene segments, in some cases, 23-mer RSS, D H Human D2, operably bound to the RSS of (c)23mer adjacent to the 3' end of the 2-2 gene segment. H 2-8 gene segments, in some cases, RSS of 23mers, D H Human D2, operably bound to the RSS of (d)23mer adjacent to the 3' end of the 2-8 gene segment. H2-15 gene segments, in some cases, RSS of 23mers, D H 2-15 include the 3' end of the gene segment, or any combination of (e)(a)~(d).

[0033] This method involves the manipulated D H A variable immunoglobulin heavy chain region containing the region may result in a nucleotide molecule containing, for example, the (human) immunoglobulin heavy chain variable region described herein, and the reconstituted (or unreconstituted) (human) immunoglobulin heavy chain region may be a non-human immunoglobulin heavy chain constant region or a portion thereof, for example, at least rodent C H The intron enhancer region and / or the rodent IgM gene may be optionally operably bound to the rodent immunoglobulin heavy chain constant region, which may include, for example, a non-human immunoglobulin heavy chain locus. In some embodiments, during recombination, such immunoglobulin heavy chain locus may include an immunoglobulin heavy chain variable domain, for example, with 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 6. Includes a reconstituted immunoglobulin heavy chain variable region coding sequence that encodes an immunoglobulin heavy chain variable domain having a complementarity-determining region 3 (CDR3) amino acid length longer than 20 amino acids, which may be the result of recombination. Thus, the reconstituted immunoglobulin heavy chain V encoding an immunoglobulin heavy chain variable domain containing CDR3, where the length of 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 HRodents, rodent cells, gene loci and / or nucleotide molecules containing 6 sequences are provided herein.

[0034] Furthermore, for example, the genome, for example, the germline genome, contains D manipulated according to the present invention. H Non-human animals, such as rodents, containing regions, for example, nucleic acids, targeting vectors and / or immunoglobulin heavy chain variable loci described herein, are also described herein. Furthermore, genomes of such non-human animals, such as rodent genomes, are also described herein. In some embodiments, a rodent germline genome, or rodent germline genome, containing an immunoglobulin heavy chain variable region described herein, wherein the immunoglobulin heavy chain variable region comprises (i) at least one unreconstituted heavy chain variable (V H ) gene segment, (ii) manipulated heavy chain variable region diversity (D H ) Region, manipulated D H The region is one or more unreconstructed D where each has a 12-mer RSS at one end and a 12-mer RSS at the other end. H A gene segment, and one or more D molecules operably bound to a 23-mer recombinant signaling sequence (RSS). H (iii) containing a gene segment and (iii) at least one unreconstituted heavy chain bond (J H ) containing gene segments, and (i)~(iii) are operably bound so that, upon recombination, the immunoglobulin heavy chain variable region contains a reconstituted heavy chain variable region sequence encoding the immunoglobulin heavy chain variable domain, and optionally the reconstituted heavy chain variable region sequence is V H (D H -D H )J H One or more D2s are formed after the recombination phenomenon, and in some cases, each is operably coupled to a 23-mer recombinant signal sequence (RSS). H At least one of the gene segments is V H (D H -D H) During the rearrangement phenomenon, one or more unreorganized D segments are adjacent to each other, each consisting of a 12-mer RSS at one end and another 12-mer RSS at the other end. H It binds to one of the gene segments. In some embodiments, one or more D segments are operably bound to the RSS of the 23-mer. H The segment is operably coupled to a 3' 23mer RSS. H Includes a gene segment. In some embodiments, one or more D segments are operably bound to the 23-mer RSS. H The segment is operably coupled to a 5' 23mer RSS. H Includes a gene segment. In some embodiments, the immunoglobulin heavy chain variable region is human V H , D H , and J H This is a human immunoglobulin heavy chain variable region containing only a gene segment. In some embodiments, the human immunoglobulin heavy chain variable region is operably bound to the immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is, for example, the endogenous immunoglobulin heavy chain constant region of a non-human animal or non-human animal genome at the endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain variable region is V in germline arrangement H V from 3-74 H Human V spanning 6-1 H Includes a gene segment. In some embodiments, the immunoglobulin heavy chain variable region is human J H It includes 6 gene segments. In some embodiments, the immunoglobulin heavy chain variable region may be two human V H Inserted into a gene segment (for example, human V H 1-2 gene segments and human V H It comprises one or more nucleotide molecules encoding one or more rodent Adam6 polypeptides (e.g., rodent Adam6 genes), which may be inserted between the 6-1 gene segments and / or in place of the human Adam6 pseudogene. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent genome is an engineered D HIt is heterozygous for the region. In some embodiments, the rodent genome is manipulated D H It is homozygous for the region. 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 may be 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 rodents, rodent genomes, or rodent cells described herein are reconstituted heavy chain VDJs and / or Vs encoding immunoglobulin heavy chain variable domains. H (D H AD H B)J H The coding sequence further includes (1) in its germline genome, for example in germline cells, operably bound to the 23-mer RSS H Manipulated D containing gene segment H (2) an immunoglobulin heavy chain locus containing the region, and in its somatic genome, for example in B cells, the reconstituted heavy chain variable region V H (D H AD H B)J H A non-human animal, such as a rodent, such as a rat or mouse, is described, including a code sequence, and the first or second D H Gene segments (each, D H A or D H B) D is operably coupled to the 23mer RSS. H A gene segment, or a part thereof (for example, a D operably bound to a 23-mer RSS) H It is derived from a gene segment, a somatically highly mutated variant thereof, and / or a sequence identical to at least a portion of its degenerate variant. In some embodiments, the B cell is a naive B cell, and / or the reconfigured heavy chain variable region coding sequence is operably bound to the IgM constant region sequence, and DH A and D H At least one of the B gene segments is operably linked to the 23-mer RSS of D H It contains at least nine consecutive nucleotides that align with the nucleotide molecule encoded in the gene segment, D H A and D H Each of the B gene segments is germline D H It contains at least five consecutive nucleotides aligned with the nucleotide molecule of the gene segment. In some embodiments, the B cell is a plasma cell or a memory B cell, and / or the reconfigured heavy chain variable region coding sequence is somatically highly mutant and / or operably bound to a non-IgM constant region sequence (e.g., IgG, IgA, IgE, etc.) and D H A and D H Each of B has at most one nucleotide mutation, affecting the first and second germline D H It shows 40% identity of the gene segment. In some embodiments, all reconstituted heavy chain VDJ and / or V in rodents. H (D H AD H B)J H At least 95% of the coding sequence has a CDR3 of at least 10 amino acids in length, and possibly all reconstituted heavy chain VDJ and / or V in rodents. H (D H AD H B)J H At least 70% of the coding sequence has a CDR3 of at least 11 amino acid length, and possibly all reconstituted heavy chain VDJ and / or V in rodents. H (D H AD H B)J H At least 15% of the coding sequence has a CDR3 of at least 14 amino acid length. In some embodiments, VDJ and / or V in rodents. H (D H AD H B)J HThe coding sequence population has CDR3 of at least 15 amino acid length, optionally at least 16 amino acid length, optionally at least 17 amino acid length, and optionally at least 18 amino acid length. In some embodiments, the reconstituted heavy chain V H (D H AD H B)J H A rodent or rodent cell expressing the coding sequence, or a nucleic acid or immunoglobulin gene locus containing the coding sequence, as described herein, and the second germline D H The gene segment is D H 3-3, and in some cases, reconstituted heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 longer than 20 amino acids. In some embodiments, a reconstituted heavy chain V H (D H AD H B)J H A rodent or rodent cell expressing the coding sequence, or a nucleic acid or immunoglobulin locus containing the coding sequence, is described herein, and the first germline D H The gene segment is D H 2-2, D H 2-8 or D H 2-15, and in some cases, reconstituted heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 longer than 20 amino acids. In some embodiments, a reconstituted heavy chain V encodes a CDR3 longer than 20 amino acids. H (D H AD H B)J H Rodents or rodent cells expressing the 6-coding sequence are described herein.

[0036] In some embodiments, a reconstituted human immunoglobulin heavy chain V is operably bound to a rodent immunoglobulin heavy chain constant region sequence. H (D HAD H B)J H A rodent genome, nucleic acid, or immunoglobulin locus containing a coding sequence is provided. In some embodiments, D H The B gene segment is human germline D H It is derived from the 3-3 gene segment. In some embodiments, D H The A gene segment is human germline D H 2-2, D H 2-8 or D H It is derived from gene segments 2-15. In some embodiments, D H A and D H Each of B has at most one nucleotide mutation, affecting the first and second germline D H Since it shows 40% identity with the gene segment, the reconstituted heavy chain is V H (D H AD H B)J H The code sequence is determined later. In some embodiments, the reconstructed heavy chain V H (D H AD H B)J H The coding sequence encodes a CDR3 longer than 20 amino acids. In some embodiments, J H The gene segment is human germline J H It is derived from 6 gene segments. In some embodiments, V H (D H AD H B)J H A rodent or rodent cell containing a 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, a reconstituted heavy chain V fused with myeloma cells is provided. H (D H AD H B)J H A hybridoma is provided that contains rodent B cells expressing a coding sequence. In some embodiments, V H (D HAD H B)J H Each array is D H A and D H The sequences identified as B each have a maximum of one nucleotide mutation, and are first and second germline D H Even when it shows 40% identity with the gene segment, D H -D H It has been confirmed that this is the result of genetic recombination.

[0037] In some embodiments, the genome is manipulated D H Non-human animals or cells containing an immunoglobulin heavy chain variable region, including the region, are provided and manipulated D H The region is operably coupled to the first and second recombinant signal sequences (RSS) with at least one D H Includes segments. In some embodiments, the first RSS is a 23-mer RSS and the second RSS is a 12-mer RSS.

[0038] In some embodiments, the genome is manipulated D H Non-human animals are provided that include a variable region of immunoglobulin heavy chains, and manipulated D H Each region is operably coupled to one or more D2s, each a 23-mer recombinant signal sequence (RSS). H Includes segments.

[0039] In some embodiments, the genome is manipulated D H Non-human cells or tissues containing an immunoglobulin heavy chain variable region are provided, and manipulated D H Each region is operably coupled to one or more D2s, each a 23-mer recombinant signal sequence (RSS). HIncludes segments. In some embodiments, the cells are of lymphoid or myeloid origin. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are selected from B cells, dendritic cells, macrophages, monocytes, and T cells. In some embodiments, the tissue is selected from fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testes, oocyte, or any combination thereof.

[0040] In some embodiments, immortalized cells produced from non-human cells as described herein are provided, such as hybridoma cells produced by fusing B cells isolated from non-human animals as described herein with myeloma cells.

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

[0042] In some embodiments, the use of non-human embryonic stem cells described herein for producing non-human animals is provided. In some embodiments, the non-human embryonic stem cells are mouse embryonic stem cells, which are manipulated as described herein. H It is used to produce mice containing an immunoglobulin heavy chain variable region that includes the region. In some embodiments, the non-human embryonic stem cells are rat embryonic stem cells and are manipulated as described herein. H Used to produce rats containing an immunoglobulin heavy chain variable region that includes the region. In some embodiments, manipulated D H Non-limiting exemplary methods for producing rats containing an immunoglobulin heavy chain variable region including the region may include the method disclosed in U.S. Patent No. 20140309487, which is incorporated herein by reference in its entirety.

[0043] In some embodiments, non-human embryos are provided that include, are made from, are obtained from, or are generated from non-human embryonic stem cells as described herein. In some embodiments, the non-human embryo is a rodent embryo, in some embodiments it is a mouse embryo, and in some embodiments it is a rat embryo.

[0044] In some embodiments, the use of non-human embryos described herein for producing non-human animals is provided. In some embodiments, the non-human embryo is a mouse embryo and is manipulated as described herein. H It is used to produce mice containing an immunoglobulin heavy chain variable region that includes the region. In some embodiments, the non-human embryo is a rat embryo and is manipulated as described herein. H It is used to produce rats containing the immunoglobulin heavy chain variable region, which includes the region.

[0045] The genome is manipulated by D H A method for producing a rodent including a region, comprising: (a) one or more D, each operably coupled to a 23mer RSS H A step of modifying the genome of rodent embryonic stem cells, for example, by including a DNA fragment containing a segment, the DNA fragment being a nucleotide molecule, a targeting vector, and / or an engineered D H A method is provided herein that includes the step of generating rodents using (a) modified rodent embryonic stem cells (b) including a region. In some embodiments, the method includes at least one D operably coupled to the RSS of 23mer H Unreconstituted D12, which includes the segment of the immunoglobulin heavy chain variable region. H The process includes modifying the region, and the unreconstructed D H Each region consists of one or more unreorganized D2s, each with a 12-mer RSS at one end and another 12-mer RSS at the other end. HThe gene segment is further included, thereby producing the rodent. In some embodiments, modification is performed on one or more unreconstituted human D H Substitution of gene segments, and in some cases, one or more J H The gene segment is operably linked to at least one D23mer RSS. H This includes substituting or deleting segments. In some embodiments, the immunoglobulin heavy chain variable region is a human immunoglobulin heavy chain variable region, for example, a human immunoglobulin heavy chain variable region is at least one V H Unreconstituted human immunoglobulin heavy chain V containing gene segments H One or more unreconstructed human D2 containing gene clusters H Unreconstituted human immunoglobulin heavy chain D containing gene segments H Region, and one unreconstructed human J H Unreconstituted human immunoglobulin heavy chain J containing gene segments H Gene cluster, unreconstituted human immunoglobulin heavy chain D H The region is operably coupled to the RSS of 23mer, with at least one D H It is modified to include a segment. In some embodiments, the modification step is (i) functional human V H Gene segment, for example, V H V from 3-74 H 6-1, including all functional V H (ii) A complete repertoire of gene segments, (ii) at least one D operably bound to the RSS of 23mers H D is replaced in the gene segment H Unreconstituted human D, excluding 7-27. H (iii) a complete repertoire of gene segments, and (iii) at least unreconstructed human J H 6 gene segments, and, if applicable, at least unrearranged J H 4 gene segments, unrearranged J H5 gene segments, and unreconstructed J H This results in a variable immunoglobulin heavy chain region containing 6 gene segments. In some embodiments, at least one human D2 is operably bound to the 23-mer RSS. H The gene segment is operably linked to the 23-mer RSS at 5'. H Includes 3-3 gene segments. In some embodiments, the modification step is (i) functional human V H Gene segment, for example, V H V from 3-74 H 6-1, including all functional V H (ii) Complete repertoire of gene segments, (ii) Unreconstructed human D H 2-2, D H 2-8, and D H Each of the 2-15 gene segments is operably linked to a 23-mer RSS at its 3' end. H Human D2-2 gene segment, operably linked to the 23-mer RSS at its 3'. H Human D2-8 gene segments and their 23-mer RSS operably linked at 3' H Unreconstructed human D19, except that it has substitutions in gene segments 2-15. H (iii) an unreconstructed human J H Gene segments, for example, unreconstructed human J H 1 gene segment, unreconstructed human J H Two gene segments, unreconstructed human J H 3 gene segments, unreconstructed human J H 4 gene segments, unreconstructed human J H 5 gene segments, and unreconstructed human J HThis results in an immunoglobulin heavy chain variable region containing a complete repertoire of 6 gene segments. In some embodiments, the immunoglobulin heavy chain variable region (a) further includes one or more rodent Adam6 genes, and optionally one or more rodent Adam6 genes include two unreconstructed V H Between gene segments, for example, unreconstructed human V H 1-2 gene segments and unreconstructed human V H 6-1 is located between gene segments and / or (b) is operably bound to an immunoglobulin heavy chain constant region, which may be an endogenous rodent immunoglobulin heavy chain constant region, e.g., the 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 comprising 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, the kits described herein are provided for use in the manufacture and / or development of drugs (e.g., antibodies or their antigen-binding fragments) for therapeutic or diagnostic purposes. In some embodiments, the kits described herein are provided for use in the manufacture and / or development of drugs (e.g., antibodies or their antigen-binding fragments) for the treatment, prevention, or improvement of diseases, disorders, or conditions.

[0048] In some embodiments, the 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 the manipulated D described herein. HIncludes a region. In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector is operably bound to one or more D23mers of the RSS. H The transgene includes a DNA fragment containing a segment. In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector further includes one or more selection markers. In some embodiments, the transgene, nucleic acid construct, DNA construct, or targeting vector further includes 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 shown in Figure 2.

[0049] In some embodiments, the use of the transgenes, nucleic acid constructs, DNA constructs, or targeting vectors described herein is provided for producing non-human animals, non-human cells, non-human embryonic stem cells, and / or non-human embryos.

[0050] In some embodiments, one or more D H Each segment is operably coupled to a 3' 23mer RSS. In some embodiments, one or more D H Each segment is operably coupled to a 5' 23mer RSS.

[0051] In some embodiments, the manipulated D H The region is a single D duct operably coupled to a 5' 23m RSS. H Includes segments. In some embodiments, the operated D H The region is a single D operably coupled to the 3' RSS. H Includes segments. One D operably coupled to a 5' 23mer RSS. H In some embodiments of the segment, one D H The segment is synthetic D H It is a segment, and in some embodiments, synthetic human D H It is a segment, and in some embodiments, human DH Synthetic human D having a sequence identical or substantially identical to segment 3-3 H It is a segment. One D operably coupled to a 3' 23mer RSS. H In some embodiments of the segment, one D H The segment is synthetic D H It is a segment, and in some embodiments, synthetic human D H It is a segment, and in some embodiments, human D H Synthetic human D having a sequence identical or substantially identical to segment 3-3 H It is a segment.

[0052] In some embodiments, the manipulated D H The region consists of three D segments, each operably coupled to a 5' 23m RSS. H Includes segments. Three D segments operably coupled to a 5' 23m RSS each. H In some embodiments of the segment, three D H The segment is synthetic D H It is a segment. Three D segments are operably coupled to a 5' 23m RSS each. H In some embodiments of the segment, three D H The segment is Human D H It is a 2 family segment. Each has three D segments operably coupled to a 5' 23m 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 Selected from 2-21 and its combinations. Each consists of three D operably coupled 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, and Human D H The answer is 2-15.

[0053] In some embodiments, the manipulated D H The region consists of three D segments, each operably coupled to a 3' RSS of 23m. H Includes segments. Three D segments operably coupled to a 3' 23m RSS each. H In some embodiments of the segment, three D H The segment is synthetic D H It is a segment. Three D segments are operably coupled to a 3' 23m RSS each. H In some embodiments of the segment, three D H The segment is Human D H It is a 2 family segment. Each has three D segments operably coupled to a 3' 23m 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 Selected from 2-21 and its combinations. Each consists of three D operably coupled 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, and Human D H The answer is 2-15.

[0054] In some embodiments, the operated D described herein H The region is multiple human D H Includes segments, multiple human D H At least one of the gene segments is operably bound to the 5' or 3' RSS, and in some embodiments, to the 5' 23mer RSS. In some embodiments, the manipulated D described herein H The region is multiple human D H Includes segments, multiple human D HAt least three gene segments are operably bound to the 5' or 3' RSS, and in some embodiments, to the 3' 23-mer RSS.

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

[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 bound to the 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 variable region of the human immunoglobulin heavy chain is V H V from 3-74 H Human V up to 6-1 H Includes a gene segment. In some embodiments, the variable region of the human immunoglobulin heavy chain is at least human J H Gene segment J H Includes 6. In some embodiments, the variable region of the human immunoglobulin heavy chain is at least human J H Gene segment J H 4, J H 5 and J H Includes 6. In some embodiments, the variable region of the human immunoglobulin heavy chain is 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 non-human animals, non-human cells, or non-human tissues, the genome lacks the endogenous Adam6 gene. In some embodiments of non-human animals, non-human cells, or non-human tissues, the genome further includes insertions of one or more nucleotide sequences encoding one or more rodent Adam6 polypeptides, and in some embodiments, one or more nucleotide sequences are first and second human V H In some embodiments, one or more nucleotide sequences are inserted between gene segments in place of the human Adam6 pseudogene, and in some embodiments, one or more nucleotide sequences are human V H Genetic segments and human D H It is inserted between gene segments. 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 provided non-human animal, non-human cell, or non-human tissue is manipulated according to the D described herein. H The region is homozygous, heterozygous, or hemizygous. In some embodiments, the non-human animal, non-human cell, or non-human tissue provided is the manipulated D described herein. H It is transgenic to the domain.

[0060] In some embodiments, the genome is manipulated D H A method is provided for producing non-human animals containing an immunoglobulin heavy chain variable region, the method comprising: (a) inserting a DNA fragment into a non-human embryonic stem cell, the DNA fragment being one or more D fragments operably bound to a 23-mer RSS H (b) a step of obtaining non-human embryonic stem cells generated in (a); (c) a step of creating a non-human animal using the non-human embryonic stem cells from (b).

