BCR transgenic mice with a consensus leader sequence

Abstract

The present invention provides transgenic animals containing some or all components of human heavy and / or light chain immunoglobulin variable region loci, methods for producing such animals, methods for producing human antibodies using such animals, and treatment methods using human antibodies produced in such animals, which contain in their genome multiple human heavy chain V gene segments all immediately preceded by the same first leader peptide-encoding sequence and / or multiple human light chain V gene segments all immediately preceded by the same second leader peptide-encoding sequence, or both. The present invention also provides polynucleotide constructs containing two or more human heavy or light chain leader / V gene segments containing identical leader peptide-encoding sequences. Such animals, constructs, and methods are used in the efficient generation of optimally diverse populations of antibodies against an antigen of interest, such as an antigen of therapeutic interest.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 949,707, filed December 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This specification also incorporates by reference in its entirety a Sequence Listing electronically submitted herewith (filename: 20201120_SEQL_13330WOPCT_GB.txt; date of creation: November 20, 2020; file size: 29 KB).

Background Art

[0003] Antibodies are increasingly being used as drugs to treat human diseases such as autoimmune diseases and cancer. The earliest such therapeutic antibodies were non - human (e.g., mouse) antibodies that induced a human anti - drug antibody response (e.g., human anti - mouse antibody - HAMA response), so repeated dosing was not possible. Subsequent generations of therapeutic antibodies were non - human antibodies with non - human constant regions replaced by human constant regions, typically "chimeric" forms of mouse antibodies, as well as "humanized" antibodies in which all sequences other than the complementarity - determining regions (CDRs) were converted to human counterparts to minimize immunogenicity in human subjects.

[0004] The newest generation of therapeutic antibodies consists entirely of sequences derived from human germline immunoglobulin sequences, such as sequences derived from transgenic mice humanized at the immunoglobulin locus in vivo, or sequences derived from phage display libraries in vitro. Finlay & Almagro (2012) Front. Immunol. 3(342):1. Transgenic mice carrying human immunoglobulin gene segments are immunized with antigens designed to elicit antibodies against therapeutic targets. The diverse selection of antibody sequences in a pool of polyclonal anti-antigen antibodies, which results and contains antibodies derived from the maximum number of different germline sequences, maximizes the possibility of finding antibodies with excellent properties, such as high target affinity and epitope diversity. The diversity of antibody sequences depends on the number of different sequence elements, such as V gene segments, D gene segments, and J gene segments for the heavy chain, and V gene segments and J gene segments for the light chain, available in the mouse germline for incorporation into the heavy and light chains, as well as nucleotide additions / deletions in CDR3 during rearrangement, and somatic hypermutation that occurs during affinity maturation of the antibody during B cell development. However, different V gene segments, D gene segments, and J gene segments are incorporated into antibodies at different frequencies, resulting in a bias of antibody sequences towards preferred germline sequences and limited sequence diversity. Finlay & Almagro (2012) Front. Immunol. 3:242. Different leader peptides associated with different V gene segments can also affect the translation and secretion of specific antibodies derived from these sequences, and may further bias the distribution and limited diversity of antibody sequences.

[0005] Next, the antibodies produced by the mouse must be recovered for the selection of preferred antibodies, such as candidate therapeutic substances. Regardless of the diversity of the antibodies produced by the mouse, only the antibodies that are efficiently recovered will be available, for example, for selection as candidate therapeutic substances. Any isolation step that biases towards some antibodies, leaving out other sequences, will further reduce the diversity of the pool of polyclonal antibodies.

Summary of the Invention

Means for Solving the Problems

[0006] There is a need for an improved method for obtaining a pool of polyclonal antibodies that enhances the diversity of antibodies from which the most desired antibodies can be selected, and an improved mouse for use in such a method. Such a method would ideally avoid bias from the initial polyclonal antibody pool in antibody production, isolation, and evaluation so as not to lose good candidate substances before they can even be examined.

[0007] The present invention provides a non-human animal having a humanized heavy-chain immunoglobulin locus for use in the production of human antibodies, the humanized heavy-chain immunoglobulin locus comprising a plurality of human heavy-chain leader / V gene segments, all of which contain the same leader peptide coding sequence. The present invention also provides a non-human animal having a humanized light-chain immunoglobulin locus for use in the production of human antibodies, the humanized light-chain immunoglobulin locus comprising a plurality of human light-chain leader / V gene segments, all of which contain the same leader peptide coding sequence. The present invention further provides a non-human animal having humanized heavy-chain and light-chain immunoglobulin loci, the heavy-chain locus comprising a plurality of human heavy-chain leader / V gene segments, all of which contain the same first leader peptide coding sequence, and the light-chain locus comprising a plurality of human light-chain leader / V gene segments, all of which contain the same second leader peptide coding sequence. In some embodiments, the first leader peptide coding sequence is different from the second leader peptide coding sequence, and in other embodiments, they are the same sequence. In various embodiments, the animal is a mouse, a rat, or a cow.

[0008] In another aspect, the present invention provides a method of producing a non-human transgenic animal as described in the preceding paragraphs, the method comprising incorporating into the genome of the non-human animal a plurality of human heavy chain leader / V gene segments, all of which contain the same first leader peptide coding sequence, and / or a plurality of human light chain leader / V gene segments, all of which contain the same second leader peptide coding sequence, wherein the first leader peptide coding sequence and the second leader peptide coding sequence may be the same sequence. In some embodiments, no additional human heavy chain or light chain leader / V gene segments other than the plurality of human heavy chain or light chain leader / V gene segments containing the same first leader peptide coding sequence are introduced into or present in the genome of the non-human animal. In some embodiments, the non-human transgenic animal contains both a plurality of human heavy chain leader / V gene segments, all of which contain the same first leader peptide coding sequence, and a plurality of human light chain leader / V gene segments, all of which contain the same second leader peptide coding sequence, where the first leader peptide coding sequence and the second leader peptide coding sequence may be the same sequence. In various embodiments, the animal is a mouse, rat, or cow.

[0009] In another aspect, the present invention provides a method for generating a human antibody or an antigen-binding fragment thereof against a target antigen, the method comprising immunizing a mouse having humanized heavy and light chain immunoglobulin loci, wherein all heavy chain leader / V gene segments contain the same first leader peptide coding sequence and all light chain leader / V gene segments contain the same second leader peptide coding sequence, with the antigen, or an antigenic fragment or derivative thereof, and recovering from said mouse a human antibody that specifically binds to the antigen, or a sequence encoding the heavy and light chain variable regions of a human antibody that specifically binds to the antigen. The recovery may be by the hybridoma method, by single B cell cloning, or by any other suitable method of obtaining the antibody or its coding nucleic sequence from cells of a non-human animal that expresses the antibody. In some embodiments, the leader peptide coding sequences for the heavy and light chain leader / V gene segments are selected from the sequences listed in Tables 2, 3, and 4.

[0010] In yet a further aspect, the present invention provides a human antibody produced by the method of the preceding paragraph.

[0011] In yet a further aspect, the present invention provides a method of treating a subject, e.g., a human subject, comprising administering a human antibody produced by the method of the preceding paragraph. In various embodiments, the present invention provides treatment of a subject having an autoimmune disease, an infectious disease, a cardiovascular disease, or cancer.

[0012] In various embodiments of the animals and methods of the present invention, the leader peptide coding sequence preceding the heavy chain V gene segment encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. In another embodiment, the leader peptide coding sequence preceding the light chain V gene segment encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. In yet another embodiment, both the leader peptide coding sequences preceding the heavy chain V gene segment and the light chain V gene segment are selected from the sequences indicated in each of the two preceding sentences. In a specific embodiment, the heavy chain leader peptide coding sequence, such as the heavy chain leader peptide sequence of SEQ ID NO: 86 (IGHV 3-23) and the light chain leader peptide sequence of SEQ ID NO: 112 (IGKV 3-20), encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71, 85, 86, and 93, and / or the light chain leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 104 and 112.

[0013] In various embodiments of the animals and methods of the present invention, the leader peptide coding sequence preceding the heavy chain V gene segment is a sequence selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. In another embodiment, the leader peptide coding sequence preceding the light chain V gene segment is a sequence selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. In yet another embodiment, both the leader peptide coding sequences preceding the heavy chain V gene segment and the light chain V gene segment are selected from the sequences indicated in each of the two preceding sentences. In a specific embodiment, the heavy chain leader peptide coding sequence, such as the SEQ ID NO: 16 or 136 (IGHV 3-23 or IGHV 3-23 genome), and the light chain leader peptide coding sequence of SEQ ID NO: 49 or 137 (IGKV 3-20 or IGKV 3-20 genome), the heavy chain leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1, 15, 16, 27, and 136, and / or the light chain leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 39, 49, and 137. In a preferred embodiment, the leader peptide genomic coding sequences of IGHV 3-23 (SEQ ID NO: 136) and IGKV 3-20 (SEQ ID NO: 137) are used for the heavy chain and light chain V gene segments, respectively.

[0014] In another aspect, the present invention provides a polynucleotide comprising a plurality (two or more) of heavy chain V gene segments or light chain V gene segments preceded by a common (identical) leader peptide coding sequence. In some embodiments, the polynucleotide comprises a single leader peptide coding sequence encoding a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71-133 and 135, such as SEQ ID NO: 71, 85, 86, or 93, immediately upstream of a plurality of naturally occurring human heavy chain V gene segments. In other embodiments, the polynucleotide comprises a single leader peptide coding sequence encoding a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71-133 and 135, such as SEQ ID NO: 104 or 112, immediately upstream of a plurality of naturally occurring human light chain V gene segments. Such polynucleotides can be, for example, synthetic, incorporated into a long-chain polynucleotide construct such as a vector, or incorporated into a chromosome.

[0015] Exemplary heavy chain leader peptide coding sequences for incorporation into the polynucleotides of the present invention are SEQ ID NOs: 16 and 136, and more specifically include SEQ ID NOs: 1, 15, 16, 27, and 136, such as SEQ ID NO: 136. Exemplary light chain leader peptide coding sequences for incorporation into the polynucleotides of the present invention are SEQ ID NOs: 49 and 137, and more specifically include SEQ ID NOs: 39, 49, and 137, such as SEQ ID NO: 137.