[0061] In some embodiments, one or more D H Each DNA fragment containing a segment is operably bound to the 3' 23mer RSS. In some embodiments, the DNA fragment is operably bound to a single D H Includes segments. In some embodiments, the DNA fragment is operably bound to a 3' 23-mer RSS of one synthetic human D H Includes segments. In some embodiments, the DNA fragment is operably bound to a 3' 23-mer RSS of one synthetic human D H It includes 3-3 segments. In some embodiments, the DNA fragment consists of three D segments, each operably linked to a 3' 23-mer RSS. H Includes segments. In some embodiments, the DNA fragment consists of three human D2 fragments, each operably linked to a 3' 23-mer RSS. H Includes segments. In some embodiments, the DNA fragment consists of three human D2 fragments, each operably linked to a 3' 23-mer RSS. H Includes segments, Human D H The segment is Human D H 2-2, Human D H 2-8 and Human D H The answer is 2-15.

[0062] In some embodiments, the DNA fragment is one or more D H The DNA fragment includes segments, each of which is operably bound to a 5' 23-mer RSS. In some embodiments, the DNA fragment is operably bound to a single D H Includes segments. In some embodiments, the DNA fragment is a single synthetic human D2 operably bound to the 23 mer of the 5'. H Includes segments. In some embodiments, the DNA fragment is operably bound to a 5' 23-mer RSS of one synthetic human D HIt includes 3-3 segments. In some embodiments, the DNA fragment is operably bound to a 5' 23-mer RSS of one synthetic human D H Includes 3-3 segments, synthetic human D H Segment 3-3 is Human D H Human D instead of 7-27 segments H It is located in the region.

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

[0064] In some embodiments, the genome is manipulated D H A method is provided for producing non-human animals containing an immunoglobulin heavy chain variable region, the method comprising: the genome of a non-human animal or a cell of a non-human animal, each operably linked to one or more D2s in a 23-mer RSS. H Operated D including segments H The process includes modifying the genome of a non-human animal or a cell of a non-human animal to include an immunoglobulin heavy chain variable region, thereby producing the non-human animal.

[0065] In some embodiments for creating non-human animals, the genome of a non-human animal or non-human animal cell is operably linked to one or more D2s at a 5' 23-mer RSS. H The segments are modified to include the segments. In some embodiments for creating non-human animals, the genome of a non-human animal or non-human animal cell is operably linked to one or more D segments in the RSS of 23' at 3' each. H It is modified to include a segment.

[0066] In some embodiments, a method for producing antibodies in non-human animals is provided. In some embodiments, a method for producing antibodies or obtaining nucleic acids encoding them includes the steps of immunizing a non-human animal (e.g., a rodent (e.g., a rat or mouse)) with an antigen, wherein the rodent is operatively bound to one or more D23-mer RSSs. H Operated D including segments H The method includes the step of causing a rodent having a germline genome containing a region to produce an immune response to an antigen, including an antibody, or a nucleic acid encoding the antigen that binds to it. In some embodiments, the method further includes the step of recovering the antibody, or the nucleic acid encoding it, from the rodent or rodent cells, such as B cells, or hybridomas. In some embodiments, one or more D H Each segment is operably coupled to a 5' 23mer RSS. In some embodiments, one or more D H Each segment is operably coupled to a 3' 23mer RSS.

[0067] In some embodiments, a method is provided for producing antibodies in non-human animals, the method comprising the steps of (a) immunizing the non-human animals with an antigen, each non-human animal having one or more D operatively bound to a 23-mer RSS H Operated D including segments H (b) a non-human animal having a genome containing an immunoglobulin heavy chain constant region; (b) a non-human animal having a genome for an antigen; (c) a non-human animal being a rodent under conditions sufficient to produce a response to the antigen; and (d) a non-human animal or non-human animal cell being recovered an antibody that binds to the antigen. 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 is one or more human V H A human immunoglobulin heavy chain variable region containing a gene segment, at least one human D operatively fused to a 23-mer RSS. HOperated D including segments H region, and at least one human J H A non-human animal containing a gene segment is provided, and when the rodent is immunized with the antigen, it becomes human D H -D H Recombination and / or J H The human immunoglobulin heavy chain variable domain is operably bound to one or more endogenous immunoglobulin constant region genes, and the antibody exhibits specific binding to the antigen, characterized by generating an antibody containing a human heavy chain variable domain including a CDR3 region generated by enhanced recombination to 6 gene segments. In some embodiments, at least one human D is operably bound to a 23-mer RSS. H The segment is Human D H Located in place of segments 7-27. In some embodiments, the human immunoglobulin heavy chain variable region is less than six human J H Includes gene segments. In some embodiments, the human immunoglobulin heavy chain variable region is six human J H It includes only one of the gene segments. In some embodiments, the human immunoglobulin heavy chain variable region is human J H It contains 6 gene segments and functional J H Lacking one gene segment, functional J H Lacking two gene segments, functional J H Lacking 3 gene segments, functional J H Lacking 4 gene segments, functional J H It lacks 5 gene segments. In some embodiments, the human immunoglobulin heavy chain variable region is six human J H It includes only three of the gene segments. In some embodiments, the human immunoglobulin heavy chain variable region is human J H 4 gene segments, human J H 5 gene segments and human J H It contains only 6 gene segments and functional J H Lacking one gene segment, functional J H Lacking two gene segments, functional J HIt lacks 3 gene segments. In some embodiments, the genome has fewer than six human J genomes. H A human immunoglobulin heavy chain variable region containing a gene segment, for example, a human immunoglobulin heavy chain variable region containing only one of six human gene segments (for example, human J H It contains 6 gene segments and functional J H Lacking one gene segment, functional J H Lacking two gene segments, functional J H Lacking 3 gene segments, functional J H Lacking 4 gene segments, functional J H Non-human animals, including the human immunoglobulin heavy chain variable region (which lacks 5 gene segments), have genomes that contain all six human J H Compared to control non-human animals containing the variable region of human immunoglobulin heavy chains, including gene segments, J H It shows enhanced recombination for 6 gene segments, for example, non-human animals, J H The genome comprises six gene segment sequences or a portion thereof, and / or a higher percentage of rearranged immunoglobulin heavy chain sequences encoding CDR3s that are at least 20 amino acids longer than those of control non-human animals. In some embodiments, the genome comprises fewer than six human J genomes. H Variable regions of human immunoglobulin heavy chains, including gene segments, e.g., six human J H A human immunoglobulin heavy chain variable region containing only three of the gene segments (e.g., human J H 4 gene segments, human J H 5 gene segments, and human J H It contains 6 gene segments and functional J H Lacking one gene segment, functional J H Lacking two gene segments, functional J H Non-human animals, including the human immunoglobulin heavy chain variable region lacking three gene segments, have genomes that contain all six human J H Compared to control non-human animals containing the variable region of human immunoglobulin heavy chains, including gene segments, J HExhibits enhanced recombination for the 6 gene segments, for example, the non-human animal is J H Contains a 6 gene segment sequence or a part thereof, and / or contains a higher percentage of rearranged immunoglobulin heavy chain sequences encoding a CDR3 at least 20 amino acids longer than that of a control non-human animal.

[0069] In some embodiments, the genome is one or more human V H A human immunoglobulin heavy chain variable region containing gene segments, at least one human D adjacent to a 23mer RSS at the 3' H An engineered D containing a segment H Region, and at least two human J H Gene segments, a non-human animal is provided, and when the rodent is immunized with an antigen, a human D H -D H The human immunoglobulin heavy chain variable region is operably linked to one or more endogenous immunoglobulin constant region genes such that antibodies are produced that contain a human heavy chain variable domain containing a CDR3 region generated by D-D recombination, and the antibodies exhibit specific binding to the antigen. In some embodiments, the engineered D H Regions each contain at least three human D H Segments adjacent to a 23mer RSS at the 3'. In some embodiments, the engineered D H Regions each contain three human D H Segments, and the three human D H Segments are 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 contains human V H From V H 6-1 up to human V H Gene segments.

[0070] In some embodiments, the antigen is a pathogen, such as a bacterial, fungal, or viral pathogen. In some embodiments, immunization of the non-human animals herein includes infecting the non-human animals with a pathogen, such as a bacterial, fungal, or viral pathogen. In some embodiments, immunization of the non-human animals herein includes administering to the non-human animals a genomic or proteinaceous substance isolated from a pathogen, such as 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 provided non-human animals, non-human cells or non-human tissues have a genome further comprising the insertion of one or more human V L gene segments and one or more human J L gene segments into the endogenous light chain locus. In some embodiments, the human V L and J L segments are V κ and J κ gene segments and are inserted into the endogenous κ light chain locus. In some embodiments, the human V κ and J κ gene segments are operably linked to a rodent Cκ gene (e.g., a mouse or rat Cκ gene). In some embodiments, the human V L and J L segments are V λ and J λ gene segments and are inserted into the endogenous λ light chain locus. 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).

[0072] In some embodiments, the use of non-human animals, non-human cells, or non-human tissues described herein in the manufacture and / or development of drugs or vaccines for pharmaceutical use, such as pharmaceutical use. In some embodiments, the use of non-human animals, non-human cells, or non-human tissues described herein in the manufacture and / or development of antibodies for administration to humans. In some embodiments, the use of non-human animals, non-human cells, or non-human tissues described herein in the manufacture of pharmaceuticals for the treatment, prevention, or improvement of diseases, disorders, or conditions.

[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 drugs for therapeutic or diagnostic purposes. In some embodiments, the non-human animals, non-human cells, or non-human tissues described herein are provided for use in the manufacture of pharmaceuticals for the treatment, prevention, or improvement of diseases, disorders, or conditions.

[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 includes a nucleotide sequence that comprises the sequence represented as sequence number 151. [Brief explanation of the drawing]

[0076] The drawings included in this specification, consisting of the following figures, are for illustrative purposes only and not intended to limit them.

[0077] [Figure 1] Figure 1 provides a non-scale general description of embodiments of the present invention, showing the regular assembly of gene segments in the DJ recombination phenomenon for immunoglobulin heavy chain variable region gene segments having unmodified DH regions (upper panel) and manipulated DH regions (lower panel). 12-mer recombination signal sequences (RSSs) are shown as unfilled triangles. 23-mer RSSs are shown as triangles with vertical stripes. Exemplary unmodified VH gene segments (unfilled squares) and DH gene segments (filled squares) are shown as unfilled squares and filled squares, respectively, and provided with common nomenclature using alphabetical letters. D gene segments (e.g., DH3-3) and unmodified JH gene segments operably bound to 23-mer RSSs are shown as squares with horizontal stripes and unfilled squares, respectively, and provided with their appropriate nomenclature. The μ0 promoter (μ0pro) is also shown. Recombination of VH gene segments into recombinant DJ gene segments is not shown.

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

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

[0080] [Figure 4] Figure 4 shows a non-scale description of non-limiting, exemplary embodiments of Cre-mediated deletion of a selective cassette at a humanized immunoglobulin heavy chain locus after electroporation and integration of the 23:DH3-3:12 / JH6 targeting vector, as described in Examples 1 and 2. Hash lines represent VH or DH gene segments contained within the heavy chain variable region locus, but not specifically shown.

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

[0082] [Figure 6] Figure 6 shows a non-scale description of non-limiting exemplary embodiments of Cre-mediated deletion of a selected cassette at a humanized immunoglobulin heavy chain locus after electroporation and integration of the 23:DH3-3:12 / JH4-6 targeting vector, as described in Examples 1 and 2. Hash lines represent VH or DH gene segments contained within the heavy chain variable region locus, but not specifically shown.

[0083] [Figure 7] Figure 7 shows a non-scale description 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 a humanized immunoglobulin heavy chain variable region locus within the genome of mouse ES cells via electroporation (EP). Hash lines represent VH or DH gene segments contained within the heavy chain variable region locus, but not specifically shown. Filled triangles represent pseudogenes.

[0084] [Figure 8] Figure 8 provides a non-scale description of exemplary, non-limiting embodiments of Cre-mediated deletion of a selection cassette at a humanized immunoglobulin heavy chain locus after 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. Hash lines represent VH or DH gene segments contained within the heavy chain variable region locus, but not specifically shown.

[0085] [Figure 9A] Figure 9A shows results related to embodiments of the present invention, graphing the percentage (y-axis) of all functional immunoglobulin (Ig) reads resulting from DH-DH recombination phenomena (bottom panel) with CDR3 of specific amino acid lengths (x-axis) isolated from animals modified with 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vectors. "S1", "S2", and "S3" represent different experimental mice, respectively.

[0086] [Figure 9B] Figure 9B shows results related to embodiments of the present invention, graphing the percentage of all Ig reads (y-axis) resulting from DH-DH recombination phenomena (bottom panel) with CDR3 of specific amino acid lengths (x-axis) isolated from animals modified with 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vectors. "S1", "S2", and "S3" represent different experimental mice, respectively.

[0087] [Figure 10A]Figure 10A shows results related to embodiments of the present invention, graphing the percentage (y-axis) of all functional immunoglobulin (Ig) readouts resulting from DH-DH recombination phenomena with CDR3 containing a specific number of cysteine ​​residues (x-axis) isolated from animals modified with 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vectors. "S1", "S2", and "S3" represent different experimental mice, respectively.

[0088] [Figure 10B] Figure 10B shows results related to embodiments of the present invention, graphing the percentage of all immunoglobulin (Ig) reads (y-axis) resulting from DH-DH recombination phenomena with CDR3 containing a specific number of cysteine ​​residues (x-axis) isolated from animals modified with 12:DH2-2:23|12:DH2-8:23|12:DH2-15:23 targeting vectors. "S1", "S2", and "S3" represent different experimental mice, respectively.

[0089] [Figure 11] Figure 11 shows results related to embodiments of the present invention, graphing typical antibody titers (y axis) in individual mice having humanized immunoglobulin heavy and κ light chain variable region loci (VI; see, for example, U.S. Patents 8,697,940 and 8,642,835; each of which is incorporated herein by reference) immunized with DNA immunogen encoding G protein-coupled receptors (GPCRs), as well as 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 (GPCR; x axis) and cells not expressing GPCRs (control; x axis).

[0090] [Figure 12A]Figure 12A shows results related to embodiments of the present invention, showing the percentage (%; y axis) of reconfigured immunoglobulin heavy chain VHDHJH gene segments, which are presumed to be the result of VHDHA-DHBJH rearrangement according to the strict criteria described in Table 7, isolated from mouse bone marrow (BM) or spleen modified with a 23:DH3-3:12 / JH6 targeting vector and heavy chain CDR3 (HCDR3) having amino acids (AA) of 5-30 amino acid length (x axis). BM cell counts and spleen cell counts are not standardized with respect to each other. n=1.

[0091] [Figure 12B] Figure 12B shows results related to embodiments of the present invention, providing an enlargement of the graph shown in panel Figure 12A. BM cell counts and spleen cell counts are not standardized with respect to each other. n=1.

[0092] [Figure 12C] Figure 12C shows results related to embodiments of the invention, representing the percentage of readings with CDR3s having an amino acid length longer than 21 in both the bone marrow or spleen of mice modified with the 23:DH3-3:12 / JH6 targeting vector. BM cell counts and spleen cell counts are not standardized with respect to each other. n=1.

[0093] [Figure 13] Figure 13 shows results related to embodiments of the present invention, representing the estimated percentage (%; y axis) of reconstructed immunoglobulin heavy chain VHDHA-DHBJH gene sequences isolated from mouse bone marrow (BM) or spleen (according to the strict criteria described in Table 7) modified with the 23:DH3-3:12 / JH6 targeting vector and heavy chain CDR3 (HCDR3) all having an amino acid (AA) length exceeding 20 amino acids (x axis). BM cell counts and spleen cell counts are not standardized with respect to each other. n=1. [Modes for carrying out the invention]

[0094] definition The scope of the present invention is defined by the claims appended to this specification and is not limited by the specific embodiments described herein. Those skilled in the art will recognize, by reading this disclosure, a variety of modifications that may be equivalent to the embodiments described herein, or that may be within the scope of the claims by other means. In general, technical terms are used in the sense understood in the art unless explicitly suggested otherwise. Explicit definitions of certain terms are provided herein and below, but the meanings of these and other terms in specific examples throughout this specification will be evident to those skilled in the art from the context. Further definitions of the following and other terms are provided throughout this specification. References cited herein or in relevant parts thereof are incorporated herein by reference in their entirety.

[0095] The use of sequential terms such as “first,” “second,” and “third” in a claim to modify a claim does not in itself imply superiority, precedence, or sequence of one claim element over another, or a temporal order in which the actions of the method are performed, but is used solely as a distinguishing mark to differentiate one claim element having a particular name from another element having the same name (other than the use of sequential terms).

[0096] In this specification and in these claims, the articles “a” and “an” should be understood to include multiple referents unless it is explicitly stated otherwise. Claims or statements containing “or” between one or more elements of a group are satisfied if one, two or more, or all elements of the group are present, used, or otherwise related to a given product or process, unless it is indicated otherwise or otherwise obvious from the context. The present invention includes embodiments in which exactly one element of a group is present, used, or otherwise related to a given product or process. The present invention also includes embodiments in which two or more, or all elements of a group, are present, used, or otherwise related to a given product or process. Furthermore, it should be understood that the present invention covers all variations, combinations, and reorders in which one or more limitations, elements, phrases, descriptive terms, etc., from one or more of the described claims are introduced into another claim (or any other related claim) that depends on the same basic claim, unless otherwise specified or unless it is obvious to a person skilled in the art that a contradiction or inconsistency would occur. Where elements as described exist (e.g., in the form of a Markush group or similar), each subgroup of the elements is also disclosed, and any element can be removed from the group. Naturally, when the present invention or an aspect of the present invention is referred to as including certain elements, features, etc., the embodiments or aspects of the present invention consist of, or substantially consist of, such elements, features, etc. For brevity, these embodiments are not specifically described herein in many terms in all cases. Naturally, any embodiment or aspect of the present invention may be expressly excluded from the claims, whether or not certain exclusions are enumerated in the specification.

[0097] As used herein, the terms “about” and “approximately” are used synonymously. Any figures used herein, with or without “about” or “approximately,” are intended to cover any normal variation as understood by those skilled in the art, e.g., + / - 5%.

[0098] Administration: This refers to the administration of a composition to a target or system (e.g., cells, organs, tissues, organisms, or related components or groups thereof). Those skilled in the art will recognize that the route of administration may vary depending on, for example, the target or system to which the composition is administered, the properties of the composition, and the purpose of administration.

[0099] For example, in some embodiments, administration to an animal subject (e.g., human or rodent) may be by bronchial administration (including bronchial infusion), oral administration, intraenteral administration, interdermal administration, intra-arterial administration, intradermal administration, gastric administration, intrathecal administration, intramuscular administration, intranasal administration, intraperitoneal administration, intrathecal administration, intravenous administration, intraventricular administration, mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous administration, sublingual administration, local administration, tracheal administration (including intratracheal infusion), transdermal administration, vaginal administration, and / or intravitreal administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) over at least a selected period. In some embodiments, antibodies produced by non-human animals disclosed herein may be administered to a subject (e.g., human subject or rodent). In some embodiments, the pharmaceutical composition includes antibodies produced by non-human animals disclosed herein. In some embodiments, the pharmaceutical composition may include buffers, diluents, excipients, or any combination thereof. In some embodiments, the pharmaceutical composition comprising antibodies produced by non-human animals as disclosed herein may be contained in a container for storage or administration, such as a vial, syringe (e.g., an IV syringe), or bag (e.g., an IV bag).

[0100] Biological activity refers to the characteristic of any drug that is active in a biological system, in vitro, or in vivo (e.g., in a living organism). For example, if a drug is present in a living organism, a drug that has a biological effect within that organism is considered biologically active.

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

[0102] Equivalent means two or more drugs, entities, situations, conditions, etc., that are not identical to each other but are similar enough to allow for a comparison that enables reasonable conclusions to be drawn based on observed differences or similarities. A person skilled in the art will understand, in context, the degree of identity required for two or more such drugs, entities, situations, conditions, etc., to be considered equivalent in a given situation.

[0103] Conservative: Conservative amino acid substitution refers to the substitution of an amino acid residue with another amino acid residue that has a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein in question, such as its ability to bind to a ligand or act as a receptor. Examples of amino acid groups with side chains having 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 acid substituents include, for example, valine / leucine / isoleucine, phenylalanine / tyrosine, lysine / arginine, alanine / valine, glutamic acid / aspartic acid, and asparagine / glutamine.

[0104] In some embodiments, a conserved amino acid substitution may be a substitution of any undenatured residue in an alanine-containing protein, for example, used in alanine scanning mutagenesis. In some embodiments, a conserved substitution with a positive value in the PAM250 log-likelihood matrix is ​​performed, as disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445 (this document is incorporated herein by reference in its entirety). In some embodiments, the substitution is a moderately conserved substitution, in which case the substitution has a non-negative value in the PAM250 log-likelihood matrix.

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

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

[0107] Disruption: This refers to the result of homologous recombination events with DNA molecules (e.g., endogenous homologous sequences such as genes or gene loci).

[0108] In some embodiments, disruption may achieve or demonstrate an insertion, deletion, substitution, exchange, missense mutation, or frameshift of a DNA sequence, or any combination thereof. Insertions may include insertions of an entire gene or a fragment of a gene (e.g., an exon), which may be of a non-endogenous origin (e.g., a heterologous sequence) or a coding sequence derived from or isolated from the specific gene of interest. In some embodiments, disruption may increase the expression and / or activity of a gene or gene product (e.g., a protein encoded by the gene). In some embodiments, disruption may decrease the expression and / or activity of a gene or gene product. In some embodiments, disruption may alter the sequence of a gene or a gene product (e.g., a encoded protein). In some embodiments, disruption may alter the sequence of a chromosome or chromosomal location within the genome. In some embodiments, disruption may cleave or fragment a gene or a gene product (e.g., a encoded protein). In some embodiments, disruption may elongate a gene or a gene product. In some such embodiments, disruption may result in the assembly of a fusion protein. In some embodiments, damage may affect the level of a gene or gene product but not its activity. In some embodiments, damage may affect the activity of a gene or gene product but not its level. In some embodiments, damage may have no significant effect on the level of a gene or gene product. In some embodiments, damage may have no significant effect on the activity of a gene or gene product. In some embodiments, damage may have no significant effect on either the level or activity of a gene or gene product. In some embodiments, a significant effect can be measured, for example, by a Student's T test, though not limited to these methods.

[0109] Endogenous locus or endogenous gene: Meaning a locus that exists in the parent or reference organism prior to introducing the modification, disruption, deletion, insertion, alteration, substitution, or exchange described herein.

[0110] In some embodiments, the endogenous locus contains, 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 modified organism. In some embodiments, the reference organism is a laboratory-bred organism (wild-type or genetically modified).

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

[0112] "Manipulated" generally means a manner in which something has been manipulated by human hands. As is common and as is understood by those skilled in the art, manipulated polynucleotides or cellular offspring are also usually considered "manipulated," even if the actual manipulation was performed on a past entity. Furthermore, as is recognized by those skilled in the art, various techniques are available through which the "manipulations" described herein can be carried out.

[0113] In some embodiments, two or more sequences that are not linked together in their natural order may be considered "manipulated" if they are manipulated by humans and directly linked to each other in the manipulated polynucleotide. In some embodiments, the manipulated polynucleotide may include a control sequence that is operatively bound to a first coding sequence in nature but not to a second coding sequence, and which is operatively bound to the second coding sequence by humans. H In the embodiment of the gene segment, D H The gene segment is manipulated by human hands to operably bind to the 23mer RSS (for example, at least one of them is adjacent to, next to, or right next to the 23mer RSS). In some embodiments, D is manipulated to operably bind to the 23mer RSS. H The gene segment is another DH Derived from a gene segment, for example, D operably bound to the 23-mer RSS H The gene segment is other D H It contains the same nucleotide sequence as the gene segment, but the difference is due to the degeneracy of the gene code and / or the substitution of a 12-mer RSS with a 23-mer RSS. H Gene segment and the manipulated D derived therefrom H Gene segments (e.g., D operably bound to the 23-mer RSS) H A gene segment can be considered a corresponding gene segment. For example, human D2 operably bound to a 23-mer RSS. H The 3-3 gene segment is a D-type gene where a 12-mer RSS is adjacent to another 12-mer RSS at one end. H It can be considered to correspond to a 3-3 gene segment, and D is operably bound to the 23-mer RSS. H 3-3 gene segments and a 12-mer RSS at one end and another 12-mer RSS at the other end adjacent to each other D H 3-3 gene segments share the same nucleotide sequence, except for differences due to degeneracy of the gene code and / or substitution of two 12-mer RSSs in a 23-mer RSS. Alternatively, or further, 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 manipulated polynucleotide. Similarly, in some embodiments, a cell or organism may be considered "manipulated" if it is manipulated so that its genetic information is altered (e.g., new genetic material that was not previously present is introduced, or genetic material that was previously present is altered or removed).

[0114] For example, in some embodiments, “Operation” may include selecting or designing (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues and / or organisms) via the use of a computer system programmed to perform analysis or comparison, or to analyze recommended and / or selected sequences by other means. Alternatively, or further, in some embodiments, “Operation” 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 polymerase chain reactions), mutation, transformation, transfection, etc., and / or the use of various arbitrary controlled mating methods. As is recognized by those skilled in the art, various established techniques of this kind (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. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; the entire work is incorporated herein by reference).

[0115] Gene: Meaning a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). For clarity, the term “gene” generally means a portion of a nucleic acid that codes for a polypeptide; the term may optionally include a regulatory sequence, which should be evident from the context to those skilled in the art. This definition is not intended to exclude the application of the term “gene” to non-protein-coding expression units, but rather to clarify that as used herein, the term often refers to a nucleic acid that codes for a polypeptide.

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

[0117] Heterogeneous: This means an agent or entity from a different source. For example, when used in relation to a polypeptide, gene, or gene product present in a particular cell or organism, the term makes it clear that the related polypeptide or its fragment, gene or its fragment, or gene product or its fragment is (1) genetically engineered by humans, (2) introduced into a cell or organism (or its precursor) by humans (e.g., by genetic engineering), and / or (3) not naturally produced by or present in the related cell or organism (e.g., related cell type or organism type). Another example includes polypeptides or their fragments, genes or their fragments, or gene products or their fragments that are not naturally associated and, in some embodiments, are non-endogenous, normally present in a particular native cell or organism, but are modified, for example, by mutation or substitution.

[0118] Host cell: This refers to a cell into which a different (e.g., exogenous) nucleic acid or protein has been introduced. A person skilled in the art will understand that such a term is used not only to refer to a specific subject cell, but also to refer to the offspring of such a cell. Since certain modifications can occur in subsequent generations due to mutation or environmental influences, such offspring may not actually 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 a prokaryotic cell or a eukaryotic cell, or includes one. In some embodiments, the host cell is a mammalian cell, or includes one. Generally, the host cell is any cell suitable for receiving and / or producing heterologous nucleic acids or proteins, regardless of the species of cell specified. Examples of cells include prokaryotic and eukaryotic (unicellular or multicellular) cells, bacterial cells (e.g., Escherichia coli), and Examples include strains of *C. coli*, *Bacillus* spp., *Streptomyces* spp., etc., mycobacterial cells, fungal cells, yeast cells (e.g., budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), *Pichia pastoris*, *Pichia methanolica*, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, nettle moth (Trichoplusia ni), etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas.

[0120] In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic cells and are 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 aforementioned cells. In some embodiments, the cells include one or more viral genes, for example, retinal cells expressing viral genes (e.g., PER.C6® cells). In some embodiments, the host cells are isolated cells or include isolated cells. In some embodiments, the host cells are part of a tissue. In some embodiments, the host cells are part of an organism.

[0121] "Humanization" refers to a molecule (e.g., nucleic acids, proteins, etc.) whose origin is non-human, and whose corresponding portion has been replaced with a corresponding portion of a corresponding human molecule so that the modified (e.g., humanized) molecule retains its biological function and / or maintains the structure that performs such retained biological function. In contrast, "human," etc., encompasses molecules that have human nucleotides or proteins that are of human origin only, e.g., molecules that contain only human nucleotides and amino acid sequences, respectively. The term "human (humanized)" is used to reflect that the human (humanized) molecule may (a) be a human molecule or (b) be a humanized molecule.

[0122] Identity: This refers to the identity determined by various algorithms known in the art that can be used to measure nucleotide and / or amino acid sequence identity in relation to sequence comparison.

[0123] In some embodiments, the identity described herein is determined using ClustalW v.1.83(slow) alignment with a Gonnet similarity matrix (MACVECTOR® 10.0.2, MacVector Inc., 2008) with a gap start penalty of 10.0 and 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, reaction vessel, or cell culture.

[0125] In vivo: This refers to events occurring within multicellular organisms, such as humans and / or non-human animals. In the context of cell-based systems, the term may also be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0126] Isolated means (1) a substance and / or entity separated from at least some of the components to which it was originally associated when it was first produced (either in a natural environment or / or a laboratory environment), and / or (2) a substance and / or entity designed, produced, prepared, and / or manufactured by human hands. Isolated substances and / or entities may be separated from about 10 or more other components to which they were originally associated. In some embodiments, isolated factors are at least about 80% pure. A substance is “pure” if it is substantially free of other components. In some embodiments, as a person skilled in the art will understand, a substance may still be considered “isolated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percentage of isolation or purity of the substance is calculated without including such carriers or excipients.

[0127] For example, in some embodiments, a naturally occurring biomolecule such as a polypeptide or polynucleotide is considered “isolated” if (a) its origin or source of derivation is not related to some or all of the components that accompany it in its natural state, (b) it substantially does not contain other polypeptides or nucleic acids of the same species that produces it in nature, or (c) it is expressed by a cell or other expression system that is not the species that produces it in nature, or otherwise related to components from there. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the one that produces it in nature is considered an “isolated” polypeptide. Or, additionally, in some embodiments, a polypeptide that has undergone one or more purification techniques may be considered an “isolated” polypeptide insofar as it is a) separated from the other components to which it accompanies it in nature and / or b) separated from the other components to which it accompanies it when it was first produced.

[0128] Non-human animals: This refers to any vertebrate organism that is not human.

[0129] In some embodiments, non-human animals are cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, non-human mammals are primates, goats, sheep, pigs, dogs, cattle, or rodents. In some embodiments, non-human animals are rodents such as rats or mice.

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

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

[0132] Operablely coupled: This means that the components described are proximal to each other in such a relationship that they can function in the manner they are intended.

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

[0134] In other embodiments, operable linkages do not require continuity. For example, unreconstructed variable region gene segments “operably linked” to each other have the ability to be reconstructed to form a re-ruptured variable region gene, where the unreconstructed variable region gene segments are not necessarily contiguous with each other. Unreconstructed variable region gene segments operably linked to each other and to contiguous constant region genes have the ability to be reconstructed to form a reconstructed variable region gene that is expressed by linking to the constant region gene as a polypeptide chain of an antigen-binding protein. A “operably linked” control sequence is linked such that expression of the coding sequence is achieved under conditions that match the control sequence. “Operatably linked” sequences include both expression regulatory sequences adjacent to the gene of interest and expression regulatory sequences that act in trans or at a distance to regulate the gene of interest.

[0135] The term "regulatory sequences" refers to polynucleotide sequences necessary for the expression and processing of the coding sequences to which they are ligated. Regulatory sequences include appropriate transcription start, 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 regulatory sequences varies depending on the host organism. For example, in prokaryotes, these regulatory sequences generally include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, these regulatory sequences typically include promoters and transcription termination sequences. The term "regulatory sequence" is intended to include elements whose presence is essential for expression and processing, but may 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 bound to a recombination signal sequence (RSS), which may be a 12-mer RSS or a 23-mer RSS (e.g., related, adjacent at one or both ends, adjacent, etc.). H Any gene segment (including a gene segment) adjacent at its lateral end may be considered unrecombined and therefore "unrecombined" gene segment. In some embodiments, the unrecombined gene segments herein are gene segments of their germline (e.g., wild-type) configuration, each adjacent at both ends by 23mer RSSs, e.g., germline V H Genetic segments and germline J H It may include gene segments. In contrast, germline D H Gene segments, for example, unreconstructed D HThe gene segment has 12-mer RSSs adjacent at each end. D is operably bound to the 23-mer RSS. H Gene segments, for example, manipulated D H The gene segment is also unrecombined and therefore contains (i) a 23-mer RSS and (ii) a 12-mer RSS. H Gene segments (e.g., D operably bound to the 23-mer RSS) H The gene segment is operably bound to another D12mer RSS. H Gene segments can be rearranged according to the 12 / 23 rules of recombination.

[0137] Physiological conditions: This includes the meaning known in the art, referring to the conditions under which a cell or organism survives and / or reproduces. In some embodiments, this term means external or internal environmental conditions that may occur naturally for an organism or cell system. In some embodiments, physiological conditions are conditions within the body of a human or non-human animal, particularly those at and / or within a surgical site. Physiological conditions typically include, for example, a temperature in the range of 20–40°C, 1 atmosphere, pH 6–8, glucose concentration of 1–20 mM, atmospheric oxygen concentration, and gravity occurring on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained to 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 polymer chain of amino acids.

[0139] In some embodiments, the polypeptide has an amino acid sequence that exists in nature. In some embodiments, the polypeptide has an amino acid sequence that does not exist in nature. In some embodiments, the polypeptide has an amino acid sequence that includes parts that exist separately in nature (i.e., from two or more different organisms, such as human and non-human parts). In some embodiments, the polypeptide has a genetically engineered amino acid sequence in the sense that it is designed and / or produced through human intervention. In some embodiments, the polypeptide may contain or consist of multiple fragments, each of which is found in the same parent polypeptide in a spatial arrangement different from that found in the polypeptide of interest (for example, fragments that are directly linked in the parent polypeptide may be spatially separated in the polypeptide of interest, or vice versa, and / or fragments may be in a different order in the polypeptide of interest than in the parent polypeptide), and the polypeptide of interest is thus a derivative of its parent polypeptide.

[0140] Recombination: Means polypeptides designed, manipulated, prepared, expressed, produced, or isolated by recombinant means, for example, polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant combinatorial human polypeptide libraries (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 transgenic to human immunoglobulin genes (e.g., mice) (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.) See al., 2014, Proc. Natl. Acad. Sci. USA 111(14):5153-5158, each of which is incorporated herein by reference, or means polypeptides prepared, expressed, produced or isolated by any other means involving cross-splicing of selected sequence elements.

[0141] In some embodiments, one or more such selected sequence elements exist in nature. In some embodiments, one or more such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements arise from, for example, the 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 contains a sequence found in the genome (or polypeptide) of the source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide contains a sequence that occurs separately in nature in two different organisms (e.g., human and non-human organisms) (i.e., derived from two or more different organisms, such as human and non-human parts). In some embodiments, the recombinant polypeptide has an amino acid sequence resulting from mutagenesis (e.g., in vitro or in vivo in non-human animals), and therefore the amino acid sequence of the recombinant polypeptide is a sequence that originates from and is related to the polypeptide sequence, but may not naturally exist in vivo in the genome of a non-human animal.

[0142] Reference: Refers to a standard or control agent, animal, cohort, individual, population, sample, sequence, or value compared to the agent, animal, cohort, individual, population, sample, sequence, or value of interest. "Reference" may refer to a "reference animal." A "reference animal" may have the modifications described herein, different modifications, or be unmodified (i.e., wild-type). Generally, as will be understood by those skilled in the art, the reference agent, animal, cohort, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent 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, a reference drug, animal, cohort, individual, population, sample, sequence, or value is tested and / or calculated substantially concurrently with the test or calculation of the drug, animal, cohort, individual, population, sample, sequence, or value under consideration. In some embodiments, the reference drug, animal, cohort, individual, population, sample, sequence, or value may be a known reference embodied in a tangible medium. In some embodiments, the reference may refer to a control.

[0144] Substantial: This refers to a qualitative condition indicating the complete or near-complete range or degree of the characteristic or property in question. Those skilled in the field of biology will understand that biological and chemical phenomena rarely, if any, proceed to completion and / or to a complete state, or achieve or avoid absolute results. Therefore, the term "substantial" is used to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0145] Substantial homology: This refers to the similarity between amino acid sequences or nucleic acid sequences. As those skilled in the art will understand, two sequences are generally considered "substantial homology" if they contain homologous residues in their corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with moderately similar structural and / or functional characteristics. For example, as those skilled in the art will know, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or having "polar" or "nonpolar" side chains. Substitution of one amino acid with another of the same type is often considered a "homologous" substitution. A general classification of amino acids is 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 the various algorithms available, 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, SF et al., 1990, J.Mol.Biol., 215(3):403-410; Altschul et al., 1996, Methods Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-402; Baxevanis, AD and BFFOuellette (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 programs generally provide an indicator of the degree of homology.

[0147] In some embodiments, two sequences are considered substantially homologous if at least 95% of their corresponding residues are homologous across a relevant interval of residues. In some embodiments, the relevant interval is a complete sequence. In some embodiments, the relevant interval consists of at least nine residues. In some embodiments, the relevant interval includes adjacent residues along the complete sequence. In some embodiments, the relevant interval includes discontinuous residues along the complete sequence, such as non-adjacent residues attracted by a folded structure of a polypeptide or a part thereof. In some embodiments, the relevant interval consists of at least ten residues.

[0148] "Substantially identical" means similarity between amino acid or nucleic acid sequences. As those skilled in the art will understand, two sequences are generally considered "substantially identical" if they contain identical residues at their corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of the various algorithms available, 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, SF et al., 1990, J.Mol.Biol., 215(3):403-410; Altschul et al., 1996, Methods Enzymol. 266:460-80; Altschul, SF et al., 1997, Nucleic Acids Res., 25:3389-3402; Baxevanis, AD and BFFOuellette (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 programs often also provide an indicator of the degree of identity.

[0149] In some embodiments, two sequences are considered substantially identical if at least 95% of the corresponding residues are identical across a relevant interval of residues. In some embodiments, the relevant interval is a complete sequence. In some embodiments, the relevant interval consists of at least 10 residues.

[0150] Targeting vector or targeting construct: This refers to a polynucleotide molecule containing a targeting region. The targeting region contains a sequence identical or substantially identical to the sequence of the target cell, tissue, or animal, and integrates the targeting construct to a location within the genome of the cell, tissue, or animal by homologous recombination. This also includes targeting regions that are targeted using site-specific recombinase recognition sites (e.g., loxP sites or Frt sites).

[0151] In some embodiments, the targeted construct described herein further includes a nucleic acid sequence or gene of particular interest, a selectable marker, a regulatory sequence and / or modulating sequence, and other nucleic acid sequences that enable recombination mediated by the external addition of proteins that assist or promote recombination involving such sequences. In some embodiments, the targeted construct described herein further includes all or part of the gene of interest, which is a heterologous gene encoding all or part of a polypeptide having a similar function to the protein encoded by the endogenous sequence. In some embodiments, the targeted construct described herein further includes all or part of the humanized gene of interest, which encodes all or part of a polypeptide having a similar function to the polypeptide encoded by the endogenous sequence. In some embodiments, the targeted construct (or targeting vector) may include a human-engineered nucleic acid sequence. For example, in some embodiments, the targeting construct (or targeting vector) may be constructed to include an engineered polynucleotide or recombinant polynucleotide comprising two or more sequences that are not bound to each other in their natural state but have been engineered by human intervention to be directly bound to each other in the engineered polynucleotide or recombinant polynucleotide.

[0152] Transgene or transgene construct: Meaning a nucleic acid sequence (e.g., a sequence encoding all or part of a polypeptide of interest) introduced into a cell by human intervention (e.g., by the method described herein). The transgene may be partially or entirely heterologous, i.e., exogenous to the transgenic animal or cell into which it is introduced. The transgene may contain one or more transcriptional regulatory sequences, such as introns or promoters, and any other nucleic acids, which may be required for the expression of the selected nucleic acid sequence. The transgene may contain one or more selection markers that enable the subsequent selection of offspring (e.g., cells) that have incorporated the transgene.

[0153] Transgenic animals, transgenic non-human animals, or Tg + : Used interchangeably herein, meaning any non-natural non-human animal, in which one or more cells of the non-human animal contain heterologous nucleic acids and / or genes encoding all or part of the polypeptide in question.

[0154] In some embodiments, heterologous nucleic acid sequences and / or genes are introduced into cells directly or indirectly by planned genetic engineering, such as by microinjection or by infection with recombinant viruses, into precursor cells. The term genetic engineering refers to the introduction of recombinant DNA molecules, rather than traditional mating techniques. These molecules may be incorporated into chromosomes or may be DNA that replicates outside of chromosomes. The term "Tg+" includes animals that are heterozygous or homozygous to heterologous nucleic acids and / or genes, and / or animals that have a single copy or multiple copies of heterologous nucleic acids and / or genes.

[0155] A vector is a nucleic acid molecule that can transport another nucleic acid with which it is associated.

[0156] In some embodiments, the vectors have the ability to replicate outside of chromosomes and / or express the nucleic acids to which they are bound in host cells, such as eukaryotic and / or prokaryotic cells. A vector capable of inducing the expression of an operably bound gene is referred to herein as an “expression vector.”

[0157] Wild-type: This has the meaning known in the art, meaning an entity with a structure and / or activity that exists in nature in a "normal" state or condition (in contrast to a mutated, diseased, modified, etc.). Those skilled in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0158] Other features, purposes, and benefits of the present invention will become apparent in the detailed description of some embodiments below. However, it should be understood that while the detailed description illustrates some embodiments of the present invention, they are presented for illustrative purposes only and not for limiting purposes. 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 a specific embodiment The present invention provides, in particular, a transgenic or engineered non-human animal having heterologous genetic material encoding one or more portions (functional fragments, binding sites, etc.) of human immunoglobulin, wherein the heterologous genetic material has a heavy chain constant (C HThe ) gene is inserted into the immunoglobulin heavy chain variable region locus so as to bind operably to the gene. Such non-human animals are intended to demonstrate the ability to produce antibodies encoded by the reconstituted V(DD)J sequence. Such non-human animals are also intended to demonstrate an antibody population characterized by a heavy chain variable region with increased CDR3 diversity compared to an antibody population with immunoglobulin heavy chain variable CDR3 diversity produced from a wild-type immunoglobulin heavy chain variable region locus (or an immunoglobulin heavy chain variable region locus occurring in nature). Thus, the non-human animals provided are particularly useful in the development of antibody-based therapies that bind to specific antigens, in particular those with low and / or poor immunogenicity or antigen binding, characterized by one or more epitopes that are undesirable to bind by conventional (or wild-type) antibodies. In particular, the present invention relates to the D in the immunoglobulin heavy chain variable region. H One or more D regions H Introducing a 23-mer recombinant signal sequence adjacent to a segment (e.g., 5' or 3'), thereby enabling D during VDJ recombination. H and D H This provides the ability to reconfigure segments, and a single D H Compared to antibodies generated from VDJ recombinants containing segments, these transgenic non-human animals represent the expression of antibodies with heavy chain variable regions, particularly the CDR3 region, which may be characterized by longer amino acid lengths. These transgenic non-human animals provide an in vivo system for identifying and developing antibodies and / or antibody-based therapies that bind disease targets beyond the targeting capabilities of established drug discovery techniques. Furthermore, these transgenic non-human animals provide a useful animal model system for developing antibodies and / or antibody-based therapies that focus on, or are designed to disrupt, core protein-protein interactions in various diseases and / or disease pathologies affecting humans.