[0016] In various embodiments, the transgenic animal or polynucleotide of the invention comprises a plurality of heavy chain V gene segments and / or light chain V gene segments selected from the group consisting of all naturally occurring human V gene segments such as human heavy chain V gene segments IGHV 3-23; IGHV 5-51; IGHV 3-7; IGHV 1-2; IGHV 1-69-1; IGHV 3-48; IGHV 1-18; IGHV 1-46; IGHV 3-21; IGHV 3-30; IGHV 3-74; IGHV 4-39; IGHV 3-9; IGHV 2-5; IGHV 1-3; IGHV 4-4; IGHV 7-4-1; IGHV 3-66; and IGHV 1-24, and / or human light chain V gene segments IGKV 1-39; IGKV 3-11; IGKV 1-33; IGKV 3-20; IGKV 4-1; IGKV 1-27; IGKV 1-5; IGKV 1-16; IGKV 1-12; IGKV 2-30; IGKV 3-15; IGKV 2-28; IGKV 1D-13; IGKV 1-17; IGKV 6-21; IGKV 1-9; and IGKV 1D-43, or a subset thereof, or the methods of the invention involve the use thereof. In other embodiments, the plurality of heavy chain human V gene segments and / or light chain human V gene segments comprise one or more non-naturally occurring V gene segments such as engineered V gene segments or mutant V gene segments. In various embodiments, in the case of the heavy chain, one or more human J gene segments and one or more human D gene segments are incorporated into the transgenic animal or polynucleotide such as all naturally occurring D gene segments and / or J gene segments, or a desired subset thereof. In other embodiments, non-naturally occurring D gene segments and / or J gene segments such as engineered or mutant D gene segments and / or J gene segments are incorporated.

[0017] In various embodiments, the present invention provides non-human animals having a humanized heavy chain immunoglobulin locus, and methods of making these animals, wherein the humanized heavy chain immunoglobulin locus comprises a plurality of human heavy chain leader / V gene segments that include more than one but a limited number of leader peptide coding sequences. Such animals can include two, three or more different leader peptide coding sequences associated with diverse V gene segments, although at least two of the leader / V gene segments include the same leader peptide coding sequence. Such embodiments do not achieve the maximum benefit of using a single leader peptide coding sequence for all leader / V gene segments and require the use of a mixture of primers for amplification, yet can present substantial advantages over the use of different leader peptide coding sequences that are naturally associated with each V gene segment. The use of sequences encoding more than one leader peptide may be necessary, for example, if no single leader peptide coding sequence works well with all of the desired V gene segments. Most of this specification relates to embodiments that include a human immunoglobulin single genomic variable region sequence for all heavy chain V gene segments and / or light chain V gene segments, although those skilled in the art will recognize that the number of sequences is preferably minimal, yet the benefits of the present invention can still be largely achieved even if more than one leader peptide coding sequence is used. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0019] A schematic diagram similar to Figures 1 and 2, excluding the light chain construct, i.e., a schematic diagram when only the D gene segment is excluded, will also be immediately apparent to those skilled in the art.

[0020] Definitions For the present disclosure to be more readily understood, certain terms are first defined. As used in this application, unless explicitly stated otherwise herein, each of the following terms shall have the meaning set forth below. Further definitions are provided throughout this application.

[0021] "administering" refers to the physical introduction of a composition containing an agent, such as an antigen or therapeutic agent, to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for the antibodies of the present invention include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal, or other parenteral routes of administration via injection or infusion. As used herein, the phrase "parenteral administration" generally means a mode of administration other than enteral and topical administration via injection, and includes, without limitation, intravenous injection and infusion, intraperitoneal injection and infusion, intramuscular injection and infusion, intraarterial injection and infusion, intrathecal injection and infusion, intralymphatic injection and infusion, intralesional injection and infusion, intra-articular injection and infusion, intraorbital injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intratracheal injection and infusion, subcutaneous injection and infusion, subepidermal injection and infusion, intra-articular injection and infusion, subcapsular injection and infusion, subarachnoid injection and infusion, intraspinal injection and infusion, epidural injection and infusion, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies of the present invention can be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or locally. Administration can also be carried out, for example, once, multiple times, and / or over one or more periods. Administration can be carried out by one or more individuals, including but not limited to a physician, nurse, another healthcare provider, or the patient himself or herself.

[0022] Reference is made to transgenic animals comprising a human immunoglobulin locus optimized for use in the methods of the invention, and as used herein, "animal" refers to any animal species suitable for the production of human antibodies. Exemplary animals that have been used to produce human antibodies include rodents such as mice and rats, as well as cows. See, for example, Bruggemann et al. (2015) Arch Immunol Ther Exp (Warsz) 63:101. Other animals may also be used. Unless otherwise indicated, the methods and examples presented herein, specifically referring to mice, will be equally applicable to other suitable animal species.

[0023] An animal for the purposes of the present disclosure is "transgenic" if its germline nucleic acid sequences are modified to include nucleic acid sequences derived from a different species, such as sequences derived from human germline sequences or artificial sequences not found in the mouse genome. Transgenic animals, such as transgenic mice of the invention, will typically contain human immunoglobulin sequences integrated into their genomes. The heterologous nucleic acid sequences can be introduced at any locus, for example, at the corresponding animal immunoglobulin locus, and can be introduced by any method.

[0024] "Introduction" of a gene construct into an animal such as a mouse can include mating or crossing animals with the desired trait so as to create progeny that carry both traits. For example, an animal carrying a transgenic human heavy chain variable region locus can be crossed with an animal carrying a transgenic human light chain variable region locus so as to create an animal capable of producing antibodies that contain human variable domains.

[0025] An "antibody" (Ab), without limitation, includes immunoglobulins, which are glycoproteins that specifically bind to an antigen, and includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or those containing antigen-binding portions thereof. Each H chain includes a heavy chain variable region (abbreviated herein as V H ), and a heavy chain constant region. The heavy chain constant region includes three domains, C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (abbreviated herein as V L ), and a light chain constant region. The light chain constant region is composed of one domain, C L . The V H region and the V L region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). Each V H and each V L are composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The variable region may be referred to herein synonymously with the "variable domain", and the constant region may be referred to herein synonymously with the "constant domain".

[0026] Depending on the context, "antibody" may refer to a pool of polyclonal antibodies, such as a plurality or a limited number of individual antibodies, or a pool of all anti-antigen antibodies recovered from immunization with an antigen.

[0027] As used herein, and as is commonly understood in the art, an antibody described as including "one" heavy chain and / or "one" light chain refers to an antibody that includes "at least one" of the recited heavy and / or light chains, and thus would include antibodies having two or more heavy chains and / or light chains. Specifically, antibodies so described would include conventional antibodies having two substantially identical heavy chains and two substantially identical light chains. Antibody chains may be substantially identical but not completely identical if they differ due to post-translational modifications such as cleavage of lysine residues at the C-terminus, alternative glycosylation patterns, etc.

[0028] Unless otherwise indicated or apparent from the context, an antibody defined by its target specificity (e.g., an "anti-CTLA-4 antibody") refers to an antibody that can bind to its human target (e.g., human CTLA-4). Such an antibody may or may not bind to CTLA-4 from other species.

[0029] Immunoglobulins can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype may be divided into subclasses: in humans, IgG1, IgG2, IgG3, and IgG4; and in mice, IgG1, IgG2a, IgG2b, and IgG3 in certain species. IgG antibodies may be referred to herein, as is apparent from the context, by the symbol gamma (γ), or simply by the symbol "G", e.g., IgG1 may be designated "γ1" or "G1". "Isotype" refers to the class of antibody (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0030] An "isolated antibody" refers to an antibody that has a different antigen specificity and is substantially free of other antibodies (e.g., an isolated antibody that specifically binds to CTLA-4 is substantially free of antibodies that specifically bind to antigens other than CTLA-4). However, an isolated antibody that specifically binds to CTLA-4 may cross-react with other antigens, such as CTLA-4 molecules from different species. Additionally, an isolated antibody may be substantially free of other cellular and / or chemical substances. For purposes of comparison, an "isolated" nucleic acid refers to a nucleic acid that is distinct, i.e., has a unique chemical identity, properties, and utility, from the nucleic acids that occur in nature. For example, an isolated DNA is distinct from natural DNA in that it is an independent portion of natural DNA and is not an essential part of the chromosome, which is a large structural complex found in nature. Further, an isolated DNA is distinct from natural DNA in that it can be used, inter alia, as a PCR primer or hybridization probe for detecting biomarker genes or mutations, for measuring gene expression, diagnosing a disease, or predicting the efficacy of a therapeutic agent. An isolated nucleic acid may also be purified using standard techniques well known in the art so as to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or cellular proteins.

[0031] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of a single molecular composition, i.e., antibody molecules whose primary sequences are essentially identical and which exhibit a single binding specificity and binding affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0032] As used herein, a "human" antibody (or antigen-binding fragment thereof) refers to an antibody (or fragment) derived from a human genomic immunoglobulin variable region sequence, including a naturally occurring germline sequence and variants thereof, such as variants that include a V gene segment preceded by the same leader peptide coding sequence according to the present invention. Derivatives of human genomic immunoglobulin variable region sequences include, for example, subtle sequence changes so as to eliminate potential amino acid sequence disadvantages in the resulting antibody. A "human" antibody is to be distinguished from an antibody derived from the immunoglobulin sequence of the germline of an animal, such as a mouse. Since the leader peptide is cleaved from the mature heavy and mature light chains of the antibody, an antibody created in the mouse of the present invention, or an antibody produced from the mouse of the present invention, will contain only the human-derived immunoglobulin variable region sequence, regardless of the origin (human, non-human, artificial) of the leader peptide sequence used. A human antibody of the present invention may contain amino acid residues not encoded by the immunoglobulin sequence of the human germline (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence. In the context of an antibody that is a potential therapeutic agent for humans isolated from a transgenic animal, if the variable domain is of human origin, the constant domain will ultimately be changed to a human constant domain in the final therapeutic antibody, and thus the antibody may be considered a human antibody regardless of the origin of the constant domain. A "fully human" antibody includes both a variable domain sequence and a constant domain sequence derived from the immunoglobulin sequence of the human germline.