[0160] As shown in the upper panel of Figure 1, recombination between immunoglobulin gene segments follows a rule commonly known as the 12 / 23 rule, where adjacent gene segments with recombination signal sequences (RSSs) are joined by an ordered process. Each RSS is a coding sequence (e.g., V H , D H or J H An RSS consists of a conserved block of seven nucleotides (heptamer; 5'-CACAGTG-3'; SEQ ID NO: 144) followed by a non-conserved region known as a spacer, which is either 12 bp or 23 bp long, and a second conserved block of nine nucleotides (nonomer; 5'-ACAAAAACC-3'; SEQ ID NO: 145). The spacer's sequence can vary, but its conserved length corresponds to one or two turns of the DNA double helix. This allows the heptamer and nonomer sequences to move to the same side of the DNA helix and bind to complexes of proteins that catalyze 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 for recombination generally promotes recombination between 12-mer RSSs and 23-mer RSSs, and prevents recombination between 23-mer RSSs and other 23-mer RSSs, or between 12-mer RSSs and other 12-mer RSSs, for example, direct germline V H and germline J H Recombination (i.e., 23mer and 23mer fusion) or germline D H and germline D H This prevents recombination (i.e., the binding of 12mers).

[0161] In some embodiments, the non-human animals described herein are each operably coupled to a 23mer RSS, and therefore D H -D H One or more D that have the ability to rearrange H An manipulated diversity cluster characterized by the presence of segments (i.e., manipulated D HIt includes an immunoglobulin heavy chain variable region containing the [field]. In some embodiments, an antibody containing a CDR3 generated from such recombination can be characterized by having increased diversity resulting from a longer amino acid length that directs direct binding to a specific antigen (e.g., virus, membrane channel, etc.). In some embodiments, the non-human animals described herein have VDJ recombination such that the V H , J H and more than one D H segments undergo recombination to create a heavy chain variable region that binds to the antigen of interest, and is operably linked to a human heavy chain variable (V H ) and joining (J H ) gene segments. In some embodiments, the engineered D H region described herein contains one or more (e.g., 1, 2, 3, 4, 5, 10 or more) human D H segments engineered to allow (or promote) D H and D H recombination at an increased frequency compared to the reference immunoglobulin heavy chain variable region locus. In some embodiments, the non-human animals described herein contain a plurality of V H and J H gene segments operably linked to one or more D H segments operably linked to a 23mer recombination signal sequence (RSS) in the immunoglobulin heavy chain variable region within the genome of the non-human animal. In many embodiments, the V H and J H segments are human V H and human J H gene segments.

[0162] <00029②> ​In some embodiments, the non-human animals described herein further include a human or humanized immunoglobulin light chain locus (e.g., κ and / or λ) such that the non-human animals produce antibodies comprising human variable regions (i.e., heavy and light chains) and non-human constant regions. In some embodiments, the human or humanized immunoglobulin light chain locus is operably bound to a rodent light chain constant region (e.g., rodent Cκ or Cλ) of human V L and J L Includes gene segments. In some embodiments, the non-human animals described herein are U.S. Patent Nos. 9,796,788; 9,969,814; U.S. Patent Publications 2011 / 0195454A1, 2012 / 0021409A1, 2012 / 0192300A1, 2013 / 0045492A1, 2013 / 0185821A1, 2013 / 0302836A1, 2018 / 01 Further comprising the immunoglobulin light chain loci described in International Patent Application Publication Nos. 25043; 2011 / 097603, 2012 / 148873, 2013 / 134263, 2013 / 184761, 2014 / 160179, 2014 / 160202, and 2019 / 113065 (each of which is incorporated herein by reference in whole).

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

[0164] VDJ rearrangement Genes involved in immunoglobulin synthesis are found in all animal cells and are located in gene segments that are sequentially positioned along the chromosome. The configuration of inheritable human gene segments, e.g., the germline arrangement of human gene segments, e.g., the order of human gene segments in the human germline genome (e.g., the genome passed on to the next generation), is found in Lefranc, M.-P., Exp. Clin. mmunogenet., 18, 100-116 (2001), which is incorporated herein by reference in its entirety and also shows the functional gene segments and pseudogenes found within the human immunoglobulin heavy chain loci in the germline arrangement. A series of recombination phenomena involving several gene components help assemble immunoglobulins from the ordered sequences of gene segments (e.g., V, D, and J). This assembly of gene segments is known to be imprecise, and therefore, immunoglobulin diversity is achieved both by the combination of different gene segments and by the formation of unique combinations through imprecise linkages. Furthermore, diversity is generated through a process known as somatic hypermutation, in which the variable region sequence of immunoglobulins is modified to increase their affinity and specificity for antigens. Immunoglobulin molecules are Y-shaped polypeptides composed of two identical heavy chains and two identical light chains, each having two structural components: one variable domain and one constant domain. The variable domains of the heavy and light chains are formed by the assembly of gene segments, while the constant domain is fused to the variable domain through RNA splicing. The mechanism of assembling (or binding) the gene segments is similar for the heavy and light chains, but only one binding 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 the heavy and light chain variable regions (referred to as VDJ recombination and VJ recombination, respectively) is induced by conserved non-coding DNA sequences adjacent to each gene segment, called recombination signal sequences (RSS), which ensure DNA rearrangement at the precise location compared to the V, D, and J coding sequences (see, e.g., Ramsden, DA et al., 1994, Nuc. Acids Res. 22(10):1785-96; the entire text is incorporated herein by reference). A representative schematic diagram of the sequences involved in VDJ recombination of heavy chain gene segments, as understood by those skilled in the art, is shown in Figure 1. Each RSS consists of a coding sequence (e.g., a V, D, or J segment), followed by a spacer (either 12 bp or 23 bp) and a conserved block of seven nucleotides (heptamers), and a second conserved block of nine nucleotides (nonamers). While considerable sequence differences are tolerated between individuals at 12 bp or 23 bp spacers, the lengths of these sequences typically do not vary. Recombination between immunoglobulin gene segments generally follows a rule referred to as the 12 / 23 rule, where gene segments adjacent to an RSS with a 12 bp spacer (or 12 mer) typically assemble with gene segments adjacent to a 23 bp spacer (or 23 mer, see, for example, Hiom, K. and M. Gellert, 1998, Mol. Cell., 1(7):1011-9, the entire work incorporated herein by reference.The RSS sequence has been reported to affect the efficiency and / or frequency of recombination with specific gene segments (see, for example, 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 these, or the whole thereof, is incorporated herein by reference). In fact, numerous reports have shown that gene segments, particularly D. H It has been noted that the use of segments is highly biased and variable among individuals. Unless otherwise indicated, or unless it is obvious to those skilled in the art that inconsistencies or discrepancies will arise, an unreconstructed gene segment is presumed to include two RSSs that are naturally related, e.g., adjacent, or possibly operably linked. In some embodiments, an unreconstructed gene segment as herein is a gene segment of its germline (e.g., wild-type) configuration, e.g., germline V, where each RSS is adjacent to a 23-mer RSS at both ends. H Genetic segments and germline J H It may include gene segments. In contrast, germline D H Gene segments, for example, unreconstructed D H Each gene segment has 12-mer RSSs adjacent to each other at its ends.

[0166] Therefore, an unreconstructed gene segment also refers to the gene segment of that germline configuration, including any RSS associated with such germline configuration. Furthermore, multiple gene segments in that germline configuration generally refer not only to each individual gene segment of that germline (e.g., unreconstructed) configuration, but also to the order and / or position of the functional gene segments. For example, see Lefranc, M.-P., Exp. Clin. Immunogenet., 18, 100-116 (2001), whose entire work is incorporated herein by reference for the germline configurations of the human V, D, and J gene segments.

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

[0168] Although not theoretically linked, further diversity in the heavy chain CDR3 repertoire may be possible through an increase in the number of bonds forming the reconstructed heavy chain variable region gene sequences, and / or an increase in the length of the CDR3 region. One mechanism that increases the number of bonds and / or the length of the CDR3 region is V H (D H AD H B)J H The subsequent D that leads to the gene sequence H -J H and VD H J H Prior to rearrangement, D H -D H In the recombination phenomenon, the first D H Segment (D H Another D (which can be commonly referred to as "A") H Segment (D H It can be through bonding to (which can be commonly referred to as "B"). In nature, D H -D H It was long believed that the recombination phenomenon would be prevented by Rule 12 / 23 (Alt, FW et al., 1984, EMBO J.3(6):1209-19, the whole of which is incorporated herein by reference). However, D H -D HRecombination phenomena follow a 12 / 23 rule (1 in 800 or approximately 0.125% of naive B cells; 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 Contrary to the findings of the previous study (see 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, BS et al., 2012, Immunol.137:56~64; each of these is incorporated herein by reference in its entirety), it has been revealed that this is the main mechanism for generating abnormally long CDR3s that occur at a very low frequency in humans and are observed in some heavy chains (see Janeway's Immunobiology., Vol. 9, Kenneth Murphy, Casey Weaver., Chapter 5, 2017; the entirety is incorporated herein by reference). However, some potential therapeutic targets (e.g., non-limitingly, viruses, cell surface receptors, type IV transmembrane proteins such as GPCRs, ion channels) mask or concave 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, possess broad neutralizing activity (e.g., broad neutralizing antibodies against HIV-1 or influenza). Increasing the frequency of heavy chains with very long HCDR3s is useful in selecting antibodies capable of reaching these hidden epitopes. While not intended to be tied to theory, one way to achieve this is to include a 23-mer RSS and D H Longer J gene segment H D to increase the frequency of recombination into 6 gene segmentsH By manipulating the segment, heavy chain V H (D H AD H B)J H The goal is to increase the frequency of reconfiguration. In mice, D H -J H The binding is performed in two ordered steps: (1) the proximal DQ52 segment (in humans, D H The closest J (mentioned as 7-27) H Segment (J H 1 or J H 2) This is thought to occur in the primary reconstruction into one of the two. Next, the strong μ0 promoter upstream of DQ52 causes the remaining J H Segment (J H 3 and J H 4) becomes more accessible by recombinant activating genes (RAG); (2) distal D H The remaining J of the segment H Segment (J H 3 or J H 4) Second reconstruction into one of the mice (J H 3-J H 4) and humans (J H 4-J H Downstream J observed in both of 6) H This is consistent with the more frequent use of the segment (Nitschke et al., 2001, J.Immunol., 166:2540-52, the whole of which is incorporated herein by reference). Although we do not wish to be tied to theory, D H 7-27 and J H 1-J H 3 (or J H 1-J H 5) A synthetic D having a 5' 23mer RSS and a 3' 12mer RSS. H Genes (for example, synthetic D H Substitution in 3-3 segments is a high frequency of V H (D H -D H )J H It is assumed that this rearrangement can occur through a three-step mechanism: (1)23(D H)J H 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 H (D H 1-1~D H 1-26) 23(D H )J H Reconstruction to (23(D H )J H V to H (The rearrangement is prevented by the 12 / 23 rule), (3) to produce the VDDJ coding sequence encoding the immunoglobulin heavy chain variable domain, V H 12(D H -D H )J H Reconfiguration to . See, for example, the lower panel of Figure 1. Also, long D that can form disulfide bonds that are thought to stabilize the long HCDR3 region (see, for example, Wang et al., 2013, Cell 153:1379~93, the whole of which is incorporated herein by reference) H D is a gene segment (containing more than 31 nucleotides that code for the two cysteines that make up the segment). H 2-2, D H 2-8, and D H Substitutions in the gene segments of 2-15, operably linked to the 12-mer RSS at the 5' end and the 23-mer RSS at the 3' end, respectively, also involve a three-step mechanism: (1) 12(D H )J H J H 4, J H 5, or J H 12 to 6: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 of 12(D H )J H (3) Reconfiguration to produce a VDDJ coding sequence encoding the immunoglobulin heavy chain variable domain, H 12(D H -D H )J H High frequency V that may result from reconstruction H (D H -D H )J H It is also assumed that recombination occurs. As described herein, the manipulated D H The region is one or more D H It is constructed by placing a 23-mer spacer at either the 5' or 3' adjacent position to the segment, thereby creating a D in the humanized immunoglobulin heavy chain variable region locus. H From D H This will allow for reconfiguration.

[0169] In some embodiments, the non-human animals described herein include an immunoglobulin heavy chain variable region locus exhibiting VDJ recombination that does not conform to Rule 12 / 23 compared to a reference non-human animal. In some embodiments, the non-human animals described herein include a wild-type D H Compared to the segment, one or more modified D H Includes one or more RSS adjacent to or adjacent to the segment. In some embodiments, one or more D of the immunoglobulin heavy chain variable regions of non-human animals described herein. H The gene segment is D H -D HEach is operably coupled to either a 5' or 3' 23-mer RSS such that the recombination frequency is increased in the non-human animal compared to the reference non-human animal. The recombination efficiency and / or frequency may, in some embodiments, be determined by the frequency of use of the gene segment in the population of antibody sequences (e.g., from an individual or a 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 whole of which is incorporated herein by reference). Thus, the non-human animals described herein are, in some embodiments, D H -D H Recombination is D in reference non-human animals. H -D H Compared to recombination, one or more D operatively coupled or adjacent to the 23mer RSS occur with increased frequency. H Includes segments. In some embodiments, the operated D described herein H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least three times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least four times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least 5 times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -DH Compared to recombination, the frequency of D increased by at least 10 times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least 20 times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least 30 times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least 40 times. H -D H Recombination is shown. In some embodiments, the operated D described herein is used. H Rodents, including the region, are referenced in D non-human animals. H -D H Compared to recombination, the frequency of D increased by at least 50 times. H -D H This shows the rearrangement.

[0170] Related In Vivo Systems This invention is based on the recognition that certain antigens are associated with low and / or poor immunogenicity and are therefore unsuitable targets for antibody-based therapies. Indeed, numerous disease targets (e.g., viruses, channel proteins) have been characterized as refractory or not leading to the development of new drugs. Accordingly, this invention is based on the creation of in vivo systems for developing antibodies and antibody-based therapies that overcome the challenges associated with established drug development technologies and / or approaches. In some embodiments, this invention relates to one or more D HRSS is 12mer D H -12mer format to 12mer D H -23mer or 23mer D H -12mer format is changed, modified, or manipulated, thereby D observed in the reference in vivo system. H From D H Compared to recombination to D at an increased frequency H From D H Manipulated D that enables rearrangement to H This disclosure provides an in vivo system characterized by the presence of immunoglobulin loci, particularly humanized immunoglobulin heavy chain variable region loci, including the region. H This paper specifically demonstrates the construction of transgenic rodents containing a variable immunoglobulin heavy chain region, and the manipulated D H The region is D with increased frequency. H -D H One or more D that enable rearrangement H One or more D segments are operably coupled to each of the 23-mer RSS segments located relative to each of the segments. H Includes segments. The methods described herein are operated D as described herein. H To generate the region, any number of D23mer RSSs, each operably coupled to a 5' or 3' RSS. H The segment (e.g., conventional or synthetic) can be adjusted to achieve the manipulated D H The region is once integrated into the immunoglobulin heavy chain variable region (i.e., V H and J H V H and J H More than one of the gene segments D HIt provides recombination with segments. In some embodiments, such heavy chain variable regions have the ability to access hard-to-reach epitopes such as viruses, channel proteins, and GPCRs.

[0171] While we do not wish to be bound by any particular theory, we believe that the data provided herein, in some embodiments, are operably linked to a 5' 23-mer RSS. H The manipulated D characterized by its inclusion H We have observed that rodents containing an immunoglobulin heavy chain variable locus including the region efficiently produce antibodies produced by V(DD)J recombination. We have found that in some embodiments the genome contains three D(DD) regions operably linked to the 3' 23-mer RSS, each of which is operably linked to the RSS. H The manipulated D characterized by its inclusion H We also noticed that rodents containing the immunoglobulin heavy chain variable locus, which includes the region, efficiently produce antibodies produced by V(DD)J recombination. H Some or all five J's upstream of the 6 gene segments H Deletion of a gene segment, J H This specification also shows that preferential recombination to 6 gene segments is also possible. In particular, J H The 6 gene segments contain 63 nucleotides, for example, 52, 53, 50, 40, and 51 nucleotides respectively, J H 1, J H 2, J H 3, J H 4, and J H It includes 10 nucleotides other than the 5 gene segment. Therefore, in at least some embodiments, this disclosure encodes, for example, a reconstituted immunoglobulin heavy chain variable region gene sequence, for example, a CDR3 of at least 20 amino acids in length, for example, a heavy chain variable domain having a CDR3 of 20 to 30 amino acids in length. H D H J H and V H (DH AD H B)J H For example, V H (D H AD H B)J H By providing rodents that generate 6 sequences, this includes the development of in vivo systems for generating antibodies and / or antibody-based therapies against intractable disease targets. In some embodiments, the reconstituted immunoglobulin heavy chain variable region gene sequences of non-human animals described herein, e.g., V 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 of at least 21 amino acids in length.

[0172] In some embodiments, (1) the germline genome, for example, in germline cells, operably bound to the 23-mer RSS H Manipulated D containing gene segment H (2) the immunoglobulin heavy chain locus containing the region, and (3) its somatic genome, e.g., in B cells, the reconstituted heavy chain V H (D H AD H B)J H Non-human animals, such as rodents, such as rats or mice, including a code sequence, are described herein and are first or second D H gene segment (i.e., each, V H (D H AD H B)J H Code array D H A or D H B) D is operably coupled to the 23mer RSS. H Includes a gene segment, or a part thereof, for example, the first or second D H The gene segment is operably linked to the 23-mer RSS. HIt has at least nine consecutive nucleotides aligned with the gene segment, and the first and second D H Each gene segment is independent of the corresponding germline D of any overlap. H It contains at least five consecutive nucleotides aligned with the gene segment.

[0173] In some embodiments, the non-human animal provided is composed of multiple human Vs arranged in a germline configuration. H , D H and J H It includes immunoglobulin heavy chain loci operably bound to non-human immunoglobulin heavy chain constant regions, enhancer and regulatory regions, characterized by the presence of gene segments. In some embodiments, the non-human animal provided is one or more human V H Gene segment, one or more human D H One or more human J operatively bound to gene segments and non-human immunoglobulin heavy chain constant regions. H Includes gene segments.

[0174] In some embodiments, the non-human animal provided is at least 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 Includes 6-1.

[0175] In some embodiments, the provided non-human animal is 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 Includes 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, are further included. In some embodiments, the non-human animal provided is human D H Includes 7-27 as well.

[0176] In some embodiments, (1) the germline genome, for example, in germline cells, operably bound to the 23-mer RSS H Manipulated D containing gene segment H (2) the immunoglobulin heavy chain locus containing the region, and (3) its somatic genome, e.g., in B cells, the reconstituted heavy chain V H (D H AD H B)J H Non-human animals, such as rodents, such as rats or mice, including a code sequence, are described herein and are first or second D H gene segment (i.e., each, V H (D H AD H B)J H Code array D H A or D H B) D is operably coupled to the 23mer RSS. H Includes a gene segment, or a part thereof, for example, the first or second D H The gene segment is operably linked to the 23-mer RSS. H It has at least nine consecutive nucleotides aligned with the gene segment, and the first and second D H Each gene segment corresponds to a germline D, independent of any overlap. H It contains at least five consecutive nucleotides aligned with the gene segment. In some embodiments, the non-human animal provided is operably bound to a 23-mer RSS of human D2. H Includes a gene segment. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains two cysteine ​​codons. In some embodiments, human D2 is operably bound to the 23-mer RSS. HThe gene segment contains at least 37 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 19 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 20 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 23 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 28 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 31 nucleotides. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment contains at least 37 nucleotides.

[0177] In some embodiments, human D23mer is operably coupled to the RSS. H The gene segment is human D H Contains 1 gene segment. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H It contains two gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H It contains three gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H It contains four gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H It contains 5 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. HThe gene segment is human D H It includes 6 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H It contains 7 gene segments.