[0033] An "antibody fragment" generally refers to the "antigen-binding portion" ("antigen-binding fragment") of a complete antibody that retains the ability to specifically bind to an antigen bound by the complete antibody, or a portion of the whole antibody that includes the Fc region of the antibody and retains the ability to bind to FcR. Exemplary antibody fragments include Fab fragments and single-chain variable domain (scFv) fragments.

[0034] As used herein, a "leader peptide sequence" typically refers to a series of amino acid residues at the N-terminus of a newly synthesized polypeptide that is cleaved before or concurrently with the secretion of the protein. A leader peptide sequence may also be referred to as, for example, a leader sequence, leader peptide, signal sequence, and signal peptide. A leader peptide sequence typically has a length of 16 to 30 amino acids. For a protein having a leader peptide, the full-length form of the sequence including the leader peptide sequence is referred to as a proprotein, whereas the sequence remaining after removal of the leader peptide is referred to as a mature protein. At the genetic level, the leader peptide coding sequence is present immediately upstream of the sequence encoding framework region 1 (FR1) of the variable domain, which is found at the 5' end of the V gene segment.

[0035] A "leader peptide coding sequence" refers to a nucleic acid sequence, typically a DNA sequence, when referring to a gene construct for integration into the genome of a non-human animal that encodes a leader peptide sequence, or a gene construct that is integrated into the former. A leader peptide coding sequence may be, for example, in the case of a naturally occurring DNA sequence that encodes the original leader peptide sequence in that organism, with or without any intron sequences present in the leader peptide coding sequence in the genome, in the case of a codon-optimized DNA sequence that encodes the leader peptide sequence, or in the case of any other DNA sequence that encodes the leader peptide sequence.

[0036] As used herein, a "V gene segment" refers to a genomic gene element that is capable of rearrangement such that, when placed within an immunoglobulin locus, it includes a sequence encoding the amino-terminal portion of the variable domain of a mature antibody heavy or light chain. Unless otherwise indicated, a "V gene segment" refers to a naturally occurring human genomic gene segment that does not include a sequence encoding a leader peptide. Janeway et al. (2001) Immunobiology, 5th Ed. at Section 4.2 and Figure 4.2. "D gene region" and "J gene region" refer to additional genomic gene elements within an immunoglobulin locus (in the light chain, only the "J gene region") that are capable of rearrangement such that they include a sequence encoding the carboxy-terminal portion of the variable domain of an antibody heavy or light chain. The transgenic animals of the present invention include in their genome one or more human immunoglobulin loci that include a plurality of V gene segments (two or more), as well as at least one D gene segment (for the heavy chain) and at least one J gene segment. A "variable region gene segment" refers to the entirety of the V gene segment, D gene segment (for the heavy chain), and J gene segment found within a variable region locus.

[0037] As used herein, the "leader / V gene segment" refers to a genetic element that includes a V gene segment immediately downstream of the sequence encoding the leader peptide. When integrated into a rearranged immunoglobulin locus, the leader / V gene segment may be transcribed into RNA and spliced to create the 5' end of the mRNA encoding the heavy or light chain of the antibody. The leader / V gene segment encodes the leader peptide and the N-terminal portion of the variable domain of the antibody chain, including the beginnings of framework 1 (FR1), complementarity-determining region 1 (CDR1), FR2, CDR2, FR3, and CDR3. The leader peptide is cleaved from the heavy and light chains in the mature secreted antibody. In some embodiments of the present invention, while the V gene sequences within these leader / V gene segments include human germline V gene sequences, the leader peptide coding sequences may be of human origin, non-human origin, or synthetic origin. See, for example, Tables 2, 3, and 4. In multiple embodiments of the present invention, for a given heavy chain locus or light chain locus, different leader / V gene segments each contain different V gene sequences but contain the same leader peptide coding sequence. Unless otherwise indicated, the nucleic acid sequence transcribed immediately upstream of a given leader / V gene segment includes the 5' untranslated region (UTR) that is naturally associated with this specific V gene segment of the original species, such as human.

[0038] As used herein, an "immunoglobulin locus", such as a "human immunoglobulin locus", refers to a genomic location that includes nucleic acid sequences necessary to assist in the rearrangement for producing a heavy or light chain of an antibody. In the context of the methods of the invention or the transgenic animals of the invention, a human heavy or light chain immunoglobulin locus can be located at or near the corresponding human heavy chain locus or animal light chain locus of the animal genome, for example, near the mouse immunoglobulin locus. A human immunoglobulin locus in a transgenic animal includes human gene elements for the variable regions of the heavy or light chain ("human immunoglobulin variable region locus"), for example, V elements, D elements, and J elements for the heavy chain variable region, and V elements and J elements for the light chain variable region, and may include one or more human gene elements for the constant region of the antibody chain. An immunoglobulin locus can include any number of V, D, or J gene segments, and one to the number occurring in nature or more than that number of functional human gene segments. Multiple embodiments of the invention include at least two V gene segments for at least the heavy chain locus or the light chain locus.

[0039] Unless otherwise indicated or apparent from the context, as used herein, "sequence" refers to a nucleic acid sequence such as a genomic DNA sequence, etc. Thus, "identical sequence" will necessarily encode the same polypeptide sequence. Unless otherwise indicated or apparent from the context, all references herein to nucleic acid sequences (such as genes or gene segments) or proteins are to the human (Homo sapiens) ortholog of this nucleic acid sequence or protein.

[0040] Referring to the heavy or light chain leader / V gene segments, as used herein, the "identical leader peptide coding sequence" refers to the N-terminal leader peptide coding sequence in a plurality of leader / V gene segments that contains the same nucleic acid sequence as the leader peptide coding sequences in all other leader / V gene segments in each of the heavy or light chains. The leader peptide coding sequences may be natural coding sequences for the leader peptide, such as those derived from specific human V gene segments, provided that they are all the same nucleic acid sequence, or may be optimized (such as codon-optimized) or otherwise modified coding sequences that encode the same leader peptide amino acid sequence. In the animals and methods of the present invention, all heavy chain leader / V gene segments will contain an identical leader peptide coding sequence (referred to as the "first" leader peptide coding sequence), and all light chain leader / V gene segments will contain an identical leader peptide coding sequence (referred to as the "second" leader peptide coding sequence). The first leader peptide coding sequence may or may not be the same as the second leader peptide coding sequence.

[0041] A sequence element is "upstream" of a second sequence element if it is closer to the 5' end of the coding strand of the polynucleotide or to the amino terminus (N-terminus) of the polypeptide than the second sequence element. A sequence element is "immediately upstream" of a second sequence element if it is directly fused to the 5' end (or N-terminus) of the second sequence element without addition or deletion at the junction. Similarly, a sequence element is "downstream" of a second sequence element if it is closer to the 3' end of the coding strand of the polynucleotide or to the carboxy terminus (C-terminus) of the polypeptide than the second sequence element, and is "immediately downstream" if it is directly fused to the 3' end (or C-terminus) of the second sequence element without addition or deletion.

[0042] As used herein, a "genomic" sequence is a sequence found within the chromosomes of germ line cells of an animal such as human (Homo sapiens) or a non-human transgenic animal of the invention. The genomic sequence may or may not contain introns. As used herein, a "no-intron" sequence is a sequence in which there are no introns and thus directly encodes a protein without the need for splicing, and thus corresponds to a spliced mRNA sequence for this protein, or equivalently, a cDNA sequence for this protein. When a sequence is derived from a genomic sequence containing one or more introns, it is typically referred to as no-intron. In various embodiments, some or all of the leader peptide coding sequence and V gene segment used in the transgenic animals, methods, and nucleic acids of the invention are no-intron sequences. In other embodiments, introns are retained in the leader peptide coding sequence and / or V gene segment.

[0043] Unless otherwise indicated, discussion herein of a sequence or gene segment refers to their copy number in a haploid genome on an individual chromosome in one genomic complement. Similarly, discussion herein of a sequence or gene segment does not take into account heterozygosity. Unless otherwise indicated, animals of the invention may be heterozygous or homozygous for a given sequence or gene element. Animals that are heterozygous for a given sequence or gene element may be selectively mated, for example, to produce homozygous progeny animals for use in eliciting antibodies against a target antigen.

[0044] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. The unregulated cell division and growth result in the formation of malignant tumors or cancerous cells that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0045] "Cell surface receptor" refers to molecules and molecular complexes that can receive signals and transmit such signals through the cell's plasma membrane.

[0046] "Effector cell" refers to cells of the immune system that express one or more FcRs and mediate one or more effector functions. Preferably, the cells express at least one type of activating Fc receptor, such as human FcγRIII, and carry out the ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils, and eosinophils.

[0047] "Effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include binding to C1q (Clq), complement-dependent cytotoxicity (CDC), binding to Fc receptors, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the binding of the Fc region to a binding domain (e.g., the variable domain of an antibody).

[0048] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternative splicing forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans), and one inhibitory receptor (FcγRIIB). The diverse properties of human FcγRs are summarized in Table 1. Most natural effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) and do not express the inhibitory FcγRIIB in mice and humans.

[0049] Unless otherwise used in the figures, "Fc region" (fragment crystallizable region), "Fc domain", or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various immune system cells (e.g., effector cells) or to the first component of the classical complement system (C1q). Thus, the Fc region is a polypeptide that includes the constant region of the antibody, excluding the immunoglobulin domain that is the first constant region. In the antibody isotypes IgG, IgA, and IgD, the Fc region is composed of two identical protein fragments derived from the second constant domain (C H2 ) and the third constant domain (C H3 ) of the two heavy chains of the antibody; IgM Fc regions and IgE Fc regions each contain three heavy chain constant domains (C HIt contains domains 2 to 4). For IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. The boundaries of the Fc region of the immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as the amino acid residues at position C226 or P230 of the heavy chain to the carboxy-terminal sequence, in which case the numbering follows the EU index in Kabat. The C H2 domain extends from around amino acid 231 to around amino acid 340, while the C H3 domain is within the Fc region and is located on the C-terminal side of the C H2 domain, that is, the C H3 domain extends from around amino acid 341 to around amino acid 447 of IgG. As used herein, the Fc region may be a natural Fc sequence or a mutant Fc. Fc may also refer to this region alone or in the context of a polypeptide of a protein containing Fc, such as a "binding protein containing an Fc region" which is also referred to as an "Fc fusion protein" (e.g., an antibody or an immunoadhesin).

[0050]

Table 1

[0051] The term "immune response" refers to a biological response within a vertebrate to foreign agents, which is a response that defends the organism against these agents and the diseases caused by them. The immune response is mediated by the action of soluble macromolecules produced by any of the immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils), and any of these cells or the liver (including antibodies, cytokines, and complement), resulting in the selective targeting, binding to, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, cells or tissues infected by pathogens, cancerous cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or human tissues.

[0052] The term "immune modulator" or "immunoregulatory factor" refers to a component of a signaling pathway that can be involved in modulating, regulating, or modifying an immune response. "Modulating", "regulating", or "modifying" an immune response refers to any change within immune system cells or any change in the activity of such cells. Such modulation includes stimulation or suppression of the immune system manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that can occur within the immune system. Both inhibitory and stimulatory immune modulators have been identified, and some of these may enhance the function of the tumor microenvironment. In a preferred embodiment of the disclosed invention, the immune modulator is located on the surface of T cells. The term "immunomodulating target" or "immunoregulatory target" refers to an immune modulator that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by such binding. Immunomodulating targets include, for example, receptors on the cell surface ("immunomodulating receptors"), and receptor ligands ("immunomodulating ligands").

[0053] "Immunotherapy" refers to the treatment of a subject suffering from, or at risk of developing or recurring with, a disease by a method involving the induction, enhancement, suppression, or other form of modification of an immune response.

[0054] "Enhancement of an endogenous immune response" means increasing the effectiveness or efficacy of an existing immune response in a subject. This increase in effectiveness and efficacy may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response, or by stimulating mechanisms that enhance the endogenous host immune response.

[0055] "Protein" refers to a chain containing at least two contiguous amino acid residues, without an upper limit on the length of the chain. One or more amino acid residues within a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or formation of disulfide bonds. The term "protein" is used interchangeably with "polypeptide" herein.

[0056] Unless otherwise indicated or apparent from the context, "subject" refers to a human to whom a therapeutic substance is administered. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, rabbits, mice, rats, and guinea pigs, avian species such as chickens, amphibians, and reptiles. In preferred embodiments, the subject is a mammal such as a non-human primate, sheep, dog, cat, rabbit, ferret, or rodent. In more preferred embodiments for any aspect of the disclosed invention, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0057] The term "treatment" or "therapy" refers to any type of intervention or process, or the administration of an active agent, that is performed on a subject for the purpose of suppressing, alleviating, improving, inhibiting, slowing, or preventing the development, progression, onset, severity, or recurrence of a disease-related symptom, complication, condition, or biochemical sign.

[0058] Common leader peptide method The present invention provides a method for generating transgenic animals for use in the production of diverse polyclonal pools of human antibodies, for example, for the selection of antibodies for use as therapeutic agents for humans. The present invention also provides transgenic animals, such as mice, including the improvement of artificial human immunoglobulin loci for use in the production of diverse polyclonal pools of human antibodies, methods for making antibodies using such transgenic animals, antibodies obtained from such transgenic animals, treatment methods using antibodies obtained from such transgenic animals, and related polynucleotide constructs. The method of the present invention involves the introduction of multiple genomic heavy and / or light chain leader / V gene segments into an animal, such as a mouse, rat, or cow, where all leader / V gene segments for a given chain contain the same leader peptide coding sequence.

[0059] The human immunoglobulin heavy chain locus contains approximately 45 functional V gene segments, 25 D gene segments, and 6 J gene segments, and the kappa light chain locus contains approximately 40 functional V gene segments and 5 J gene segments. Lucas (2003) Encyclopedia of Life Science 1-8. The combinatorial assortment of these germline gene elements results in baseline levels of antibody sequence diversity, which is enhanced by junctional mutations (e.g., deletions, as well as the addition of N nucleotides and P nucleotides) and somatic hypermutation, resulting in an array of antibodies with bewildering levels of sequence diversity. This sequence diversity is advantageous for exposing antibodies to any number of potential pathogens, but it introduces complexity into the isolation and purification of these polyclonal antibody pools. A seeker of individual antibodies with excellent properties for a given purpose will wish to screen a large population of antibodies with the greatest possible sequence diversity. This requires, for example, not only the generation of diverse pools of polyclonal antibodies within transgenic animals, but also the recovery of the entire antibody population produced by the animals. Antibody recovery methods may introduce a selection step in which certain sequences are preferentially retained or lost, reducing the sequence diversity within the recovered polyclonal antibody pool. It would be desirable to design from the outset a system for generating and recovering pools of polyclonal antibodies that facilitates the unbiased generation and recovery of polyclonal antibody pools for use in subsequent selection steps.

[0060] Transgenic animals such as mice are engineered to express human germline immunoglobulin genes for the generation of antigen-binding domains of human antibodies for use as therapeutic agents. Such animals contain multiple human leader / V gene segments, as well as D gene segments and J gene segments, to allow for combinatorial diversity. Such mice enhance efficient somatic hypermutation and, thus, these human variable domain loci are expressed in combination with one or more constant domain sequences that are native to the host animal, such as a mouse, and may be sequences at the endogenous mouse locus, to enhance affinity maturation of antibodies during development in the host animal. Murphy et al. (2014) Proc. Nat'l Acad. Sci. (USA) 111:5153. The animals are injected with the antigen of interest one or more times according to an immunization protocol that induces the production of antigen-specific antibodies. The resulting polyclonal population of antibodies is then recovered, for example, by fusion of spleen cells from the immunized mice with myeloma cells that form hybridoma cells in which the antibodies are isolated and studied. However, the fusion method and the antibody production process constitute a de facto selection process that enriches for some sequences at the expense of others in the resulting pool of antibodies, which can result in a pool with reduced antibody diversity.

[0061] Alternatively, B cells derived from immunized mice may be isolated and individually sequenced to obtain the heavy and light chain sequences of the antibody for direct cloning in methods known as single B cell cloning. See, for example, Tiller et al. (2008) J. Immunol. Meth. 329:112; Wardemann & Busse (2019) Expression Cloning of Antibodies from Single Human B Cells. In: Kueppers R. (eds) Lymphoma. Methods in Molecular Biology, vol. 1956. Humana Press, New York, NY. Cloning and sequencing of single B cell antibodies minimizes the number of steps between the full antibody diversity in the immunized animal and the final pool of polyclonal antibodies by directly screening the B cell population in the animal.

[0062] In either case, the variable domain sequences of the antibody are obtained by polymerase chain reaction (PCR) using primers that hybridize to sequences on the 5' and 3' sides of the heavy and light chain variable domains. The PCR primers must be designed to accommodate the fact that the sequences flanking the variable domains differ between the antibodies in the pool due to the use of different V genes and isotypes. Primarily, when targeting only IgG antibodies, the sequence of the 3' (reverse) primer may be based on, for example, a conserved sequence at the 5' end of the CH1 domain of all IgG constant domains. However, the primer at the 5' end must correspondingly accommodate antibodies derived from any of approximately 45 leader / V gene segments with diverse leader peptide coding sequences.

[0063] One approach is to use a mixture of primers that each anneal to a leader peptide coding sequence naturally associated with a different V gene segment, as illustrated in FIG. 1. FIG. 1 presents a schematic representation of the human immunoglobulin heavy chain variable domain locus, typically found in the transgenic animals of the invention used to generate human antibodies. FIG. 1 is not representative of the invention. An array of gene segments for the heavy chain variable locus in the animal genome is shown, where each leader / V gene segment contains a distinct leader peptide coding sequence associated with this specific V gene segment. FIG. 1 further presents a schematic representation of two PCR reactions used to recover the variable region sequences of fully rearranged antibodies from the B cells of immunized animals. In PCR1, a mixture of a forward primer (forward primer 1) and a reverse primer (reverse primer 1) is used to amplify the variable region. Since the leader peptide coding sequences for the leader / V gene segments are different, a mixture of primers is required for use as the forward primer. The diversity in the use of V gene segments is a highly important factor in the diversification of the potential binding epitopes, specificities, and affinities of a population of polyclonal antibodies. Forward primer 1 hybridizes to the 5' end of the leader peptide coding sequence of the problematic leader / V gene segment, which is a known sequence for each individual leader / V gene segment, thus amplifying all leader / V gene segments that contain the leader peptide coding sequence. Reverse primer 1 hybridizes to a sequence within the 5' region of the CH1 domain that is conserved among all IgG isotypes (IgG1, IgG2, IgG3, IgG4).

[0064] Next, a second (nested) PCR step (PCR2) is performed on the product of PCR1. Similar to PCR1, PCR2 uses a mixture of a forward primer (forward primer 2) and a reverse primer (reverse primer 2), or at most, a mixture of a few reverse primers where each primer has a 5’ “tail” region such that additional sequences are added only outside the variable region. The forward primer 2 must be a mixture of primers for the same reason that the forward primer 1 was a mixture of primers, i.e., the sequences to which it hybridizes (FR1) differ between V gene segments, and it is necessary to include primers for all possible germline FR1 sequences to ensure amplification of the full diversity of antibody sequences. The reverse primer 2 hybridizes to the framework region 4 (FR4) of the variable region, which is somewhat conserved among human V gene segments, so the reverse primer 2 may be a single sequence or a small mixture of sequences. The product of PCR2 is the variable region with an artificial 5’ “tail” replacing the leader peptide coding sequence and an artificial 3’ “tail” replacing the constant domain / Fc sequence. These 5’ and 3’ tails are designed to anneal to upstream and downstream constructs containing the optimal promoter and leader peptide coding sequence as well as the desired Fc sequence, respectively, as shown.