[0178] In some embodiments, human D23mer is operably coupled to the RSS. H The gene segment is human D H Includes a 1-1 gene segment. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H Includes 2-2 gene segments. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H Includes 3-3 gene segments. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H It includes a 4-4 gene segment. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H It includes a 5-5 gene segment. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H It includes a 6-6 gene segment. In some embodiments, human D2 is operably bound to the 23-mer RSS. H The gene segment is human D H Includes 1-7 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 2-8 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 3-9 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. HThe gene segment is human D H Includes 3-10 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 5-12 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 6-13 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 2-15 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 3-16 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 4-17 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 5-18 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 6-19 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 1-20 gene segments. In some embodiments, human D2 is operably bound to a 23-mer RSS. H The gene segment is human D H Includes 2-21 gene segments. In some embodiments, human D23 operably bound to the RSS of 23mer. H The gene segment is human D H Includes 3-22 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D HIncludes 6-25 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 1-26 gene segments. In some embodiments, human D23 operably bound to the RSS of 23mer. H The gene segment is human D H Includes 1-14 gene segments. In some embodiments, human D23 operably bound to the RSS of 23mer. H The gene segment is human D H Includes 4-11 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 4-23 gene segments. In some embodiments, human D23 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 5-24 gene segments. In some embodiments, human D2 is operably bound to the RSS of 23mer. H The gene segment is human D H Includes 7-27 gene segments.

[0179] In some embodiments, the non-human animal provided is a human D, which is typically arranged in the order found in the unreconstructed human genome variable loci. H Includes a complete or substantially complete repertoire of gene segments, and a complete or substantially complete repertoire of human D H One of the gene segments was engineered to operably bind to the 23-mer RSS of D H It is replaced with a gene segment. In some embodiments, a complete or substantially complete repertoire of human D H One of the gene segments was engineered to operably bind to the 23-mer RSS of the corresponding D H The gene segment is replaced, for example, wild-type D H The 2-2 gene segment was engineered to operably bind to the 23-mer RSS. H2-2 gene segments are replaced. In some embodiments, the complete or substantially complete repertoire of human D H One of the gene segments was engineered to operably bind to the 23-mer RSS of another D H The gene segment is replaced, for example, wild-type D H The 7-27 gene segment was engineered to operably bind to the 23-mer RSS. H 3-3 gene segments are replaced. In some embodiments, the human D is commonly arranged in the order found in unreconstructed human genome variable loci. H D of the complete or substantially complete repertoire of gene segments H The 1-1 gene segment is engineered to operably bind to a 23-mer RSS, for example, the 3' 23-mer RSS, corresponding to or another D H It is replaced by a gene segment. In some embodiments, it is commonly arranged in the order found in unreconstructed human genome variable loci. H D of the 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 2-21 gene segments or any combination thereof are manipulated to operably bind to a 23-mer RSS, e.g., a 3' 23-mer RSS, corresponding to or another D H It is replaced by a gene segment. In some embodiments, it is commonly arranged in the order found in unreconstructed human genome variable loci. H D of the 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 is engineered to operably bind to a 23-mer RSS, for example, a 3' 23-mer RSS, corresponding to or another D H It is replaced by a gene segment.

[0180] In some embodiments, the non-human animal provided is at least human J H Gene segment J H Includes 6. In some embodiments, the non-human animal provided is at least human J H Gene segment J H 4, J H 5 and J H Includes 6. In some embodiments, the non-human animal provided is 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 includes one or more non-human immunoglobulin heavy chain constant region genes, such as 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 includes 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 bound to one or more non-human immunoglobulin heavy chain enhancers (i.e., enhancer sequences or enhancer regions). In some embodiments, the human V H , D H and J H The gene segment is operably bound to one or more non-human immunoglobulin heavy chain regulatory regions (or regulatory sequences). In some embodiments, the human V H , D H and J H The gene segment is operably bound 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 non-human animal provided does not contain (or lacks) the endogenous Adam6 gene. In some embodiments, the non-human animal provided does not contain or lacks the endogenous Adam6 gene (or Adam6 coding sequence) at the same germline genomic location found in the germline genome of a congeneral wild-type non-human animal. In some embodiments, the non-human animal provided does not contain or lacks the human Adam6 pseudogene. In some embodiments, the non-human animal provided includes an insertion of at least one nucleotide sequence encoding one or more non-human (e.g., rodent) Adam6 polypeptides. The insertion is outside the manipulated immunoglobulin heavy chain locus described herein (e.g., at the 5' end) within the manipulated immunoglobulin heavy chain locus or another location (e.g., a randomly introduced non-human Adam6 coding sequence) in the germline genome of the non-human animal, cell, or tissue. H (Upstream of the gene segment) In some embodiments, the non-human animal provided does not contain or lacks the functionally endogenous Adam6 pseudogene.

[0183] In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein contain endogenous non-human V in the antibody molecule. H The region, in whole or in part, is not detectably expressed. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein contain endogenous non-human V in the antibody molecule. H Domain (for example, V H , D H and / or J H ) does not contain (or lacks or contains deletions of) one or more nucleotide sequences that encode in whole or in part. In some embodiments, the non-human animals, non-human cells or non-human tissues provided herein contain endogenous non-human V H , D H and J H The non-human animal has a germline genome that contains a deletion in whole or in part in a gene segment. In some embodiments, the non-human animal provided is fertile.

[0184] In some embodiments, the non-human animal provided further includes an immunoglobulin κ light chain locus characterized by the presence of multiple human Vκ and Jκ gene segments located in the germline structure, inserted upstream of a non-human Cκ gene segment, and operably bound. In some embodiments, the manipulated immunoglobulin κ light chain locus includes at least a human Vκ gene segment appearing in a proximal variable cluster (or proximal arm, or proximal duplication) of the human immunoglobulin κ light chain locus. In some embodiments, the manipulated immunoglobulin κ light chain locus includes 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 manipulated immunoglobulin κ light chain locus includes 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 manipulated immunoglobulin κ light chain locus includes human Jκ gene segments Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In some embodiments, the manipulated immunoglobulin κ light chain locus includes 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 bound to one or more non-human immunoglobulin κ light chain enhancers (i.e., enhancer sequences or enhancer regions). In some embodiments, the human Vκ and Jκ gene segments are operably bound to the mouse Igκ light chain intron enhancer region (Igκ Ei or Eiκ). In some embodiments, the human Vκ and Jκ gene segments are operably bound 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 bound to the mouse Igκ light chain 3' enhancer region (Igκ3'Ei or 3'Eiκ). In some embodiments, the human Vκ and Jκ gene segments are operably bound to mouse Eiκ and operably bound to mouse 3'Eκ. In some embodiments, the human Vκ and Jκ gene segments are operably bound to one or more non-human immunoglobulin κ light chain enhancers (or enhancer sequences or regions) and one or more non-human immunoglobulin κ light chain regulatory regions (or regulatory sequences). In some embodiments, the engineered immunoglobulin κ light chain locus contains the same non-human immunoglobulin κ light chain enhancer regions (or enhancer sequences) that appear in the wild-type immunoglobulin κ light chain locus. In some embodiments, the engineered immunoglobulin κ light chain locus contains non-human Igκ light chain enhancer regions (or enhancer sequences) that appear in the wild-type immunoglobulin κ light chain locus of a different species (e.g., a different rodent species).

[0186] In some embodiments, the manipulated immunoglobulin κ light chain locus described herein does not contain (i.e., lacks) the human VpreB gene (or human VpreB gene coding sequence).

[0187] In some embodiments, the non-human Cκ gene of the manipulated immunoglobulin κ light chain locus includes, for example, a rodent Cκ gene such as a mouse Cκ gene or a rat Cκ gene. In some embodiments, the non-human Cκ gene of the manipulated immunoglobulin κ light chain locus is or includes a mouse Cκ gene derived from a genetic background including the 129 line, BALB / c line, C57BL / 6 line, mixed 129xC57BL / 6 line 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 the endogenous non-human Vκ region in whole or in part in the antibody molecule. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not contain (or lack, or contain deletions thereof) one or more nucleotide sequences that encode the endogenous non-human Vκ region in whole or in part in the antibody molecule. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that contains deletions of the endogenous non-human Vκ and Jκ gene segments in whole or in part.

[0189] In some embodiments, the non-human animal provided further comprises a wild-type or inactivated (e.g., 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 the endogenous non-human Vλ region in whole or in part in the antibody molecule. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein do not contain (or lack, or contain) one or more nucleotide sequences that encode the endogenous non-human Vλ region in whole or in part in the antibody molecule. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that contains deletions of the endogenous non-human Vλ and Jλ gene segments in whole or in part. In some embodiments, the non-human animals, non-human cells, or non-human tissues provided herein have a germline genome that contains deletions of the endogenous non-human Vλ, Jλ, and Cλ gene segments in whole or in part.

[0191] Guidance for the generation of targeting vectors, non-human cells, and animals having such manipulated immunoglobulin loci can be found in U.S. Patents 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 entirety. Those skilled in the art will recognize the various technologies known in the art for accomplishing such genetic manipulation and / or manipulation of non-human (e.g., mammalian) genomes, or for manipulating such sequences for introduction into the germline genomes of non-human animals.

[0192] DNA constructs In many cases, polynucleotide molecules containing the immunoglobulin gene segments described herein, in particular, one or more D molecules each operably bound to a 5' or 3' 23-mer RSS. H The segment is inserted into a vector, preferably a DNA vector, to replicate the polynucleotide molecule in a suitable host cell.

[0193] Depending on the size, D H Segments can be cloned directly from genome sources available from commercially available sources, or they can be designed in silico based on publicly available sequences from GenBank. Alternatively, bacterial artificial chromosome (BAC) libraries can provide immunoglobulin sequences. BAC libraries contain an average insertion size of 100–150 kb and can hold insertions 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 whole of which is incorporated herein by reference). For example, human and mouse genomic BAC libraries have been constructed and are commercially available (e.g., Invitrogen, Carlsbad, California). The genomic BAC library can also be used as a source of immunoglobulin sequences and 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, may be cloned and contained within one or more YACs. If multiple YACs are used and contain regions of overlapping homology, they can be recombined within a yeast host strain to produce a single construct presenting the entire locus. YAC arms can be further modified in mammalian selection cassettes by introducing the constructs into embryonic stem cells or embryos using methods known in the art and / or methods described herein.

[0195] DNA constructs can be prepared using methods known in the art. For example, DNA constructs can be prepared as part of a large plasmid. Such preparations enable the cloning and selection of the correct construct using efficient methods known in the art. One or more D2s operably bound to the 5' or 3' 23-mer RSS as described herein.H DNA fragments containing segments can be located between convenient restriction enzyme sites on the plasmid so that they can be easily isolated from the rest of the plasmid sequence for integration into the desired animal.

[0196] In some embodiments, the methods used in plasmid preparation and transformation of host organisms are known in the Art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination methods, see Molecular Cloning: A Laboratory Manual, Part 2, edited by Sambrook, J. et al., Cold Spring Harbor Laboratory Press: 1989; the entire work is incorporated herein by reference.

[0197] Production of non-human animals In the variable region of immunoglobulin heavy chains in the genomes of non-human animals, D H and D H One or more D that enable rearrangement H Non-human animals expressing antibodies having heavy chain CDR3 diversity, characterized by long amino acid lengths resulting from the integration of 23-mer RSSs adjacent to a segment, are provided. Suitable examples described herein include rodents, in particular mice. One or more D H In many embodiments, the segment is heterogeneous D H segment (for example, human D H (Segment) includes. Compared to wild-type or reference non-human animals, D is present at an increased frequency. H and D H D, which can be reconfigured H Non-human animals, embryos, cells, and targeting constructs for producing non-human animals, non-human embryos, and cells containing segments are also provided.

[0198] In some embodiments, one or more D H The segment is a diversity cluster of immunoglobulin heavy chain variable regions in the genomes of non-human animals (i.e., D HModified to be adjacent to (or operably bound to) the 5' or 3' 23mer RSS within the region. In some embodiments, the D of the immunoglobulin heavy chain variable region H The region (or part thereof) is not deleted (i.e., intact). In some embodiments, the D of the immunoglobulin heavy chain variable region H Each region (or a portion thereof) is operably coupled to one or more D2s of 5' or 3' 23' RSS. H The segment is modified, destroyed, deleted, manipulated, or replaced. In some embodiments, D H One or more composite D regions, each operably coupled to a 5' or 3' 23-mer RSS. H Replaced by segments, and in some embodiments, one or more conventional D H The gene segment is the D of the immunoglobulin heavy chain variable region. H The region is neither deleted nor substituted. In some embodiments, D H The region is conventional D H A segment (i.e., one or more D segments operably coupled to a 5' 12mer RSS and a 3' 12mer RSS, respectively) H (segment) and manipulated D H One or more D12s operably ligated to the gene segment (i.e., the RSS of 23mers, each 5' or 3') H It contains both of the segments. In some embodiments, D as described herein H The region is synthetic D H This is the region. In some embodiments, D H The domain is Human D H This is the region. In some embodiments, D H The region is Mouse D H This is the region. In some embodiments, the operated D described herein H The region (or part thereof) is the D that was operated on. H A region (or part thereof) contains one or more V H Gene segment and / or one or more J HIt is inserted into the variable region of the immunoglobulin heavy chain so that it can be operably bound to the gene segment. In some embodiments, the manipulated D H The region is inserted into one of two copies of the immunoglobulin heavy chain variable region, and the manipulated D H This produces non-human animals that are heterozygous in the region. In some embodiments, manipulated D H A non-human animal homozygous for the region is provided. In some embodiments, the manipulated D H A non-human animal heterozygous for the region is provided.

[0199] In some embodiments, the non-human animals described herein each have one or more D2s operably linked to a 5' or 3' 23-mer RSS within their genome. H D containing segments H It contains randomly integrated human immunoglobulin heavy chain variable regions, including the region. Therefore, such non-human animals are manipulated D H It may be described as having a human immunoglobulin heavy chain transgene containing the region. Manipulated D H The region can be detected using a variety of methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele (GOA) acquisition or allele (LOA) loss assays. In some such embodiments, the non-human animals described herein are manipulated D as described herein. H It is heterozygous for the region. In some embodiments, the non-human animals described herein are manipulated D as described herein. H It is homozygous for the region. In some embodiments, the non-human animals described herein are the manipulated D described herein. H It is hemizygous to the region. In some embodiments, the non-human animals described herein are manipulated D as described herein. H It contains one or more copies of the domain.

[0200] In some embodiments, manipulated non-human animals as described herein HThe region is associated with (or operably coupled with) at least one D23mer RSS of 5'. H Includes segments. In some embodiments, manipulated D of non-human animals as described herein H The region is associated with (or operably coupled with) at least one D of the 23-mer RSS of 3'. H Includes segments. In some embodiments, manipulated D of non-human animals as described herein. H The region is associated with (or operably bound to) at least human D2 at the 23-mer of the 5'. H Includes 3-3 segments. In some embodiments, manipulated D of non-human animals as described herein. H The region is associated with (or operably bound to) at least human D2 at the 23-mer of the 3'. H Includes 2 segments, Human D H The two segments are human D H 2-2, Human D H 2-8, Human D H 2-15 and Human D H It is selected from the group consisting of 2-21.

[0201] In some embodiments, manipulated non-human animals as described herein H Each region is associated with (or operably coupled with) one more D than the 5' 23mer RSS. H Includes segments. In some embodiments, manipulated D of non-human animals as described herein H Each region is associated with (or operably coupled with) one more D than the 23-mer RSS of 3'. H Includes segments. In some embodiments, manipulated D of non-human animals as described herein. H Each region is associated with (or operably bound to) the 3' 23-mer RSS of human D H 2-2, Human D H 2-8, and Human D H Includes segments 2-15.

[0202] Human V operably bound 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 the gene segment H A composition and method for producing a non-human animal that expresses an antibody containing a heavy chain variable region including a CDR3 region having an amino acid sequence encoded by a segment, wherein the genome is manipulated D H A composition and method for producing a non-human animal containing an immunoglobulin heavy chain variable region, wherein the manipulated D H Each region is associated with (or operably coupled with) one or more D2s of 5' or 3' 23mer RSS. H Compositions and methods comprising segments are provided. In some embodiments, compositions and methods are also provided for producing 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 also provided for producing non-human animals that express such antibodies under the control of heterologous enhancers and / or heterologous regulatory sequences. The method involves adding one or more D segments in the genome of a non-human animal so that an antibody comprising an immunoglobulin heavy chain resulting from V(DD)J recombination is expressed. H Segments, and wild-type D H Compared to the segment, D increased in frequency. H and D H This includes the step of inserting other sequences that enable recombination.

[0203] In some embodiments, the method involves one or more J H D has a 5' 23-mer RSS and a 3' 12-mer RSS operably bound to the gene segment. H The process includes inserting DNA containing a gene segment. As described herein, D H The gene segment is located downstream of the μ0 promoter sequence. In some embodiments, the method is as follows: HThe gene segment has a 23-mer RSS at 5' and a 12-mer RSS at 3' so that it can access the RAG gene (e.g., RAG-1 and / or RAG-2) during recombination. H The process includes inserting the segment at a position associated with the μ0 promoter sequence. H The genetic material, including the segment and adjacent RSS, is inserted into the genome of a non-human animal, thereby creating adjacent D H Gene segment and V H and J H D H The manipulated D containing the segment and required RSS H It is possible to generate non-human animals that have a specific region.

[0204] In some embodiments, the method each has six J H Three D segments having a 5' 12-mer RSS and a 3' 23-mer RSS operably bound to the gene segment. H The process includes inserting DNA containing a gene segment. As described herein, D H Each gene segment is associated with multiple D2s, each consisting of 12-mer RSSs at the 5' and 3' ends. H It is located within the gene segment. In some embodiments, the method is associated with the 5' 12mer RSS and the 3' 23mer RSS, respectively. H 2-2, D H 2-8 and D H The process includes inserting each of the 2-15 gene segments into a diversity cluster having multiple other DH gene segments associated with conventional or wild-type RSS. H Genetic material, including gene segments and adjacent RSSs, is inserted into the genome of a non-human animal, thereby creating adjacent D H Gene segment and V H and J H D H The manipulated D containing the segment and required RSS HIt is possible to generate non-human animals that have a specific region.

[0205] In appropriate cases, D H The sequence corresponding to (or encoding) the segment may be modified to include codons optimized for expression in non-human animals (see, for example, U.S. Patents 5,670,356 and 5,874,304, each of which is incorporated in whole by reference). The codon-optimized sequence is a synthetic sequence and preferably encodes the same polypeptide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by the non-codon-optimized parent polynucleotide. In some embodiments, D H The sequence corresponding to (or encoding) the segment may include a sequence modified to optimize codon utilization for a specific cell type (e.g., rodent cells). For example, a D sequence to be inserted into the genome of a non-human animal (e.g., a rodent). H The codons of the sequences corresponding to the segments may be optimized for expression in non-human animal cells. Such sequences may be described as codon-optimized sequences.

[0206] D operably coupled to a 5' or 3' 23mer RSS H Segment D H Insertion into the region is performed by the D H The segment is V H and J H Gene segment (for example, multiple V H and J H By utilizing relatively few genomic modifications to enable operable binding to gene segments, this method results in the expression of antibodies containing heavy chains characterized by longer amino acid lengths of CDR3.

[0207] Methods for generating transgenic non-human animals, including knockout and knock-in, are well known in the art (see, for example, Gene Targeting: A Practical Approach, edited by Joyner, Oxford University Press, Inc., (2000); the entire text is incorporated herein by reference). For example, the generation of transgenic rodents may involve disruption of the locus of one or more endogenous rodent genes (or gene segments) and, in some embodiments, one or more D genes operably ligated to the RSS of each 23mer into the rodent genome at the same location as the endogenous rodent genes (or gene segments). H The introduction of segments may also be included. In some embodiments, one or more D segments are operably coupled to the RSS of 23mer each. H The segment is a randomly inserted immunoglobulin heavy chain locus in the rodent genome. H It is introduced into the region. In some embodiments, one or more D are operably coupled to the RSS of 23mer each. H The segment is the D locus of the endogenous immunoglobulin heavy chain gene in the rodent genome. H In the region introduced, and in some embodiments, the endogenous immunoglobulin heavy chain locus is modified, altered, or manipulated to contain human gene segments (e.g., V and / or J) operably bound to one or more constant region genes (e.g., human or mouse).