[0065] The use of primer mixtures for forward primer 1 and forward primer 2 adds complexity, cost, and can interfere with amplification efficiency and specificity. Primer mixtures necessarily require the synthesis of many different oligonucleotides to ensure efficient amplification of the perfect complement of the human leader / V gene segment. Then, the relative concentrations of these primers must be optimized to ensure equivalent amplification efficiency for a variety of different leader peptide coding sequences to avoid amplification bias that could result in the loss of sequences with low amplification efficiency.

[0066] The use of a mixture of primers can also result in low-level amplification. For example, forward primer 1 is used for PCR1, which is an initial amplification of the variable region from a complex mixture that includes all nucleic acid sequences of the host cell from which the antibody sequence is obtained. The presence of a number of different primer sequences within forward primer 1 increases the likelihood of spurious amplification products resulting from priming in genomic DNA sequences that are only accidentally or nearly complementary to the primer. PCR2 is performed on a relatively purified nucleic acid, i.e., the product of PCR1, but still suffers from the same cost and optimization issues as PCR1.

[0067] Figure 2 presents a schematic representation of one embodiment of a new and improved set of gene elements for use in transgenic animals used to generate human antibodies. The array of gene segments differs from the array of gene segments in Figure 1 in that all leader / V gene segments are the same color (black) because each leader / V gene segment contains the same leader peptide coding sequence. Since all leader / V gene segments have the same leader peptide coding sequence, only a single primer sequence is required for forward primer 1 and forward primer 2 in each of PCR1 and PCR2. This eliminates the need for a complex mixture of primers, ensures uniform amplification of all V gene segments, and thereby ensures the maximum diversity of sequences within the resulting polyclonal pool of antigen-specific antibodies.

[0068] As illustrated in Figure 2, the product of PCR2 contains a leader peptide coding sequence, as well as a variable region sequence with an artificial 5' extension and an artificial 3' extension. These 5' and 3' extensions are designed to anneal with upstream and downstream constructs, respectively, containing an optimal promoter sequence and a desired Fc sequence. Such gene constructs are building blocks for the polynucleotides and transgenic animals of the present invention, the main feature of which is a common leader peptide coding sequence associated with each V gene segment. Specific details for the exemplary PCR protocol of Figure 2 are not limiting of the present invention, which can be carried out by any PCR amplification method suitable for exploiting the presence of the common leader peptide coding sequence associated with each V gene segment.

[0069] The use of a common leader peptide sequence for all antibodies within a pool of polyclonal antibodies can provide additional advantages with respect to uniformity across different V gene segment sequences for all aspects of antibody production. A common germline leader peptide coding sequence can increase the uniformity of gene rearrangement between V gene segments. A uniform sequence for the leader peptide coding portion of the mRNA can enhance the uniformity of translation, and thus enhance the expression of antibodies, for example, during affinity maturation in host cells and in transient expression in vitro for screening. A uniform signal peptide on the nascent antibody chain can increase the uniformity of protein processing and secretion.

[0070] The advantages resulting from the use of a uniform polypeptide sequence can be achieved regardless of the DNA sequence encoding it, and can be achieved regardless of whether the DNA sequence encoding the leader peptide is the same among all leader / V gene segments, or substantially all leader / V gene segments. However, in a PCR reaction, additional advantages resulting from the use of a uniform DNA sequence, such as the ability to use a single upstream primer, require that the same DNA sequence be used in all leader / V gene segments, or substantially all leader / V gene segments, to encode the leader peptide.

[0071] The benefits of the present invention arise primarily from the efficiency of having a common leader peptide coding sequence for multiple human V gene segments, but additional human leader / V gene segments that do not share the same leader peptide coding sequence may also exist in non-human transgenic animals. Such additional V gene segments would not necessarily interfere with the benefits resulting from the use of a common leader peptide coding sequence for other V gene segments. However, in most embodiments, there are no human leader / V gene segments other than the human leader / V gene segments that contain a common leader peptide coding sequence.

[0072] Leader peptide The leader peptide, also referred to as the signal peptide, is a short stretch of about 12 - 30 amino acids at the N-terminus of a secretory protein that targets the nascent polypeptide chain to the endoplasmic reticulum. Since the leader peptide is cleaved from the proprotein during the secretion process to create the mature protein, these amino acid sequences do not affect the activity of the protein after secretion. Leader peptides are generally diverse within the sequence, following a highly variable N-terminal region and a central region of 7 - 15 hydrophobic residues, and consisting of a stretch of about 2 - 9 small polar residues that form a motif cleaved by signal peptidase. Holden et al. (2005) J. Biol. Chem. 280:17172.

[0073] Human leader peptide sequences and leader peptide coding sequences, such as those associated with the V gene segments of the human heavy and light chains, are available in public databases, are known in the art, and are exemplified in Tables 2 and 3 and the Sequence Listing. Leader peptide coding sequences associated with commonly used human V gene segments can be selected because they are known to function well in the context of human antibody expression, particularly when subsequent production of the selected antibody in human cells is anticipated. A number of such human V gene leader peptide coding sequences for both the heavy and light chains are presented in Tables 2 and 3.

[0074] Alternatively, leader peptide sequences derived from transgenic host animals in which antibodies are raised, such as mice, can be selected under the prediction that such leader peptides function in the animal during antibody generation, such as during affinity maturation. Exemplary mouse immunoglobulin gene leader peptide sequences are presented in Table 4.

[0075] In addition, leader peptides of human non-immunoglobulin proteins, leader peptides from other species, and artificial / synthetic leader peptides can all be used to utilize efficient leader peptide sequences, regardless of their origin. For example, an artificial leader peptide sequence called secrecon (SEQ ID NOs: 61 and 124) was created based on a computational model. Barash et al. (2002) Biochem. Biophys. Res. Commun. 294:835. Another leader sequence, the Gaussia luciferase leader peptide (SEQ ID NOs: 67 and 130), was obtained from luciferase produced by Gaussia princeps (WO2017 / 068142). Leader peptides and leader peptide coding sequences derived from silkworm, virus, and various human non-immunoglobulin genes are presented in Table 4 and the Sequence Listing.

[0076] In one aspect, the invention does not rely on the specific selection of leader peptides and leader peptide coding sequences, but simply on the use of the same leader peptide coding sequence for all V gene segments. In another aspect, specific leader peptide coding sequences are selected for use in the polynucleotides and transgenic animals of the invention based on desired properties such as efficient amplification of antibody genes and subsequent antibody production in a desired expression system, such as a transgenic animal in which antibodies are elicited for a cell line, e.g., human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells.

[0077] Exemplary leader peptide coding sequences for use in various embodiments of the present invention are set forth in Tables 2, 3, and 4 below, which are presented in the Sequence Listing submitted herewith and are incorporated herein by reference in their entirety. The exemplary leader peptide coding sequences are presented in SEQ ID NOs: 1-70, 134, 136, and 137, along with the corresponding amino acid sequences in SEQ ID NOs: 71-133 and 135. These tables present the name of the gene from which the leader sequence is obtained or derived, as well as the sequence identifier numbers for the amino acid sequence of the leader peptide and the coding DNA sequence. Tables 2 and 3 also present the sequence reference numbers for obtaining the corresponding human genomic sequences from the ImMunoGeneTics [IMGT®] immunoglobulin sequence database. The genomic sequences include not only the coding sequences of the leader peptides, but also the introns associated with the sequences encoding the specific human leader peptides, i.e., the sequences encoding IGHV 3-23 (SEQ ID NO: 136) and IGKV 3-20 (SEQ ID NO: 137), which are presented in Table 2. The incorporation of such intron-containing genomic sequences, not just the coding sequences, may have advantages when incorporated into the transgenic mice of the present invention.

[0078]

Table 2

[0079]

Table 3

[0080]

Table 4

[0081] V gene segment In the art, human heavy chain leader / V gene segments are known and are available, for example, in Genbank accession numbers: AB019437 to AB019441. Matsuda et al. (1998) J. Exp. Med. 188:2151. See also LeFranc (2001) Exp. Clin. Immunogenet. 18:100. A subset of human heavy chain V gene segments for a particular purpose consists of IGHV genes 1-2, 1-3, 1-18, 1-46, 1-69, 2-5, 2-26, 3-7, 3-9, 3-11, 3-21, 3-23, 3-30, 3-33, 3-48, 3-66, 3-72, 3-74, 4-4, 4-28, 4-31, 4-30-4, 4-34, 4-39, 4-59, 4-61, 5-51, and 7-4-1. These gene segments, as well as human heavy chain D gene segments (IGHD), human heavy chain J gene segments (IGHJ), and human heavy chain constant region gene segments (IGHC) for use in the polynucleotides, methods, and transgenic animals of the present invention, can be obtained from publicly available sequence databases using the accession numbers presented in LeFranc (2001) Exp. Clin. Immunogenet. 18:100. See also OMIM number: 147070 (immunoglobulin heavy chain variable gene cluster; IGHV) for the description of variable regions, V gene segments, D gene segments, and J gene segments, and OMIM number: 147100 (IgG heavy chain locus; IGHG1) for the description of an exemplary heavy chain C gene segment (IgG).

[0082] In the art, human light chain kappa leader / V gene segments are known and are available in public databases such as Genbank. The kappa light chain locus is located at 2p12 (genomic coordinates 2:74,800,000 - 83,100,000) on human chromosome 2. The human light chain kappa V gene segments (IGKV) of interest include approximately 40 naturally occurring kappa V gene segments disclosed in LeFranc (2001) Exp. Clin. Immunogenet. 18:161. A subset of human light chain kappa V gene segments for a particular purpose consists of 1-5, 1-9, 1-12, 1-16, 1-17, 1-27, 1-33, 1-39, 1D-13, 1D-43, 2-28, 2-29, 2-30, 3-11, 3-15, 3-20, 3D-7, 4-1, and 6-21, which are IGKV genes. These gene segments, as well as the human light chain kappa J (IGKJ) segments and human light chain constant region gene segments (IGKC) for use in the nucleic acids, methods, and transgenic animals of the present invention, can be obtained from public sequence databases using the accession numbers presented in LeFranc (2001) Exp. Clin. Immunogenet. 18:161. Also, see OMIM number: 146980 (immunoglobulin kappa light chain variable gene cluster; IGKV) for the description of the variable region kappa V gene segments and kappa J gene segments, and OMIM number: 147200 (immunoglobulin kappa light chain constant region; IGKC) for the description of the kappa C gene segments.

[0083] The sequences of human V genes are available in Genbank and are readily accessible to those skilled in the art in public databases such as, inter alia, the ImMunoGeneTics [IMGT (registered trademark)] Immunoglobulin Sequence Database. Lefranc, M.-P. et al. (1999) Nucleic Acids Res., 27:209-212; Ruiz, M. et al. (2000) Nucleic Acids Res., 28:219-221; Lefranc, M.-P. (2001) Nucleic Acids Res., 29:207-209; Lefranc, M.-P., Nucleic Acids Res. (2003) 31:307-310; Lefranc, M.-P. et al. (2004) In Silico Biol., 5, 0006 [Epub], 5:45-60 (2005); Lefranc, M.-P. et al. (2005) Nucleic Acids Res., 33:D593-597; Lefranc, M.-P. et al. (2009) Nucleic Acids Res., 37:D1006-1012; Lefranc, M.-P. et al. (2015) Nucleic Acids Res., 43:D413-422 (2015). Such databases will enable those skilled in the art to obtain the coding sequences of all human V gene segments and their naturally associated leader peptide coding sequences. In addition to genomic sequences containing introns, the annotation for each V gene segment will enable the separation of the leader peptide coding sequence from the adjacent framework and CDR coding sequences and will enable the construction of simple coding sequences (without introns) if such sequences are desired. In one embodiment, the invention typically uses a human V gene associated with a leader peptide coding sequence that retains one or more introns derived from human germline sequences, such as an intron found typically within the sequence encoding the C-terminus of the leader peptide sequence.

[0084] Sequence variant In some embodiments, the gene element is derived from or based on a human gene, and the leader peptide coding sequence and / or V gene segment sequence includes a complete human genomic sequence that includes introns. In alternative embodiments, the gene element is derived from or based on a human gene, and the genomic leader peptide coding sequence and / or V gene segment sequence includes only the nucleic acid sequence encoding the polypeptide, and thus includes the corresponding sequence of the native spliced mRNA (or corresponding cDNA). The use of such intronless nucleic acid constructs has the advantage of reducing the size of the gene elements used in the construction of the variable region locus of the present invention.

[0085] In addition to the naturally occurring genomic sequences for the leader peptide coding sequences and the native splice products of these sequences, the nucleic acids of the present invention also include codon-optimized DNA sequences that encode leader peptides. Different organisms and different cells are known to preferentially use certain codons that encode a given amino acid residue. Athey et al. (2017) BMC Bioinform. 18:391. A codon-optimized sequence is a nucleic acid sequence in which the codons are modified to optimize protein expression, in this case, for a leader peptide within a cell in which it is expressed, such as a human cell or a Chinese hamster ovary (CHO) cell. See, for example, Mauro (2018) BioDrugs 32:69. Such codon-optimized nucleic acid constructs have the advantage of improving the efficiency of translation of the heavy or light chain while retaining the function of the leader peptide sequence within the heavy chain polypeptide or the light chain polypeptide, since the amino acid sequence is invariant. The leader peptide coding sequence for the osteonectin leader (SEQ ID NO: 54) is an example of such a codon-optimized sequence and has a 7 base pair change compared to the genomic sequence found at residues 10728-10778 of NG_042174.1. Experiments must be performed to determine that such codon optimization does not interfere with the gene rearrangement required to generate a functional antibody chain or result in unacceptably low levels of amplification during the process of sequencing the variable region of the antibody.

[0086] Method for producing a transgenic animal having an improved human immunoglobulin locus for generating a diverse pool of polyclonal antibodies The transgenic animals of the present invention, which contain immunoglobulin variable domain gene loci of human heavy and light chains in their genomes such that all leader / V gene segments for a particular chain contain a common identical leader peptide coding sequence, are essentially created as follows. Briefly, following the selection of a single leader or set of leaders, germline sequences for the IGHV and IGKV genes are selected such that they contain a promoter, 5' UTR sequence, coding sequence, recombination sequences, and flanking germline sequences based on functional annotation. The leader sequence is exchanged within the context of the germline sequence such that it does not disrupt the functionality of the sequences upstream or downstream. The variable regions are generated synthetically and assembled into a single construct using standard molecular biology techniques including, but not limited to, recombineering, golden gate assembly, and restriction enzyme-based ligation to generate arrays of variable domains. The array is then assembled into a further synthetic IGH or IGK sequence, or a germline IGH or IGK sequence, by the aforementioned techniques for generating targeting vectors. The targeting vector contains a positive drug selection sequence, homology arm sequences, and / or recombination sequences and is electroporated into embryonic stem cells together with a recombinase or nuclease construct. Drug selection is performed using standard procedures and individual clones are screened by internal PCR and external PCR, TLA sequencing, or genome-wide sequencing to confirm site-specific integration. Positive clones are further screened for 40XY g band karyotyping, chimeras are injected by standard blastocyst injection and transferred into pseudopregnant females. Offspring are genetically analyzed by any of the following techniques to confirm integration: PCR, Southern blot, TLA sequencing, or genome-wide sequencing. The progeny are maintained in heterozygosity or homozygosity and outcrossed with related alleles for downstream use.

[0087] Transgenic animals having an improved human immunoglobulin locus for the generation of diverse pools of polyclonal antibodies In another aspect, the invention provides transgenic animals, such as transgenic mice, having in their genomes a human heavy chain variable region and a human light chain variable region locus comprising a plurality of different leader / V gene segments that share a leader peptide coding sequence that is common for all given strands. Such animals may also include D gene segments (for heavy chains) and J gene segments that are capable of rearrangement by the leader / V gene segments to form rearranged heavy chain variable regions and light chain variable regions. The human variable region gene segments in the transgenic animals of the invention are typically naturally occurring sequences, including allotype variants, although engineered sequences may also be used. The human variable region gene segments may be located at the locus of the corresponding non-human variable region gene segments or at an exogenous locus. The transgenic animals of the invention may include the entire repertoire of human variable region gene segments, such as the entire repertoire of human V gene segments, or a subset of such segments.

[0088] Such animals may further comprise constant region gene segments capable of rearrangement by variable regions so as to form the heavy and light chains of a full-length antibody. In some embodiments, the constant region gene segments are derived from the human germline, and in other embodiments, the constant region gene segments are native to a transgenic animal, e.g., a mouse, and include mouse constant region genes. Transgenic animals containing a fully humanized immunoglobulin locus have the advantage of directly producing fully human antibodies that can be selected for use as therapeutic agents for humans without sequence modification. Transgenic animals containing a humanized variable region locus and an endogenous constant region produce chimeric antibodies that need to be modified to introduce human constant regions before they are suitable for use as therapeutic agents for humans, but the endogenous constant regions on the antibodies have the advantage of directing more efficient class switching and affinity maturation during the immune response in the transgenic animal. Murphy et al. (2014) Proc. Nat'l Acad. Sci. (USA) 111:5153.

[0089] The transgenic animals of the invention may contain a subset of such segments, such as the entire repertoire of human constant region gene segments, or only the IgG constant region gene segments. The human constant region gene segments of the transgenic animals of the invention are typically naturally occurring sequences that include allotype variants, but engineered sequences such as variants engineered to increase or decrease binding to certain Fcγ receptors, such as activating Fcγ receptors, may also be used.

[0090] In addition to the human variable region locus, the transgenic animals of the present invention may contain endogenous immunoglobulin variable region gene segments of the transgenic host animal. Such non-human gene segments may be in their native orientation in the transgenic animal, may be preferentially inactivated, or may be silent, or may be inverted so as to impair their function. Lee et al. (2014) Nat. Biotechnol. 32:356.

[0091] Method for generating antibodies using the transgenic animals of the present invention In another aspect, the present invention provides a method for generating antibodies using the transgenic animals of the present invention. The transgenic animals of the present invention, which contain the variable region immunoglobulin gene locus of the human heavy chain and / or human light chain together with a plurality of heavy chain and / or light chain leader / V gene segments having the same leader peptide coding sequence, can be immunized with the antigen of interest according to immunization protocols known in the art. Chen & Murawsky (2018) Front. Immunol. 9:460; Asensio et al. (2019) mAbs 11:870. For example, the protein antigen may be presented as a soluble protein, as a peptide fragment expressed on the surface of cells, as a DNA expression construct, or as a series or combination thereof. The antigen may be presented, for example, in a single administration or a series of administrations under a prime / boost protocol, such as multiple times per week for 4 weeks, or once every 4 weeks for 12 weeks. The antigen may be administered, for example, subcutaneously (e.g., to the plantar surface of the mouse foot or the base of the tail), or intraperitoneally. The antigen may be administered with an adjuvant such as alum, complete Freund's adjuvant (CFA), Seppic Montanide ISA50, or alhydrogel / muramyl dipeptide (ALD / MDP), or may be administered without these.

[0092] After an appropriate interval for the increase in antibody titer and affinity maturation, the antibody sequences are obtained from mice by conventional means such as the formation of hybridomas and the sequencing of the heavy and light chain variable domains for clones that produce antibodies with the desired properties, or the cloning and sequencing of single B cell antibodies.

[0093] When an antibody is produced in an animal such as a mouse that is intended for therapeutic use in humans and produces a chimeric antibody with a constant domain sequence derived from an animal, the human variable domain is reformatted into a construct that provides a human constant domain sequence.

[0094] Antibodies produced using the transgenic animals of the present invention In another aspect, the present invention provides antibodies produced using a method for generating the transgenic animals of the present invention. The human antibodies of the present invention may have an immunoglobulin locus humanized in both the variable and constant regions and be obtained from transgenic animals that directly yield fully human antibodies, or may be humanized only in the variable region and, when used as a therapeutic agent for humans, the constant region may be replaced by a human constant region using methods known in the art, and may be obtained from transgenic animals that provide chimeric human / animal antibodies.