[0208] The genome is composed of one or more D2s, each of which is operably linked to a 23-mer RSS of either 5' or 3'. H A manipulated diversity cluster (i.e., D) that includes segments. H Manipulated D for generating rodents, including an immunoglobulin heavy chain variable region (including the region) HA schematic diagram (non-scale) of representative targeting vectors for constructing regions and their integration into rodent embryonic stem (ES) cells is provided in Figure 2. Typical strategies and methods for inserting such vectors into immunoglobulin heavy chain variable regions in the genome of rodent ES cells are provided in Figures 3–8. In each of Figures 2–8, the NotI restriction enzyme recognition sites shown are appropriate, and the names and approximate locations (dotted lines) of various primer / probe sets (see Table 4) are shown (non-scale) for the various alleles shown. Unless otherwise specified, unfilled symbols and lines represent human sequences, and filled symbols and black lines represent mouse sequences. The following abbreviations are used for each element in the diagram: 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 explained in Figure 2, one D is operably coupled to the 5' 23mer RSS. H The segment has segments (Figure 2, top and middle) and three segments D each operably coupled to a 3' 23m RSS H Multiple D (Figure 2, bottom) H DNA fragments containing segments are prepared using VELOCIGENE® technology (e.g., U.S. Patent No. 6,586,251 and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659; the entire text is incorporated herein by reference) and molecular biological techniques known in the art. In Figure 2, unless otherwise indicated, unfilled 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-scale relative positions of the 6 gene segments are present, and any remaining unnamed gene segments are D H When it is a gene segment, it is shown. Generally, it is shown by a dotted line. H The region is unreconstructed human D H It includes the complete repertoire of gene segments (see, for example, www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=locus&species=human&group=IGH; the whole is incorporated herein by reference), with the following exception: the two targeting vectors above have a 23-mer RSS at the 5' end and a 12-mer RSS at the 3' end adjacent to each other. H The last D is replaced by a sequence containing 3-3 gene segments. H The 7-27 gene is missing (indicated as an unfilled arrow); the targeting vector below has a 12-mer RSS at the 5' end and a 23-mer RSS at the 3' end adjacent to each other. H 2-2 gene segment, 12-mer RSS at the 5' end and 23-mer RSS at the 3' end are adjacent D H The 2-8 gene segment, and the adjacent 12-mer RSS at the 5' end and the 23-mer RSS at the 3' end are located in D H Unreconstituted human D2, substituted with the 2-15 gene segment. H 2-2 gene segment, unreconstructed human D H 2-8 gene segments, and D H 2-15 gene segment missing (manipulated D H 2-2, D H 2-8, and D HEach of the 2-15 gene segments is shown as an unfilled arrow. The unfilled bar graph is 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 Represents the positions of unique artificial 40mers homologous to the primer and probe sequences located in the targeting vector. The sequences of the unique artificial 40mers, indicated as "1", "2", "10", "16", "8", and "18", are described as SEQ ID NOs. 73, 74, 75, 76, 77, and 78, respectively.

[0210] DNA fragments are assembled with homologous arms for precise targeted insertion into the humanized immunoglobulin heavy chain variable region locus (Figures 3, 5, 7). A selection cassette (e.g., neomycin) adjacent to a site-specific recombination recognition site (e.g., loxP) is included in the targeting vector to facilitate screening for appropriate insertion into ES cell clones 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 is then processed into the D H The region is human V at 5'. H The genomic DNA is human J at 3'. H The design ensures that genomic DNA and non-human (e.g., rodent) genomic heavy chain constant region DNA (e.g., intron enhancer and IgM constant region genes) are adjacent. The final targeting vector for integration into the genome of non-human cells (e.g., rodent embryonic stem cells) is V H Genomic DNA (for example, one or more V H (Containing gene segments), manipulated D H area, 3'J HIt contains genomic DNA and non-human (e.g., rodent) genomic heavy chain constant region DNA, and all of these, once incorporated into the genome of a non-human animal, V H Gene segment, manipulated D H Region and J H The targeting vectors are operably bound to allow recombination between gene segments. Once assembled, the targeting vectors are linearized and electroporated into rodent ES cells.

[0211] The targeting vector is the sequence contained in the targeting vector (i.e., the manipulated D H The region) has one more D H It is introduced into rodent (e.g., mouse) embryonic stem cells to produce the ability of non-human cells or non-human animals (e.g., mice) to express antibodies containing CDR3 having amino acids encoded by the segment.

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

[0213] Immunoglobulin loci containing human variable region gene segments are publicly known in the art, for example, U.S. Patents No. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, and 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 No. 9,475,559, each of which is incorporated herein by reference in whole, and U.S. Patent Application Publications 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287, and 2015 / 0113668, each of which, The entirety of these is incorporated herein by reference and can be found in PCT applications published 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 are engineered D cells that can be reconstituted in mouse progenitor B cells. H This includes endogenous complete human immunoglobulin transgenes containing the region (Alt et al., 1985, Immunoglobulin genes in transgenic mice, Trends Genet 1:231-236, the whole is incorporated herein by reference). In these embodiments, the manipulated D HThe complete human immunoglobulin transgene, including the region, may be inserted (randomly), and the endogenous immunoglobulin gene may also 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 them is incorporated by reference, the whole is incorporated), for example, the endogenous immunoglobulin heavy chain and kappa light chain loci are inactivated, for example, by targeted deletion of a small but important portion of each endogenous locus, followed by a randomly integrated giant transgene, or by the introduction of the human immunoglobulin gene locus as a minichromosome (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; the entire article is incorporated herein by reference).

[0215] In some embodiments, the manipulated D HThe heavy and light chain loci of human or humanized immunoglobulins, which include the region, are located at the endogenous immunoglobulin heavy and light chain loci, respectively. Methods for large-scale insight gene substitutions of mouse germline immunoglobulin variable loci at human germline immunoglobulin variable loci while maintaining the mouse's ability to reproduce have already been reported. See, for example, U.S. Patents 6,596,541 and 8,697,940, each of which is incorporated herein by reference in whole. Specifically, precise substitutions of six megabases in both the mouse heavy chain and κ-light chain immunoglobulin variable loci with their human counterparts have been described, while preserving the mouse constant region. As a result, mice were generated with precise substitutions of equivalent human germline immunoglobulin variable sequences across their entire germline immunoglobulin variable repertoire, while preserving the mouse constant region. The human variable region is conjugated to the mouse constant region, reconstructed, and forms a chimeric human-mouse immunoglobulin locus expressed at physiologically appropriate levels. The expressed antibodies are "reverse chimeras," meaning they contain both a human variable region sequence and a mouse constant region sequence. These mice, which express antibodies containing both human or humanized variable regions and mouse constant regions, and which possess humanized immunoglobulin variable regions, are called VELOCIMMUNE® mice.

[0216] VELOCIMMUNE® humanized mice exhibit a complete, fully functional humoral immune system that is 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. The antibody populations in these mice reflect isotype distributions resulting from normal class switching (e.g., normal isotype cis-switch). Immunizing VELOCIMMUNE® mice produces 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 naturally affinity-matured human immunoglobulin variable region sequences for producing pharmaceutically acceptable antibodies and other antigen-binding proteins. H Human V gene segment H Substitutions in gene segments have also been shown to produce immune responses containing humanized immunoglobulin variable domains. See, for example, Tien et al., (2016) Cell 166:1471-84, the entire work of which is incorporated herein by reference. By precisely substituting a mouse immunoglobulin variable sequence with a human immunoglobulin variable sequence, the human immunoglobulin variable sequence is operably bound to an endogenous non-human stationary region gene sequence in a reverse chimeric manner, thereby generating VELOCIMMUNE® mice.

[0217] Mice modified in a reverse chimeric manner have human (humanized) variable regions (e.g., including (D), J, and one or more human V gene segments) operably bound to endogenous constant regions at endogenous immunoglobulin loci, for example, (a) In the endogenous heavy chain gene locus: (i) Unreconstituted human (humanized) immunoglobulin heavy chain variable regions operably bound to the endogenous heavy chain constant region, the unreconstituted human (humanized) immunoglobulin heavy chain variable regions are multiple unreconstituted human heavy chain variable regions V HGene segments (e.g., all functional human unreconstructed human V) H (Genetic segment), one or more unreconstituted immunoglobulin heavy chains D H Gene segments, and one or more unreconstituted immunoglobulin heavy chains J H Including gene segments, In some cases, one or more unreconstituted immunoglobulin heavy chains D H Gene segments and one or more unreconstituted immunoglobulin heavy chains J H The gene segment is one or more unreconstituted human immunoglobulin heavy chain D H Gene segment (e.g., all functional human D H (Genetic segment) and / or one or more unreconstituted human immunoglobulin heavy chains J H Gene segment (e.g., all functional human J H (Genetic segment) (ii) A restrictive, unreconstituted human (humanized) heavy chain variable region operably bound to an endogenous heavy chain constant region, the restrictive, unreconstituted human (humanized) heavy chain variable region being one or more unreconstituted immunoglobulin heavy chain D H Gene segments and one or more unreconstituted immunoglobulin heavy chains J H A single, unreconstituted human heavy chain variable region V operably bound to a gene segment. H Essentially consisting of gene segments, and in some cases one or more unreconstituted immunoglobulin heavy chains D H Gene segments and one or more unreconstituted immunoglobulin heavy chains J H Each gene segment contains one or more unreconstituted human immunoglobulin heavy chain D H Gene segments and / or one or more unreconstituted human immunoglobulin heavy chains J H Gene segment, (iii) a histidine-modified, unreconstructed human (humanized) heavy chain variable region operably bound to the endogenous heavy chain constant region, the histidine-modified, unreconstructed human (humanized) heavy chain variable region comprising an unreconstructed immunoglobulin heavy chain variable gene sequence comprising the substitution of at least one non-histidine codon with a histidine codon or the 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 unreconstructed human (humanized) heavy chain variable region operably bound to the endogenous heavy chain constant region, the endogenous heavy chain constant region comprising (1) an intact endogenous IgM gene encoding an IgM isotype associated with the 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 that can covalently bind to the light chain constant domain; and / or (b) In the endogenous light chain locus: (i) Unreconstituted human (humanized) immunoglobulin light chain variable regions operably bound to the endogenous light chain constant region, the unreconstituted human (humanized) immunoglobulin light chain variable regions are multiple unreconstituted human light chain variable regions V L Gene segments (e.g., all functional human unreconstructed human V) L (Genetic segment) and one or more unreconstituted immunoglobulin light chains J L Including gene segments, In some cases, one or more unreconstituted immunoglobulin light chains J L The gene segment is one or more unreconstituted human immunoglobulin light chain J L Gene segment (e.g., all functional human J H (L gene segment) In some cases, the endogenous immunoglobulin light chain locus is the endogenous immunoglobulin light chain kappa (κ) locus, and the unreconstructed human (humanized) immunoglobulin light chain variable region includes the human variable κ (Vκ) and binding κ (Jκ) gene segments, and the endogenous light chain constant region is the endogenous κ chain constant region sequence, and / or the endogenous immunoglobulin light chain locus is the endogenous immunoglobulin light chain lambda (λ), and the unreconstructed human (humanized) immunoglobulin light chain variable region includes the human variable λ (Vλ) gene segment and binding λ (Jλ) gene segment, and the endogenous light chain constant region The endogenous λ chain constant region sequence, optionally comprising (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 bound to (c) and the rodent immunoglobulin light chain constant (Cλ) gene segment, and 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 common light chain coding sequence containing a reconstituted human (humanized) light chain variable region sequence operably bound to the endogenous light chain constant region, the reconstituted human (humanized) light chain variable region sequence is immunoglobulin light chain J L Gene segment and reconstituted human light chain variable region V L Including gene segments, (iii) A restrictive, unreconstituted human (humanized) light chain variable region operably bound to an endogenous light chain constant region, the restrictive, unreconstituted human (humanized) light chain variable region bound to one or more unreconstituted human immunoglobulin light chains (J L ) Two or fewer unreconstituted human immunoglobulin light chain variable (V) operably bound to the gene segment L ) including gene segments, (iv) A non-histidine modified human (humanized) light chain variable region operably bound to an endogenous light chain constant region, the non-histidine modified human (humanized) light chain variable region comprising a non-histidine modified human (humanized) immunoglobulin light chain variable gene sequence in the complementarity-determining region 3 (CDR3) coding sequence, the substitution of at least one non-histidine codon with a histidine codon, or the insertion of at least one histidine codon; or (v) A mouse modified to include a histidine-modified reconstituted human (humanized) light chain variable region operably bound to an endogenous light chain constant region, the histidine-modified reconstituted human (humanized) light chain variable region comprising a reconstituted human (humanized) immunoglobulin light chain variable gene sequence in which at least one non-histidine codon is replaced with a histidine codon, or at least one histidine codon is inserted, in the complementarity-determining region 3 (CDR3) coding sequence, In some cases, the mouse can also (i) The mouse has a human (humanized) immunoglobulin heavy chain locus containing the functional ADAM6 gene so that it exhibits the wild-type reproductive capacity of a non-human animal; and / or (ii) In some cases, at least 10% of the rearranged variable region genes include exogenous terminal deoxynucleotidyltransferase (TdT) genes to increase the diversity of antigen receptors, such that they include additions not present in the template. That mouse has already been described. For example, 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 Publications 20110195454, 20120021409, and 201 See Patent Applications Nos. 20192300, 20130045492, 20150289489, 20180125043, 20180244804, PCT Patent Application Publications Nos. 2019 / 113065, 2017210586, and 2011163314, Lee et al., (2014) Nature Biotechnology 32:356, the entirety of which is incorporated herein by reference.

[0218] In some embodiments, the present invention relates to a genome, in which a bioseries genome is Human V H Gene segment, the human-modified D of the present invention H Genetic regions, and human J H An endogenous immunoglobulin locus containing a variable immunoglobulin heavy chain region including a gene segment, the variable immunoglobulin heavy chain region is operably bound to a constant region, and / or The invention includes a genetically modified non-human animal comprising an endogenous chain locus containing a human VL gene segment and a human JL gene segment, the immunoglobulin light chain variable region being operably bound to a constant region.

[0219] In some embodiments, non-human animals, such as rodents, such as rats or mice, have one or more human V genes in their genome. H , D H , and J H One or more endogenous V at the endogenous immunoglobulin heavy chain locus having segments H , D H , and J H Includes segment substitution, one or more human V H , D H , and J H The segment is operably coupled to a 23-mer RSS of human D H Including gene segments, for example, endogenous non-human light chain loci, endogenous immunoglobulin heavy chain genes; in cases, non-human, for example, rodents, for example, mouse or rat, operably bound to unreconstituted or reconstituted human V L and human J L Segment, or human immunoglobulin light chain constant (C L It is operablely bound to the ) region gene.

[0220] In certain embodiments, a genetically modified non-human animal is a D gene manipulated in its genome, for example, in its germline genome. HAn immunoglobulin variable region comprising one or more unreconstructed human immunoglobulin variable region gene segments containing a region, and an immunoglobulin locus (endogenous or exogenous) containing an immunoglobulin constant region containing an immunoglobulin constant region, wherein one or more unreconstructed human immunoglobulin variable region gene segments are operably bound to the immunoglobulin constant region gene.

[0221] Generally, a genetically modified immunoglobulin locus includes an immunoglobulin variable region (including an immunoglobulin variable region gene segment) operably bound to the immunoglobulin constant region. In some embodiments, the genetically modified immunoglobulin locus includes an engineered D operably bound to the heavy chain constant region gene. H The gene-modified immunoglobulin locus includes one or more unreconstructed human immunoglobulin heavy chain variable region gene segments, including the region. In some embodiments, the gene-modified immunoglobulin locus includes an unreconstructed human immunoglobulin variable region κ gene segment operably bound to the κ chain constant region gene. In some embodiments, the gene-modified immunoglobulin locus includes an unreconstructed human immunoglobulin variable region λ gene segment operably bound to the κ chain constant region gene. In some embodiments, the gene-modified immunoglobulin locus includes an unreconstructed human immunoglobulin variable region λ gene segment operably bound to the λ chain constant region gene.

[0222] In certain embodiments, non-human animals have an engineered D gene operably bound to the endogenous heavy chain constant region at the endogenous heavy chain locus. H The immunoglobulin variable region includes an unreconstituted human (humanized) immunoglobulin heavy chain variable region, and the immunoglobulin variable region contains one or more unreconstituted human Ig heavy chain variable region gene segments. In some embodiments, one or more unreconstituted human Ig variable region gene segments contain 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 unreconstituted human D2 segments operably bound to the 23-mer RSS) H (segment), and one or more immunoglobulin heavy chain binding (J H ) segment (in some cases, one or more unreconstituted human J H (segment) included. In some embodiments, the unreconstructed human Ig variable region gene segment is multiple unreconstructed human V H Segment, one or more unreconstructed (human) D H Segments (e.g., one or more unreconstructed (human) D segments operably coupled to a 23-mer RSS) H (segment) and one or more unreconstituted (human) J H Includes a segment. In some embodiments, the unreconstructed human Ig variable region gene segment contains at least 3 V H Gene segment, at least 18 V H Gene segment, at least 20 V H Gene segment, at least 30 V H Gene segment, at least 40 V H Gene segment, at least 50 V H Gene segment, at least 60 V H Gene segment, at least 70 V H Gene segment, or at least 80 V H Includes a gene segment. In some embodiments, the unreconstructed human Ig gene segment is functional human D H Including all gene segments, functional human D H At least one of the gene segments is modified to operably bind to the 23-mer RSS. In some embodiments, the unreconstructed human Ig gene segment is functional human J HThis includes all gene segments. Exemplary variable regions including the Ig heavy chain gene segment are provided, for example, in Macdonald et al., Proc.Natl.Acad.Sci.USA 111:5147~52 and Supplemental Information, the entirety of which is incorporated herein by reference.

[0223] In some embodiments, the non-human animals provided herein include a restrictive, unreconstructed human (humanized) heavy chain variable region operably bound to an endogenous heavy chain constant region containing at least a non-human IgM gene at an endogenous heavy chain locus, wherein the restrictive, unreconstructed human (humanized) heavy chain variable region is a single human V H gene segment, multiple D H One or more unreconstituted (human) D gene segments (e.g., one or more operably linked to a 23-mer RSS) H Human D, including gene segment H (gene segment) and multiple J H Gene segment (e.g., human J H Characterized by gene segments, restrictive immunoglobulin heavy chain loci can reconstruct and form multiple distal rearrangements, each rearrangement being a single human V H Gene segment, D H One of the segments, and J H Derived from one of the segments, each reconfiguration encodes a different heavy chain variable domain (for example, described in U.S. Patent Application Publication 20130096287, which is incorporated herein by reference in its entirety). In some embodiments, a single human V H The gene segment is V H 1-2 or V H The number is 1-69.

[0224] In certain embodiments, non-human animals include an unreconstituted human (humanized) immunoglobulin light chain variable region operably bound to an endogenous light chain constant region at an endogenous light chain locus. In some embodiments, the unreconstituted human (humanized) immunoglobulin light chain variable region includes an unreconstituted human Igκ variable region gene segment. In some embodiments, the unreconstituted human (humanized) immunoglobulin variable region includes multiple unreconstituted human Vκ segments and one or more unreconstituted human Jκ segments. In some embodiments, the unreconstituted human immunoglobulin variable region gene segment includes all human Jκ segments. In some embodiments, the immunoglobulin variable region gene segment includes four functional Vκ segments and all human Jκ segments. In some embodiments, the immunoglobulin variable region gene segment includes 16 functional Vκ segments and all human Jκ segments (e.g., all functional human Vκ and Jκ segments). In some embodiments, the unreconstituted human immunoglobulin variable region gene segment includes all human JVκ segments and all human Jκ segments. Exemplary variable regions, including the Igκ gene segment, are provided, for example, in Macdonald et al., Proc.Natl.Acad.Sci.USA 1 11:5147~52 and Supplementary Information, the entirety of which is incorporated herein by reference.

[0225] In some embodiments, a restrictive, unreconstructed human (humanized) light chain variable region operably coupled to an endogenous light chain steady region is defined as having two or fewer human V L Gene segments and multiple J L Characterized in that it includes a gene segment (for example, the dual light chain mouse, or DLC, described in U.S. Patent No. 9,796,788, which is incorporated herein by reference in its entirety). In some embodiments, V L The gene segment is the Vκ gene segment. In some embodiments, V LThe gene segment is the Vλ gene segment. In some embodiments, the Vκ gene segment is IGKV3-20 and IGKV1-39. In some embodiments, the non-human animal includes, strictly speaking, two unreconstituted human Vκ gene segments and five unreconstituted human Jκ gene segments operably bound to the mouse light chain constant region at the mouse endogenous κ light chain locus, and optionally, the two strictly unreconstituted human Vκ gene segments are the human Vκ1-39 gene segment and the human Vκ3-20 gene segment, and the five unreconstituted human Jκ gene segments are the human Jκ1 gene segment, the human Jκ2 gene segment, the human Jκ3 gene segment, the human Jκ4 gene segment, and the human Jκ5 gene segment, and the unreconstituted human kappa light chain gene segments have the ability to reconstitute and encode the human variable domain of the antibody, and optionally, the non-human animal does not include an endogenous Vκ gene segment that has the ability to reconstitute to form an immunoglobulin light chain variable region.

[0226] In certain embodiments, an unreconstituted human (humanized) immunoglobulin light chain variable region operably bound to an endogenous light chain constant region contains an unreconstituted human Igλ variable region gene segment. In some embodiments, the unreconstituted human immunoglobulin variable region gene segment includes multiple human Vλ segments and one or more human Jλ segments. In some embodiments, the unreconstituted human immunoglobulin variable region gene segment includes 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 unreconstituted human immunoglobulin variable region gene segment includes all of the human Vλ segments. In some embodiments, the unreconstituted human immunoglobulin variable region gene segment includes all of the human Jλ segments. Exemplary variable regions containing Igλ gene segments are provided, for example, in U.S. Patents 9,035,128 and 6,998,514, which are incorporated herein by reference in their entirety. In some embodiments, an unreconstituted human (humanized) immunoglobulin light chain variable region operably bound to an 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 bound 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, an unreconstituted human (humanized) immunoglobulin light chain variable region operably bound to an endogenous light chain constant region includes an unreconstituted human Igλ variable region gene segment operably bound to an endogenous (e.g., rodent, e.g., rat or mouse) Cκ gene, such that a non-human animal expresses an immunoglobulin light chain containing human λ variable domain sequences derived from Vλ and Jλ gene segments fused to an endogenous κ constant domain, for example, see U.S. Patent No. 9,226,484, the whole of which is incorporated herein by reference.