[0095] Antibodies obtained by immunization of the transgenic animals of the present invention initially contain a pool of polyclonal antibodies of different sequences, from which individual antibodies can be selected for the desired properties. The pool of polyclonal antibodies of the present invention is derived from a diverse set of V gene segments and / or is more homogeneous than a pool of antibodies obtained from transgenic mice with conventional humanized immunoglobulins that associate a native leader peptide coding sequence with each V gene segment, and will contain individual antibodies derived from any given set of different V gene segments. The antibodies can be selected for any number of desired properties, or combinations of properties, depending on the intended use of the antibody.

[0096] Increased use and recovery of V genes results in increased sequence diversity, enabling the selection of antibodies without the sequence disadvantages that can have a detrimental impact on stability, developability, and production yields. For example, antibody stability and homology can be improved by selecting antibodies lacking sequences known to be susceptible to glycosylation (e.g., N-x-S / T and N-x-C), deamination (e.g., NG and NS motifs), isomerization (e.g., DG and DS motifs), and oxidation (e.g., W, F, M, or C residues). Lu et al. (2019) MAbs 11:45. Such sequence disadvantages can be engineered to be excluded from antibodies without being selected, but such sequence modifications must be investigated because they can interfere with binding to the antigen.

[0097] Increased use of V genes can also result in improved epitope diversity, i.e., increased variation at different loci on the antigen to which the antibody binds. Increased epitope diversity can enable the selection of antibodies with improved properties including, but not limited to, high affinity, pH-sensitive binding, cross-reactivity between species, cross-reactivity with related antigen sequences, specificity for blocking a specific binding partner, the ability to block the binding of two or more binding partners, the ability to bind without blocking the binding of one or more binding partners (so-called "non-blocking" antibodies), binding to antigens when expressed on the cell surface, binding to antigens on the cell surface that do not induce transmembrane signaling, binding to antigens on the cell surface that induce maximal transmembrane signaling, binding to antigens simultaneously with a second anti-antigen antibody, binding to denatured antigens, binding to antigens in tissue sections, etc. For example, cross-reactivity with human and animal antigens would be useful in therapeutic antibodies since the antibody could be used directly in the animal's toxicity model. Non-blocking antibodies can be used in therapeutic applications where the therapeutic mechanism does not require or is unable to reduce binding to a binding partner. Antibodies that can bind to antigens on the cell surface can be required for therapeutic methods that require such binding, such as delivery of a cytotoxic payload or transmembrane signaling. Antibodies that can bind to denatured antigens, antigens deposited on a plate, or can bind simultaneously with other anti-antigen antibodies can be used in a variety of assays such as ELISA, immunohistochemistry (IHC), and flow cytometry. Antibodies that specifically block only one interaction, or antibodies that block two or more interactions, can be used in therapeutic contexts where the specific pattern of blockade is mechanistically preferred.

[0098] Treatment method using an antibody produced using the transgenic animal of the present invention In another aspect, the present invention provides a method of treating, for example, human diseases using therapeutic antibodies obtained using the transgenic animals of the present invention. The antibodies of the present invention raised against a therapeutic target can be used to treat the corresponding disease associated with this target. For example, inflammatory cytokines can be used to generate antagonist antibodies that can be used to treat autoimmune and inflammatory disorders. See, for example, Singh et al. (2018) Curr. Clin. Pharmacol. 13:85. Such targets include, but are not limited to, IL-1β, IL-2, IL-4, IL-5, IL-6R, IL-13, IL-12 (p40 subunit), IL-17, IL-23 (p19 subunit), TNF-α, or any of their receptors. Typically, immuno-oncology targets, which are cell surface receptors involved in mediating the immune response against tumors, can be used to generate antibodies that can be used to treat cancer. Such targets include, but are not limited to, CTLA-4, PD-1, PD-L1, LAG3, TIM-3, TIGIT, ICOS, CD27, KIRm4-1BB (CD137), OX40 (CD134), and CD96. Tumor antigens and tumor-specific cell surface markers can also be used to generate antibodies that can be used to treat cancer. Such targets include, but are not limited to, HER-2, EGFR, VEGF, VEGFR2, fucosyl-GM1, mesothelin, CD19, CD20, CD30, CD33, CD38, CD52, and SLAMF7.

[0099] Examples of cancers that can be treated using the immunotherapies of the present disclosure include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or uveal malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal area cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, hematologic malignancies, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal / ureteral cancer, renal pelvic cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma cell types, pituitary adenoma, Kaposi sarcoma, epidermoid cancer, squamous cell carcinoma, environmentally induced cancers including cancers induced by asbestos, metastatic cancers, and any combination of the foregoing cancers.

[0100] Other cancers include hematologic malignancies including, for example, multiple myeloma, B cell lymphoma, Hodgkin lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T lymphoblastic lymphoma, and any combination of the foregoing cancers.