[0228] In some embodiments, the immunoglobulin variable region, which includes an unreconstructed human immunoglobulin variable region gene segment, also includes a human immunoglobulin variable region intergene sequence. In some embodiments, the immunoglobulin variable region includes a non-human (e.g., rodent, rat, mouse) Ig variable region intergene sequence. In some embodiments, the intergene sequence is an endogenous species-derived intergene sequence.

[0229] In some embodiments, the immunoglobulin variable region is a reconstituted light chain variable region (a general light chain variable region). In some embodiments, the reconstituted Ig light chain variable region gene is a human reconstituted Ig light chain variable region gene. Typical reconstituted Ig light chain variable regions are provided, for example, in U.S. Patents 9,969,814, 10,130,181, and 10,143,186, and U.S. Patent Publications 20120021409, 20120192300, 20130045492, 20130185821, 20130302836, and 20150313193, which are incorporated herein by reference in their entirety. In some embodiments, a non-human organism containing a general light chain variable region ("general light chain" organism) is used to produce a bispecific antibody. In some embodiments, the common light chain coding sequence comprises a single reconstituted human immunoglobulin light chain Vκ / Jκ sequence operably bound to an endogenous light chain constant region, the single reconstituted human immunoglobulin light chain Vκ / Jκ sequence being either (i) a human Vκ1-39 / Jκ5 sequence containing a human Vκ1-39 gene segment fused to a human Jκ5 gene segment, or (ii) a human Vκ3-20 / Jκ1 sequence containing a human Vκ3-20 gene segment fused to a human Jκ1 gene segment.

[0230] In some embodiments, the immunoglobulin variable region is a light-chain and / or heavy-chain immunoglobulin variable region, including the insertion and / or substitution of a histidine codon designed to introduce pH-dependent binding properties to antibodies produced in such non-human organisms. In some of these embodiments, the histidine codon is inserted into and / or substituted into a nucleic acid sequence encoding CDR3. Various such light-chain and / or heavy-chain immunoglobulin loci are provided in U.S. Patents 9,301,510, 9,334,334, and 9,801,362, and U.S. Patent Application Publication 20140013456, which are incorporated herein by reference in their entirety. In some embodiments, the histidine-modified reconstituted human (humanized) light chain variable region operably bound to the endogenous light chain constant region comprises a single reconstituted human immunoglobulin light chain variable region gene sequence including human Vκ and Jκ segment sequences, wherein the Vκ segment sequence is derived from the human Vκ1-39 or Vκ3-20 gene segment, and the single reconstituted human immunoglobulin light chain variable region gene sequence comprises the 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 non-histidine-modified reconstituted human (humanized) heavy chain variable region operably bound to the endogenous heavy chain constant region comprises a complementarity-determining region 3 (CDR3) coding sequence (e.g., modified to operably bind to the RSS of 23mer (human) D H The gene segment includes an unreconstructed human (humanized) immunoglobulin heavy chain variable gene sequence, which includes at least one substitution of a non-histidine codon with a histidine codon, or at least one insertion of a histidine codon. In some embodiments, the unreconstructed human (humanized) immunoglobulin heavy chain variable gene sequence is an unreconstructed human V H , operably coupled to the 23mer RSS H Unreconstructed human D2 including segments H or composite D H, and unreconstituted human J H Unreconstituted human D2, containing a gene segment and optionally operably linked to a 23-mer RSS. H or composite D H Gene segment or D H The gene segment includes the substitution of at least one non-histidine codon with a histidine codon, or the insertion of at least one histidine codon. In some embodiments, the histidine-modified, unreconstituted human (humanized) light chain variable region operably bound to the endogenous heavy chain constant region is unreconstituted V L and unreconstructed J L Includes gene segments. In some embodiments, the unreconstructed human (humanized) light chain variable regions include two or fewer unreconstructed human V L (For example, two or fewer Vκ gene segments) and one or more unreconstructed human J L (For example, containing the Jκ) gene segment, and containing two or fewer human V L Each gene segment contains, in its CDR3 coding sequence, at least one substitution of a non-histidine codon with a histidine codon, or at least one insertion of a histidine codon. In some embodiments, two or fewer unreconstructed human Vκ gene segments are human Vκ1-39 and human Vκ3-20 gene segments, each containing one or more substitutions of a non-histidine codon with a histidine codon, and the human Vκ and Jκ gene segments are capable of reconstruction, and the human Vκ and human Jκ gene segments encode one or more histidine-containing human light chain variable domains at positions selected from the group consisting of 105, 106, 107, 108, 109, 111 (according to IGMT numbering) and combinations thereof, where one or more histidines result from one or more substitutions.

[0231] In some embodiments, the immunoglobulin constant region includes 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 an endogenous species-derived heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a mouse or 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 as well as non-human (e.g., endogenous species-derived, mouse, rat) CH2 and / or CH3 regions. In some embodiments, the heavy chain constant region gene is a Cμ, Cδ, Cγ (Cγ1, Cγ2, Cγ3, Cγ4), Cα, or Cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region gene encodes a mutated CH1 region so that non-human animals express antibodies of the heavy chain only (see, for example, U.S. Patent No. 8,754,287, U.S. Patent Application Publication 2015 / 0289489, which is incorporated herein by reference in its entirety). In some embodiments, for example, where the objective is to generate a heavy chain to produce a bispecific antibody (in common or dual light-chain organisms), the Fc domain of the heavy chain includes modifications to promote heavy chain heterodimerization and / or inhibit heavy chain homodimerization. Such modifications are provided, for example, in U.S. Patents No. 5,731,168, 5,807,706, 5,821,333, 7,642,228 and 8,679,785, and U.S. Patent Application Publication 2013 / 0195849, which is incorporated herein by reference in its entirety.

[0232] In some embodiments, the immunoglobulin constant region includes a light chain constant region gene. In some embodiments, the light chain constant region gene is a κ constant region gene. In some embodiments, the light chain constant region gene is a λ constant region gene. In some embodiments, the light chain constant region gene is an endogenous species-derived light chain constant region gene. 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 containing the human variable region gene segment, and the immunoglobulin constant region gene to which the variable region gene segment is operably bound, 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 bound is an endogenous constant region gene.

[0234] In some embodiments, one or more endogenous immunoglobulin loci in the genome of a non-human animal provided herein, or a portion of one or more endogenous loci (e.g., a variable region and / or a constant region), are inactivated. Endogenous immunoglobulin variable region gene agents and portions thereof may be inactivated using any method known in the art, but not limited to, including deletion of the locus or a portion thereof from the genome of an organism, substitution of the locus or a portion thereof with a different nucleic acid sequence, inversion of a portion of the locus, and / or relocation of a portion of the locus to another location in the genome of a non-human organism. In some embodiments, the inactivation of the locus is only partial inactivation. In some embodiments, the variable region of the locus is inactivated, but the constant region retains its functionality (e.g., because it is operably bound to a non-endogenous variable region gene segment).

[0235] In some embodiments, the genetically modified non-human animal contains an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus or a portion thereof is inactivated by deletion, substitution, migration 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, substituted, migrated and / or inverted includes the J segment of the variable region. In some embodiments, the endogenous immunoglobulin heavy chain locus or a portion thereof is inactivated by deletion, substitution, migration 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, substituted, migrated and / or inverted includes the Oμ gene of the endogenous constant region.

[0236] In some embodiments, the genetically modified non-human animal contains an inactivated endogenous immunoglobulin κ chain locus. In some embodiments, the endogenous immunoglobulin κ chain locus or a portion thereof is inactivated by deletion, substitution, migration and / or inversion of at least a portion of the endogenous variable region of the endogenous κ chain locus. In some embodiments, at least a portion of the variable region of the endogenous κ chain locus that is deleted, substituted, migrated and / or inverted includes the J segment of the variable region. In some embodiments, the endogenous immunoglobulin κ chain locus or a portion thereof is inactivated by deletion, substitution, migration and / or inversion of at least a portion of the endogenous constant region of the endogenous κ chain locus. In some embodiments, at least a portion of the constant region of the endogenous κ chain locus that is deleted, substituted, migrated and / or inverted includes the Cκ gene of the endogenous constant region.

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

[0238] In various embodiments, immunoglobulin locus modifications do not affect the reproductive capacity of non-human animals. In some embodiments, the heavy chain loci include functional, e.g., endogenous ADAM6a, ADAM6b, or both, and the gene modification does not affect the expression and / or function of endogenous ADAM6a, ADAM6b, or both. In some embodiments, the genome of the genetically modified non-human animal further includes ectopically located functional, e.g., endogenous ADAM6a, ADAM6b, or both. Typical non-human animals expressing exogenous ADAM6a and / or ADAM6b are described in U.S. Patents 8,642,835 and 8,697,940, each of which is incorporated herein by reference in whole.

[0239] In some embodiments, genetically modified non-human animals further contain and express exogenous terminal deoxynucleotidyltransferase (TdT) genes due to increased antigen receptor diversity. A typical non-human animal expressing exogenous TdT is described in PCT Patent Application Publication 2017210586, which is incorporated herein by reference in its entirety.

[0240] In some embodiments, the genome of the non-human animal provided further includes one or more human immunoglobulin heavy and / or light chain genes (see, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, and U.S. Patent Application Publications 2013 / 0096287A1 and 2018 / 0125043A1, and PCT Application Publication 2019 / 113065, each incorporated herein by reference in its entirety). Alternatively, manipulated D H The region can be introduced into different modified lower embryonic stem cells, such as the VELOCIMMUNE® strain (see, for example, U.S. Patent No. 8,502,018 or U.S. Patent No. 8,642,835, the whole of which is incorporated herein by reference). In some embodiments, the non-human animals described herein can be prepared by introducing the targeting vector described herein into cells derived from a modified strain. For example, the targeting vector described herein may be introduced into the non-human animals described in U.S. Patent Nos. 8,642,835 and 8,697,940, the whole of which is incorporated herein by reference, and the non-human animals express antibodies having a complete human variable region and a mouse constant region. In some embodiments, the non-human animals described herein are prepared to further include human immunoglobulin genes (variable region genes and / or constant region genes). In some embodiments, the non-human animals described herein are prepared to further include the manipulated D described herein. H The genetic material includes regions and genetic material of heterologous (e.g., human) origin, and the genetic material encodes one or more human heavy chain variable regions and / or light chain variable regions, either in whole or in part.

[0241] The non-human animals described herein may, in many cases, be prepared as described above or by methods known in the art to include additional human or humanized genes, depending on the intended use of the non-human animal. Such additional human or humanized gene genetic material may be introduced as described above or by reproductive techniques known in the art using other desired genetically modified subgenos, through further modification of the genome of genetically modified cells (e.g., embryonic stem cells).

[0242] For example, operated D as described herein H Non-human animals including the region are incorporated herein by reference in their entirety by U.S. Patent Application Publication No. 2011-0195454. A1, No. 2012-0021409 A1, No. 2012-0192300 A1, No. 2013-0045492 A1, No. 2013-0185821 A1, No. 2013-0198880 A1, No. 2013-0302836 A1, No. 2015-0059009 A1; further including one or more modifications (e.g., via hybridization or multiple gene targeting strategies) described as International Patent Application Publication Nos. 2011 / 097603, 2012 / 148873, 2013 / 134263, 2013 / 184761, 2014 / 160179, and 2014 / 160202.

[0243] Transgenic founder non-human animals have manipulated D in their genomes. H The presence of the region and / or in the tissues or cells of non-human animals D H -D H Identification can be made based on the expression of antibodies containing the CDR3 region, which contains amino acids resulting from the recombination. Next, using transgenic founding non-human animals, the manipulated D H They were crossbred with further non-human animals that possessed the region, thereby producing one or more manipulated copies of each D. H A series of non-human animals having regions can be generated. Furthermore, manipulated D HTransgenic non-human animals possessing the region can be further crossbred with other transgenic non-human animals possessing other desired transgenes (e.g., human immunoglobulin genes).

[0244] Transgenic non-human animals may also be generated to contain selected systems that enable the control or direction of transgene expression. Typical systems include the Cre / loxP recombinase system of bacteriophage P1 (e.g., see Lakso, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236, the whole of which is incorporated herein by reference) and the FLP / Frt recombinase system of budding yeast (O'Gorman, S. et al., 1991, Science 251:1351-1355, the whole of which is incorporated herein by reference). Such animals may, for example, have one selected modification (e.g., manipulated D H This can be provided by constructing a "double" transgenic animal by crossing two transgenic animals, one containing an introduced gene that includes a specific region, and the other containing an introduced gene that encodes a recombinase (e.g., Cre recombinase).

[0245] Mouse (i.e., Human V) H and J H Manipulated D, which is operably bound to gene segments, all of which are operably bound to one or more mouse heavy chain constant region genes. H In mice with a region, the manipulated D H Embodiments utilizing the region are discussed extensively in this specification, but the manipulated D H Other non-human animals including the region are also provided. In some embodiments, such non-human animals are endogenous V H and J H Manipulated D operably bound to gene segments H Includes the region. In some embodiments, such non-human animals are humanized. H and J H Manipulated D operably bound to gene segmentsH This includes the region. Such non-human animals include, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, uncastrated males, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys), and others, which have increased frequency of D as wild-type immunoglobulin heavy chain loci disclosed herein. H -D H This includes any organism that can be genetically modified to express an antibody having CDR3 containing amino acids resulting from recombination. For example, in non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are used to produce non-human animals containing genetic modifications. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblasts or induced pluripotent cells), introducing the genetically modified genome into suitable cells, such as enucleated oocytes, using somatic cell nuclear transfer (SCNT), and implanting the modified cells (e.g., modified oocytes) 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, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., the CRISPR / Cas system) to modify the manipulated D described herein. H The process includes modifying the genome to include a region. Guidelines for methods of modifying germline genomes of non-human animals can be found, for example, in U.S. Patent Publications 2015-0376628A1, 2016-0145646A1, and 2016-0177339A1, which are incorporated herein by reference in their 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, e.g., small mammals of the superfamily Derboloid or Muroidoid. 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 superfamily Muroidoid. In some embodiments, the genetically modified animals described herein are derived from families selected from the families of the family Myctophidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (purebred mice and rats, gerbils, spiny mice, maned mice), Dolichophidae (tree mice, rock mice, white-tailed rats, Madagascar rats and mice), Glididae (e.g., spiny dormice), and Muridae (e.g., blind mice, bamboo mice, and highland mole mice). In some embodiments, the genetically modified rodents described herein are selected from purebred mice or rats (Muridae), gerbils, spiny mice, and maned mice. In some embodiments, the genetically modified mice described herein are derived from members of the Muridae family. In some embodiments, the non-human animals described herein are rodents. In some embodiments, the rodents described herein are selected from mice and rats. In some embodiments, the non-human animals described herein are mice.

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

[0249] In some embodiments, the non-human animal described herein is a rat. In some embodiments, the rat described herein is selected from Wistar rat, LEA strain, Sprague Dawley strain, Fisher strain, 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, Fisher, F344, F6, and Dark Agouti.

[0250] method Various in vitro and in vivo technologies have been developed to generate antibody-based therapeutics. In detail, in vivo technologies have characterized the generation of transgenic animals (i.e., rodents) possessing human immunoglobulin genes, in which the genes are either randomly integrated into the animal's genome (see, e.g., U.S. Patent No. 5,569,825, the entirety of which is incorporated herein by reference) or precisely positioned at endogenous immunoglobulin loci operably bound to the animal's endogenous immunoglobulin constant region (see, e.g., U.S. Patents No. 8,502,018, 8,642,835, 8,697,940, and 8,791,323, the entirety of which is incorporated herein by reference). Both approaches have yielded good results in generating antibody therapeutic candidates with potential for human use. Furthermore, both approaches are superior to in vitro approaches in that antibody candidates are selected from an in vivo-generated antibody repertoire, and this selection includes the selection of affinity and specificity for the antigen within the internal environment of the host immune system. Thus, the antibodies bind to naturally presented antigens (within the relevant biological epitopes and on the surface) rather than to artificial environments or in silico predictions that may accompany in vitro technologies. Despite the robust antibody repertoire obtained from in vivo technologies, antibodies against complexes (e.g., viruses, channel polypeptides) or cytoplasmic antigens remain challenging. Moreover, 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] Therefore, the present invention, in particular, provides for novel 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 diversity of CDRs, particularly CDR3, generated from (recombination to) one or more D2s operably bound to a 5' or 3' 23-mer RSS HThis is based on the understanding that it can be fabricated using segments. By having a 5' or 3' 23-mer RSS, such an operated D H Compared to immunoglobulin heavy chain loci lacking gene segments, D H Recombination between segments increases. Such added diversity can direct binding to specific antigens (e.g., viruses, channel polypeptides). H gene segment, at least two D H gene segment, and J H During the 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. Furthermore, D H From D H This CDR3 resulting from recombination contains further diversity due to the 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 produced by conventional VDJ recombination.

[0252] The non-human animals provided may be used to produce human antibodies, which include 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 non-human animals provided are immunized with the antigen of interest (e.g., a virus or channel polypeptide, whole or in part) under conditions and for a sufficient amount of time to induce an immune response to the antigen of interest. The 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 to 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 non-human animals provided include one or more human variable domains derived from one or more human variable region nucleotide sequences isolated from the non-human animals. In some embodiments, anti-drug antibodies (e.g., anti-idiotype antibodies) may be produced in the non-human animals provided.

[0253] The non-human animals described herein provide improved in vivo systems and sources of biological materials (e.g., cells) for producing human antibodies useful for various assays. In some embodiments, the provided non-human animals are used to develop therapies 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 therapies 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 therapeutic agents (e.g., antibodies, siRNA, etc.) that bind to one or more viruses, channels, or G protein-coupled receptor (GPCR) polypeptides. In some embodiments, the provided non-human animals are used to screen and develop candidate therapeutic agents (e.g., antibodies, siRNA, 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 properties of antagonists to one or more GPCR polypeptides or one or more human channel polypeptides. In some embodiments, a provided non-human animal is used to determine the epitopes or multiple epitopes of one or more candidate therapeutic antibodies that conjugate one or more human GPCR polypeptides or one or more human channel polypeptides.

[0254] In some embodiments, the pharmacokinetic profile of the antibody is determined using a provided non-human animal. 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 antibody may be administered via any preferred route of administration, including parenteral and oral routes. Parenteral routes include, for example, intravenous, intra-arterial, intra-portal, intramuscular, subcutaneous, intraperitoneal, intrathecal, subarachnoid, lateral ventricle, intracranial, intrathoracic, or other routes of administration. Oral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, oral cavity, vaginal, and ocular. Administration may also be by continuous infusion, local administration, sustained release from implants (gel, membrane, etc.), and / or intravenous injection, for example, using intravenous fluid bags. Blood is isolated from 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, including but not limited to total IgG, anti-therapeutic antibody response, and agglutination, may be used to determine the pharmacokinetic properties of the administered candidate therapeutic antibody using feed obtained from the non-human animals described herein.

[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 as a result of cellular changes or on the cell surface density of the target antigen (in the case of a cell surface receptor) 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 period of time, the effect on the target antigen-dependent process (or interaction), such as ligand-receptor interaction or antigen-related signaling, is analyzed.

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

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

[0258] In some embodiments, the non-human animal-derived cells provided may be isolated ad-hoc and used, or maintained in culture over multiple generations. In some embodiments, the non-human animal-derived cells provided may be immortalized (e.g., via the use of a virus) and maintained indefinitely in culture (e.g., in a continuous culture).

[0259] In some embodiments, the non-human animals described herein provide an in vivo system for generating antibody variants that bind to human target antigens. Such variants include antibodies that have desired functionality, specificity, and low cross-reactivity to a common epitope shared by two or more human target antigens. In some embodiments, the provided non-human animals are used to generate a panel of antibodies for producing 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 transplanted into different Fc regions based on desired effector function 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 a drug or vaccine. In some embodiments, a candidate drug or vaccine is delivered to one or more provided non-human animals, which may then be monitored to determine one or more of the immune response to the drug or vaccine, the safety properties of the drug or vaccine, or the effect on one or more symptoms of a disease or condition and / or a disease or condition. Typical methods used to determine safety properties include measuring toxicity, optimal dose concentration, antibody (i.e., anti-drug) response, efficacy of the drug or vaccine, and potential risk factors. Such drug or vaccine may be improved and / or developed in such non-human animals.

[0262] The effectiveness of a vaccine may be determined in several ways. Briefly, the non-human animals described herein are vaccinated using methods known in the art, and then the vaccine is loaded or administered to already infected non-human animals. The effectiveness of the vaccine may be determined by measuring the response of the non-human animals to the vaccine by monitoring the non-human animals (or cells isolated therefrom) and / or performing one or more assays thereon. The response of the non-human animals to the vaccine is then compared to 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 viral neutralization assay. Briefly, the non-human animals described herein are immunized, and serum is collected at various postimmunization dates. Serial dilutions of the serum are pre-incubated with the virus, during which time antibodies in the serum that are specific to the virus bind to the virus. The virus / serum mixture is then added to tolerant cells, and infectivity is determined by a plaque assay or microneutralization assay. If the antibodies in the serum neutralize the virus, there will be fewer plaques or lower relative luciferase units compared to the control group.

[0264] The non-human animals described herein provide an improved in vivo system for the development and characterization of antibody-based therapies for use in cancer and / or inflammatory diseases. Inflammation has been associated with cancer (for example, outlined in Grivennikov, SI et al., 2010, Cell 140:883-99; Rakoff-Nahoum, S., 2006, Yale J. Biol. Med. 79:123-30, the whole of which is incorporated herein by reference). Indeed, the developing tumor environment is characterized in part by the infiltration of various inflammatory mediators. Furthermore, persistent inflammation can lead to developing cancer with a higher probability. Therefore, 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 therapies. In some embodiments, a non-human animal or control non-human animal (e.g., having different or no genetic modifications than those described herein, i.e., wild-type) may be transplanted with a tumor (or tumor cells) and subsequently administered 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 may comprise a combination therapy, such as the administration of two or more monospecific antibodies administered sequentially or simultaneously. The tumor may be left untreated for a sufficient time to establish itself at one or more locations within the non-human animal before being administered one or more candidate therapeutics. Tumor cell proliferation, growth, survival, etc., may be measured both before and after administration of the candidate therapeutics. The cytotoxicity of the candidate therapeutics may also be measured in the non-human animal, if necessary.

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

[0266] Other features of the present invention will become apparent in the process of describing the following exemplary embodiments, which are given for illustrative purposes and are not intended to limit them. [Examples]

[0267] The following examples are provided to those skilled in the art to illustrate how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. Unless otherwise indicated, temperatures are given in Celsius and pressures are atmospheric pressure or near atmospheric pressure.

[0268] Example 1. Design and construction of targeting vectors This embodiment describes the construction of a targeting vector for insertion into the genome of a non-human animal, such as a rodent (e.g., mouse). In particular, the method described in this embodiment involves the genome being manipulated D H Each region is operably coupled to one or more D2s of 5' or 3' 23' RSS. H Manipulated heavy chain diversity (D) including segments HThis describes the generation of targeting vectors for insertion into the genome of rodents (e.g., mice) that include an immunoglobulin heavy chain variable region containing the ) region. In this example, three targeting vectors are described: operably bound to the 5' 23mer RSS and one human J H Proximal (or 3')D operably coupled to the segment H Operated D including segments H A first targeting vector containing a region, operably bound to the 5' 23-mer RSS, and three human J H Proximal (or 3')D operably coupled to the segment H Operated D including segments H A second targeting vector containing the region, and six human J, each associated with the 3' 23-mer RSS. H Three D segments operably coupled H Operated D including segments H A third targeting vector containing the region (Figure 2). Each of these targeting vectors was generated and separately inserted into the variable region of the humanized immunoglobulin heavy chain (Figures 3, 5, 7). D was manipulated as described below. H The region is heavy chain variable (V H ) and heavy chain bonds (J H ) Segments are operably coupled, and when VDJ rearrangement, D H -D H Antibodies containing CDR3, which was derived from recombinant DNA, were expressed.

[0269] For insertion into the variable region of immunoglobulin heavy chains, one or more human D23s associated with a 5' or 3' RSS, respectively. H Targeting vectors containing segments were generated using VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251, and Valenzuela et al., 2003, Nature Biotech. 21(6):652-659, the entire text of which is incorporated herein by reference) and molecular biological techniques known in the art. Any DH Segment, D H A 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 vectors (Figure 2) In short, the first targeting vector is applied to multiple human D H Segments, and Human D H Synthetic human D located at location 7-27 H Constructed using a genomic DNA fragment containing a 3-3 segment. A donor for in vitro Cas9 / GA modification was prepared by novel synthesis (Blue Heron Bio), and the 5' to 3' segment was modified: (a)D H (b) A 100 bp homologous arm starting 350 bp upstream of the 12-mer RSS at 5' of 7-27, (b) AgeI and XhoI sites for insertion of a neomycin-resistant cassette, (c) D H (d) 250bp region upstream of the 12 RSS at the 5' end of 7-27, (d) 23mer RSS at the 5' end (J H 4 (derived from) and the 12-mer RSS (D) at the 3' end H Synthetic human D (derived from 3-3) H 3-3 segments, and (3)J H It contains a 100 bp homologous box starting 3 bp downstream of 5. The loxp-UbC-Em7-Neo-loxp cassette, for example, allows selection of neomycin resistance genes with loxP site-specific recombination recognition sites adjacent to AgeI and XhoI sites in E. coli and mouse ES cells, through synthesis D H It was placed approximately 250 bp upstream and downstream of the segment. This 23:D contains neomycin cassette. H 3-3:12 / J HUsing six donors (SEQ ID NO: 61), the BAC was modified in vitro with Cas9 / GA using two Cas9:gRNA complexes. Prior to modification, the BAC was sequenced identically to the chimeric IgH locus in VELOCIMMUNE® mice (e.g., J H The VELOCIMMUNE® mouse exhibits an exemplary, non-limiting endogenous immunoglobulin heavy chain locus, heterozygous for 6394 alleles lacking a gene segment and containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence, and for 1460 alleles containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence (see Figures 3 and 4). Specifically, BAC is heterozygous for the most proximal human V H Gene (V H Starting with 6-1), 27 human D H All 6 human genes H The gene, mouse IgH intron enhancer (Eμ), mouse IgM switch region (Sμ), and 1460 alleles including the first four exons of the mouse IgM gene were identical. The BAC also contained a spectinomycin (spec) resistance cassette and approximately 29kb of V H It contained human intergenetic sequences upstream from gene 6-1. Human-mouse binding was human J H The 6 genes have a 222 bp 3' end and a 490 bp 5' end in the mouse Eμ enhancer. GA-mediated 23:D3-3:12 / J H Insertion of 6 donor cells into BAC is D H Substitution and J in the 23:D3-3:12 segment, which was manipulated in the 7-7 gene segment. H 1, J H 2, J H 3, J H 4, and J H The final version 23:D3-3:12 / J includes deletions of 5 gene segments. H Six targeting vectors were generated (Figure 2). Table 1 is 23:D. H 3-3:12 / J H We provide the sequences of the gRNAs, primers, and probes used to determine the precise construction of the 6 targeting vectors. [Table 1]

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

[0272] B.23:D H 3-3:12 / J H 4-6 Targeting vectors (Figure 2) In a similar manner, the first targeting vector (23:D H 3-3:12 / J H 4-6) Multiple human D H Segments, and Human D H Synthetic human D located at location 7-27 H It was constructed using the same genomic DNA fragment containing 3-3 segments. However, this second targeting vector was constructed using six human J H Three of the segments (i.e., J H 4, J H 5, J H It included 6). Final 23:D H 3-3:12 / J H 4-6 To generate targeting vectors, donors were used to modify the BAC, which has the same sequence as the chimeric IgH gene locus of VELOCIMMUNE® mice (e.g., J HThe VELOCIMMUNE® mouse exhibits an exemplary, non-limiting endogenous immunoglobulin heavy chain locus, heterozygous for 6394 alleles lacking a gene segment and containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence, and for 1460 alleles containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence (see Figures 3 and 4). The donor locus is located at 5' to 3: (a) a 100 bp homologous arm starting at 350 bp upstream of the 12-mer RSS at the 5' end of D7-27, (b) AgeI and XhoI sites for neomycin resistance cassette insertion, (c) a 250 bp region upstream of the 12-mer RSS at the 5' end of D7-27, and (d) a 23-mer RSS at the 5' end (J H 4 (derived from) and the 12-mer RSS (D) at the 3' end H Synthetic human D (derived from 3-3) H 3-3 segments, and (e)J H It contained a 100bp homologous box starting 3bp downstream of 3. The loxp-UbC-Em7-Neo-loxp cassette, for example, a neomycin resistance gene with a loxP site-specific recombination recognition site adjacent to it, was synthesized by ligation to the AgeI and XhoI sites to enable selection in E. coli and mouse ES cells. H It was placed approximately 250 bp upstream of the segment. This 23:D3-3:12 / J contains the nucleotide sequence described as SEQ ID NO: 52, including the neomycin cassette. H Using 4-6 donors, we modified BACs identical to 1460 alleles (Figures 3 and 4). Specifically, the BACs were modified to match the most proximal human V H Gene (V H Starting with 6-1), 27 human D H All 6 human genes H The gene, mouse IgH intron enhancer (Eμ), mouse IgM switch region (Sμ), and 1460 alleles including the first four exons of the mouse IgM gene were identical. The BAC also contained a spectinomycin (spec) resistance cassette and approximately 29kb of V HIt contained human intergenetic sequences upstream from gene 6-1. Human-mouse binding was human J H The 3' portion of the 6 genes is 222 bp, and the 5' portion of the mouse Eμ enhancer is 490 bp. In vitro Cas9 / GA-mediated 23:D3-3:12 / J using two Cas9:gRNA complexes. H Insertion of 4-6 donor cells into BAC is D H Substitution and J in the 23:D3-3:12 segment, which was manipulated in the 7-7 gene segment. H 1, J H 2, and J H The final version 23:D3-3:12 / J includes deletions of three gene segments. H 4-6 targeting vectors (Figure 2) were generated. Table 2 is 23:D H 3-3:12 / J H We provide the sequences of the gRNAs, primers, and probes used to determine the precise construction of the 6 targeting vectors. [Table 2]

[0273] 23:D3-3:12 / J H 4-6 Targeting vectors are also linearized with NotI and are heterozygous to the variable region of humanized immunoglobulin heavy chains (i.e., multiple human V vectors operably bound to the constant region of rodent immunoglobulin heavy chains, including rodent heavy chain enhancers and regulatory regions). H , D H , and J H A first heavy chain allele (1460 het) containing a segment and 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 incorporated herein by reference in its entirety); and multiple human V H and D H Segment, J HMouse embryonic stem cells having a genome homozygous (HO) for a humanized endogenous κ locus, containing a second heavy chain allele (6394het) comprising a rodent immunoglobulin heavy chain constant region including a region deletion and rodent heavy chain enhancer and regulatory region, and an inserted nucleotide sequence encoding one or more mouse Adam6 genes (e.g., U.S. Patents 8,642,835 and 8,697,940, each incorporated herein by reference in its entirety), and a complete repertoire of human immunoglobulin light chain Vκ and Jκ gene segments operably bound to the endogenous immunoglobulin heavy chain Cκ region (1293), were electroporated (see Figure 5). 6797 alleles were electroporated, and 1460 alleles were electroporated, and 23:D3-3:12 / J containing a neomycin cassette. H These are generated by appropriate homologous recombination with 4-6 targeting vectors (Figure 5). Using these manipulated mouse ES cells heterozygous for the 6394 and 6797 alleles, efficient screening of positive ES clones (see below) and subsequent cre-mediated removal of drug resistance cassettes are promoted. Hereafter, deletions of the 6394 and 6797 alleles will be 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 vectors (Figure 2) In short, the third targeting vector consists of three D vectors, each having 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 Multiple D including 2-15) H It was constructed using DNA fragments containing human segments. First, a BAC (e.g., J) with the same sequence as the chimeric IgH gene locus of VELOCIMMUNE® mice was constructed. HThe VELOCIMMUNE® mouse exhibits an exemplary, non-limiting endogenous immunoglobulin heavy chain locus, heterozygous for 6394 alleles lacking a gene segment and containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence, and for 1460 alleles containing a humanized variable region operably bound to the endogenous mouse immunoglobulin heavy chain constant region sequence (see Figures 3 and 4). Specifically, BAC is heterozygous for the most proximal human V H Gene (V H Starting from 6-1), the first human D for selection in E. coli and mouse ES cells H Segment (D H 1-1) A loxp-UbC-Em7-Neo-loxp cassette, 512 bp upstream of the gene, adjacent to a loxP site-specific recombination recognition site, modified using bacterial homologous recombination (BHR) for insertion into a neomycin resistance gene, comprising all 27 human D13 genes. H Genes, all 6 types of human J H The BAC was identical to 1460 alleles, including the mouse IgH intron enhancer (Eμ), the mouse IgM switch region (Sμ), and the first four exons of the mouse IgM gene. This obtained BAC was then modified with three donors using in vitro Cas9 / GA. 1. From 5' to 3', D H A 50bp homologous box starting 1419bp upstream of the 5' end of the 2-2 RSS, with multiple cloning sites (MreI-NsiI-EcoRI-KpnI-MreI), Human V H A modified D2-2 gene having a 12-mer RSS at the 3' end that is replaced by a 23-mer RSS at the putative 3' end derived from 1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC;T->A, G->C;SEQ ID NO: 151), and containing a 50bp homologous box ending 863bp downstream of the 23-mer RSS at the 3' end of D2-2, H The first donor for modification 2-2. D H The nucleotide sequence of this first donor for the 2-2 modification is described as SEQ ID NO: 70.H The region consists of four direct repeats totaling approximately 10kb, making the design of unique primers and probes for screening extremely challenging. To overcome this problem, two unique 40bp sequences were inserted; one 74bp downstream of the 5' homologous box and the other 40bp upstream of the 3' homologous box. These unique 40mers were used as binding sites for PCR / sequencing primers and Taqman probes, indicated by the unfilled rectangles "1" and "2" in Figure 2, containing the nucleotide sequences described as SEQ ID NO: 73 and SEQ ID NO: 74, respectively. 2. From 5' to 3', D H The modified D2-8 gene contains a 50bp homologous box starting 552bp upstream of the 12 RSS at the 5' end of D2-8, multiple cloning sites (MreI-NsiI-EcoRI-KpnI-MreI), a modified D2-8 gene having a 12-mer RSS at the 3' end substituted with a 23-mer RSS at the putative 3' end derived from human VH1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC;T->A, G->C;SEQ ID NO: 151), and a 50bp homologous box ending 867bp downstream of the 23-mer RSS at the 3' end of D2-8. H Second donor for modification 2-8. D H The nucleotide sequence of this second donor for modification 2-8 is described as SEQ ID NO: 71. H The region consists of four direct repeats totaling approximately 10kb, making the design of unique primers and probes for screening extremely challenging. To overcome this problem, two unique 40bp sequences were inserted; one 74bp downstream of the 5' homologous box and the other 40bp upstream of the 3' homologous box. These unique 40mers were used as binding sites for PCR / sequencing primers and Taqman probes, indicated by the unfilled rectangles “10” and “16” in Figure 2, containing the nucleotide sequences described as SEQ ID NOs. 75 and SEQ ID NOs. 76, respectively. 3. From 5' to 3', D HA 50bp homologous box starting 391bp upstream of the 12 RSS at the 5' end of 2-15, with multiple cloning sites (MreI-NsiI-EcoRI-KpnI-MreI), Human V H A modified D2-15 gene having a 12-mer RSS at the 3' end that is substituted with a 23-mer RSS at the putative 3' end derived from 1-69 (CACAGTGTGA AAACCCACAT CCTGAGAGTG ACACAAACC;T->A, G->C;SEQ ID NO: 151), and containing a 50bp homologous box ending 867bp downstream of the 23-mer RSS at the 3' end of D2-15, D H Third donor for modification 2-15. D H The nucleotide sequence of this third donor for the 2-2 modification is described as SEQ ID NO: 72. H Because the region consists of four direct repeats totaling approximately 10kb, designing unique primers and probes for screening is extremely challenging. To overcome this problem, two unique 40bp sequences were inserted; one 74bp downstream of the 5' homologous box and the other 40bp upstream of the 3' homologous box. These unique 40mers were used as binding sites for PCR / sequencing primers and Taqman probes, indicated by the unfilled rectangles "8" and "18" in Figure 2, and contain the nucleotide sequences described as SEQ ID NO: 77 and SEQ ID NO: 78, respectively. After in vitro Cas9 / GA modification in three donor groups, the final targeting vector was a modified human D2 segment with three adjacent human variable region DNA, neomycin selection cassette, and a 12-mer RSS at 5' and a 23-mer RSS at 3'. H Domain, Six Human J H It contained a 49kb 5' homologous arm containing the segment, and a 24kb 3' homologous arm containing the mouse heavy chain intron enhancer (Ei) and mouse IgM constant region gene (Figure 2). Table 3 is 12:D H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J H1-6 We provide the sequences of the gRNAs, primers, and probes used to determine the precise construction of the targeting vectors. [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 1-6 Targeting vectors are then straightened with NotI, and the functional human V H Complete repertoire of gene segments, D H D excluding 7-27 H Domain deletion, and functional human J H Mouse embryonic stem cells with a genome that is homozygous for the humanized immunoglobulin heavy chain variable region containing the complete repertoire of human immunoglobulin light chain Vκ and Jκ gene segments, and operably bound to the endogenous mouse immunoglobulin heavy chain Cκ region (1293), were electroporated (see Figure 7). The 20187 allele was electroporated, and the 6011 allele was electroporated using 12:D containing a neomycin cassette. H 2-2:23|12:D H 2-8:23|12:D H 2-15:23 / J HThese are generated by appropriate homologous recombination with 1-6 targeting vectors (Figure 7). Using these manipulated mouse ES cells, efficient screening of positive ES clones (see below) and subsequent cre-mediated removal of the neomycin drug resistance cassette are promoted, and thereafter, the removal of the 20187 allele is replaced with 20188 (Figure 8).

[0276] Example 2. ES cell screening In this example, the genome is manipulated to increase heavy chain diversity (D H This demonstrates the creation of non-human animals (e.g., rodents) containing the immunoglobulin heavy chain variable region, including the ) region, and the manipulated D H Each region is operably coupled to one or more D23mer RSS. H Includes segments.

[0277] The proper assembly of targeting vectors, as described in Example 1, which target the insertion of DNA fragments into the diversity cluster of humanized immunoglobulin heavy chain loci contained with BAC DNA, was confirmed by sequencing and polymerase chain reaction during the construction of each targeting vector. Target BAC DNA was confirmed by polymerase chain reaction, and then embryonic stem (ES) cells were introduced via electroporation and subsequently cultured 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 D H Using primers / probes that detect the correct integration of gene segments (Table 4; F: forward primer, P: probe, R: reverse primer), we screened for correct targeting using TAQMAN® and karyotype analysis, as previously described (Valenzuela et al., above; Frendewey, D. et al., 2010, Methods Enzymol. 476:295-307, the entire text of which is incorporated herein by reference).

[0278] Using the VELOCIMOUSE® method (DeChiara,™ et al., 2010, Methods Enzymol. 476:285~294; DeChiara,™, 2009, Methods Mol. Biol., 530:311~324; Poueymirou et al., 2007, Nat. Biotechnol., 25:91~99, the entire text is incorporated herein by reference), target ES cells were injected into uncompressible 8-cell stage Swiss Webster embryos and manipulated D H It is heterozygous for the region, D H -D H We generated F0 generation mice from healthy, fully ES cells that express antibodies containing heavy chain variable regions, including the CDR3 region, generated through recombinant DNA. Heterozygous male F0 mice were mated with C57Bl6 / NTac females to produce F1 heterozygotes, and these F1 heterozygotes were then crossed with each other to produce F2 generation homozygous and wild-type mice.

[0279] For example, to remove any lox-added selection cassette introduced by an unremoved targeting construct at the ES cell stage or in embryo, the drug selection cassette may be optionally removed by subsequent addition of a recombinase (e.g., by Cre treatment) by mating with a Cre-deficient mouse line (e.g., see International Patent Application Publication 2009 / 114400, which is incorporated herein by reference in its entirety). Optionally, the selection cassette may be retained within the mouse. The selection cassette manipulated in 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 Feature determination of mice modified with 1-6 targeting vectors. immunophenotype

[0281] 23:D H 3-3:12 / J H 6 or 23:D H 3-3:12 / J H Immunophenotypic analysis of animals modified with 4-6 targeting vectors was performed on heterozygous mice for each of the 6800 or 6795 modified alleles (see Figures 4 and 6). 12:D 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 1-6 targeting vectors was performed on mice homozygous for the 20188 modification allele. B cell development in these animals was analyzed by fluorescence-activated cell sorting (FACS). Briefly, the spleen and leg bones (femur and tibia) were collected from the following locations: VELOCIMMUNE® mice (n=3, 26% C57BL / 6, 23% 129S6 / SvEvTac, 51% Balb / cAnNTac, see, e.g., U.S. Patent Nos. 8,502,018 and 8,642,835), 6643het / 6800het / 1293ho mouse (23:D H 3-3:12 / J H Mod...

Claims

[Claim 1] An invention such as a nucleotide molecule comprising an engineered heavy chain diversity (DH) gene segment, which comprises at least one DH gene segment operably bound to a 23-mer recombinant signal sequence (RSS).

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