[0101] A polynucleotide comprising a plurality of human V gene segments, preceded by the same leader peptide coding sequence In another aspect, the invention provides a DNA construct comprising a polynucleotide, typically a plurality (i.e., two or more) of human heavy or light chain leader / V gene segments, wherein each leader / V gene segment contains the same leader peptide coding sequence. Such polynucleotides can be incorporated into larger polynucleotides that further include one or more D gene segments (for heavy chain variable region constructs) and one or more J gene segments. The heavy chain variable region locus can be incorporated into even larger polynucleotides that further include one or more human constant region gene segments, such as human IgG gene segments, such as human IgG1, IgG2, IgG3, or IgG4. The light chain variable region locus can be incorporated into even larger polynucleotides that further include one or more human constant region gene segments, such as the human κ light chain constant region sequence or the human λ light chain constant region sequence. The present disclosure relates to, for example, the following. [Item 1] A method for producing a non-human transgenic animal having a human immunoglobulin variable region locus, comprising introducing a plurality of human heavy chain leader / V gene segments into the genome of the non-human animal, wherein each of the plurality of human heavy chain leader / V gene segments contains the same first leader peptide coding sequence. [Item 2] The method according to item 1, wherein no additional human heavy chain leader / V gene segments other than the plurality of human heavy chain leader / V gene segments containing the same first leader peptide coding sequence are introduced into the genome of the non-human animal. [Item 3] A method for producing a non-human transgenic animal having a human immunoglobulin variable region locus, comprising introducing a plurality of human light chain leader / V gene segments into the genome of the non-human animal, wherein each of the human light chain leader / V gene segments contains the same second leader peptide coding sequence. [Item 4] The method according to item 3, wherein no additional human light chain leader / V gene segments other than the plurality of human light chain leader / V gene segments containing the same second leader peptide coding sequence are introduced into the genome of the non-human animal. [Item 5] The method according to item 1 or 2, further comprising introducing a plurality of human light chain leader / V gene segments into the genome of the non-human animal, wherein each of the human light chain leader / V gene segments contains the same second leader peptide coding sequence. [Item 6] The method according to item 3 or 4, further comprising introducing a plurality of human heavy chain leader / V gene segments into the genome of the non-human animal, wherein each of the human heavy chain leader / V gene segments contains the same first leader peptide coding sequence. [Item 7] The method according to item 5 or 6, wherein the first leader peptide coding sequence is not the same as the second leader peptide coding sequence. [Item 8] The method according to any one of items 1 to 7, wherein the first leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Item 9] The method according to item 8, wherein the first leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. [Item 10] The method according to item 8, wherein the first leader peptide sequence is selected from the group consisting of SEQ ID NOs: 71, 85, 86, and 93. [Item 11] The method according to item 9, wherein the first leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1, 15, 16, 27, and 136. [Item 12] The method according to item 10, wherein the first leader peptide sequence is SEQ ID NO: 86. [Item 13] The method according to item 11, wherein the first leader peptide coding sequence is SEQ ID NO: 16 or 136. [Item 14] The method according to item 13, wherein the first leader peptide coding sequence is SEQ ID NO: 136. [Item 15] The method according to any one of items 3 to 14, wherein the second leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Item 16] The method according to item 15, wherein the second leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. [Item 17] The method according to item 15, wherein the second leader peptide sequence is selected from the group consisting of SEQ ID NOs: 104 and 112. [Item 18] The method according to item 16, wherein the second leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 39, 49, and 137. [Item 19] The method according to item 17, wherein the second leader peptide sequence is SEQ ID NO: 112. [Item 20] The method according to item 18, wherein the second leader peptide coding sequence is SEQ ID NO: 49 or 137. [Item 21] The method according to item 20, wherein the second leader peptide coding sequence is SEQ ID NO: 137. [Item 22] The method according to any one of items 1 to 21, wherein the non-human animal is a mouse, a rat, or a cow. [Item 23] The method according to item 22, wherein the non-human animal is a mouse. [Item 24] The method according to any one of items 1 to 23, comprising introducing two or more naturally occurring human heavy chain V gene segments into a non-human animal. [Item 25] The method according to item 24, comprising introducing into a non-human animal the human heavy chain V gene segments IGHV 3-23; IGHV 5-51; IGHV 3-7; IGHV 1-2; IGHV 1-69-1; IGHV 3-48; IGHV 1-18; IGHV 1-46; IGHV 3-21; IGHV 3-30; IGHV 3-74; IGHV 4-39; IGHV 3-9; IGHV 2-5; IGHV 1-3; IGHV 4-4; IGHV 7-4-1; IGHV 3-66; and IGHV 1-24, and not introducing other heavy chain V gene segments. [Item 26] The method according to item 24, comprising introducing into a non-human animal all naturally occurring human heavy chain V gene segments. [Item 27] The method according to any one of items 1 to 26, further comprising introducing into a non-human animal one or more human heavy chain D gene segments and one or more human J gene segments. [Item 28] The method according to item 27, comprising introducing into a non-human animal all naturally occurring human heavy chain D gene segments. [Item 29] The method according to item 27 or 28, comprising introducing into a non-human animal all naturally occurring human heavy chain J gene segments. [Item 30] The method according to any one of items 1 to 29, comprising introducing into a non-human animal two or more naturally occurring human light chain V gene segments. [Item 31] The method according to item 30, comprising introducing into a non-human animal the human light chain V gene segments IGKV 1-39; IGKV 3-11; IGKV 1-33; IGKV 3-20; IGKV 4-1; IGKV 1-27; IGKV 1-5; IGKV 1-16; IGKV 1-12; IGKV 2-30; IGKV 3-15; IGKV 2-28; IGKV 1D-13; IGKV 1-17; IGKV 6-21; IGKV 1-9; and IGKV 1D-43, and not introducing other light chain V gene segments. [Item 32] The method according to item 30, comprising introducing into a non-human animal all naturally occurring human heavy chain V gene segments. [Item 33] The method according to any one of items 3 to 32, further comprising introducing into a non-human animal one or more human light chain J gene segments. [Item 34] The method according to item 33, comprising introducing into a non-human animal all naturally occurring human light chain J gene segments. [Item 35] The method according to any one of claims 1 to 34, further comprising introducing one or more human constant domain gene segments into a non-human animal. [Claim 36] The method according to claim 35, comprising introducing one or more human IgG constant domain gene segments into a non-human animal. [Claim 37] A non-human transgenic animal whose genome contains a plurality of human heavy chain leader / V gene segments, each of the human heavy chain leader / V gene segments containing the same first leader peptide coding sequence. [Claim 38] The non-human transgenic animal according to claim 37, wherein no additional human heavy chain leader / V gene segments other than the plurality of human heavy chain leader / V gene segments containing the same first leader peptide coding sequence are present in the genome of the non-human transgenic animal. [Claim 39] A non-human transgenic animal whose genome contains a plurality of human light chain leader / V gene segments, each of the human light chain leader / V gene segments containing the same second leader peptide coding sequence. [Claim 40] The non-human transgenic animal according to claim 39, wherein no additional human light chain leader / V gene segments other than the plurality of human light chain leader / V gene segments containing the same second leader peptide coding sequence are present in the genome of the non-human transgenic animal. [Claim 41] The non-human transgenic animal according to claim 39 or 40, whose genome further contains a plurality of human heavy chain leader / V gene segments, each of the human heavy chain leader / V gene segments containing the same first leader peptide coding sequence. [Claim 42] The non-human transgenic animal according to claim 41, wherein the first leader peptide coding sequence is not the same as the second leader peptide coding sequence. [Claim 43] The non-human transgenic animal according to any one of claims 37 to 42, wherein the first leader peptide coding sequence encodes a leader peptide selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Claim 44] The non-human transgenic animal according to claim 43, wherein the first leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. [Claim 45] The non-human transgenic animal according to claim 43, wherein the first leader peptide sequence is selected from the group consisting of SEQ ID NOs: 71, 85, 86, and 93. [Item 46] The non-human transgenic animal according to item 44, wherein the first leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1, 15, 16, 27, and 136. [Item 47] The non-human transgenic animal according to item 43, wherein the first leader peptide coding sequence is SEQ ID NO: 86. [Item 48] The non-human transgenic animal according to item 44, wherein the first leader peptide coding sequence is SEQ ID NO: 16 or 136. [Item 49] The non-human transgenic animal according to item 48, wherein the first leader peptide coding sequence is SEQ ID NO: 136. [Item 50] The non-human transgenic animal according to any one of items 37 to 49, wherein the second leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Item 51] The non-human transgenic animal according to item 50, wherein the second leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 1 to 70, 134, 136, and 137. [Item 52] The non-human transgenic animal according to item 50, wherein the second leader peptide sequence is selected from the group consisting of SEQ ID NOs: 104 and 112. [Item 53] The non-human transgenic animal according to item 51, wherein the second leader peptide coding sequence is selected from the group consisting of SEQ ID NOs: 39, 49, and 137. [Item 54] The non-human transgenic animal according to item 52, wherein the second leader peptide sequence is SEQ ID NO: 112. [Item 55] The non-human transgenic animal according to item 53, wherein the second leader peptide coding sequence is SEQ ID NO: 49 or 137. [Item 56] The non-human transgenic animal according to item 53, wherein the second leader peptide coding sequence is SEQ ID NO: 137. [Item 57] The non-human transgenic animal according to any one of items 37 to 56, which is a mouse, a rat, or a cow. [Item 58] The non-human transgenic animal according to item 57, which is a mouse. [Item 59] The non-human transgenic animal according to any one of items 37 to 58, the genome of which contains two or more naturally occurring human heavy chain V gene segments. [Item 60] The non-human transgenic animal according to item 59, whose genome contains the human heavy chain V gene segments IGHV 3-23; IGHV 5-51; IGHV 3-7; IGHV 1-2; IGHV 1-69-1; IGHV 3-48; IGHV 1-18; IGHV 1-46; IGHV 3-21; IGHV 3-30; IGHV 3-74; IGHV 4-39; IGHV 3-9; IGHV 2-5; IGHV 1-3; IGHV 4-4; IGHV 7-4-1; IGHV 3-66; and IGHV 1-24, and does not contain other human heavy chain V gene segments. [Item 61] The non-human transgenic animal according to item 59, whose genome contains all naturally occurring human heavy chain V gene segments. [Item 62] The non-human transgenic animal according to any one of items 37 to 61, whose genome further contains one or more human heavy chain D gene segments and one or more human J gene segments. [Item 63] The non-human transgenic animal according to item 62, whose genome contains all naturally occurring human heavy chain D gene segments. [Item 64] The non-human transgenic animal according to item 62 or 63, whose genome contains all naturally occurring human heavy chain J gene segments. [Item 65] The non-human transgenic animal according to any one of items 39 to 64, whose genome contains two or more naturally occurring human light chain V gene segments. [Item 66] The non-human transgenic animal according to item 65, whose genome contains the human light chain V gene segments IGKV 1-39; IGKV 3-11; IGKV 1-33; IGKV 3-20; IGKV 4-1; IGKV 1-27; IGKV 1-5; IGKV 1-16; IGKV 1-12; IGKV 2-30; IGKV 3-15; IGKV 2-28; IGKV 1D-13; IGKV 1-17; IGKV 6-21; IGKV 1-9; and IGKV 1D-43, and does not contain other human light chain V gene segments. [Item 67] The non-human transgenic animal according to item 65, whose genome contains all naturally occurring human heavy chain V gene segments. [Item 68] The non-human transgenic animal according to any one of items 39 to 67, whose genome further contains one or more human light chain J gene segments. [Item 69] The non-human transgenic animal according to item 68, the genome of which contains all naturally occurring human light chain J gene segments. [Item 70] The non-human transgenic animal according to any one of items 37 to 69, the genome of which further contains one or more human heavy chain constant domain gene segments. [Item 71] The non-human transgenic animal according to item 70, the genome of which contains one or more human IgG heavy chain constant region gene segments. [Item 72] A method for producing a human antibody or an antigen-binding fragment thereof, comprising: a. Administering an antigen of interest to a non-human transgenic animal according to any one of items 39 to 71; b. After step (a), obtaining nucleic acid sequences encoding the antigen-binding domains of the heavy and light chains of the antibody specific for the antigen, produced by the non-human transgenic animal; c. Expressing an antibody specific for the antigen, or an antigen-binding fragment thereof, from a gene construct containing one or both of the nucleic acid sequences obtained in step (b); and d. Isolating or purifying the antibody or antigen-binding fragment expressed in step (c). A method comprising the above steps. [Item 73] The method according to item 72, wherein the non-human transgenic animal is a mouse. [Item 74] A method for treating a subject having a disease, comprising administering to the subject an antibody or an antigen-binding fragment thereof produced by the method according to item 72 or 73. [Item 75] The treatment method according to item 74, wherein the subject is a human. [Item 76] The treatment method according to item 74 or 75, wherein the disease is an autoimmune disease, an infectious disease, a cardiovascular disease, or cancer. [Item 77] A polynucleotide comprising two or more human heavy or light chain leader / V gene segments containing the same leader peptide coding sequence. [Item 78] The polynucleotide according to item 77, wherein the two or more human heavy chain leader / V gene segments contain two or more different naturally occurring human V gene segments, and each leader / V gene segment contains the same first leader peptide coding sequence, and the first leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Item 79] The polynucleotide according to item 78, wherein the first leader peptide sequence is selected from the group consisting of SEQ ID NOs: 71, 85, 86, and 93. [Item 80] The polynucleotide according to item 79, wherein the first leader peptide sequence is SEQ ID NO: 86. [Item 81] The polynucleotide according to item 80, wherein two or more human light chain leader / V gene segments contain two or more different naturally occurring human V gene segments, and each leader / V gene segment contains the same second leader peptide coding sequence, and the second leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. [Item 82] The polynucleotide according to item 81, wherein the second leader peptide sequence is selected from the group consisting of SEQ ID NOs: 104 and 112. [Item 83] The polynucleotide according to item 82, wherein the second leader peptide sequence is SEQ ID NO: 112.

Example

[0102] The present invention is further illustrated by the following examples, which should not be construed as limiting. The content of all figures, as well as all references, patents, and patent application publications cited herein are hereby incorporated by reference into this specification explicitly. [Example 1]

[0103] Selection of the optimal leader peptide coding sequence Optimal leader peptide sequences, and optimal leader peptide coding sequences, for use in the methods, constructs, and animals of the present invention, were selected as follows. Briefly, an initial panel of potential leader peptide sequences was selected based on conservation and consensus of sequences within the group of all leader sequences, frequency and use in the human antibody repertoire, and maximization of sequence diversity across the initial panel. Also, both the amino acid sequences and DNA sequences were ranked based on the morbidity of their entire human antibody repertoire, and approved antibody drugs, such that such selection suggests sequences suitable for biologically desired sequences, commercial antibody-producing cell lines and methods, and use in therapeutic agents for humans. Subsequently, the panel of leader peptides was assayed for efficiency of recombinant protein expression by a corresponding assay comparing combinations of heavy chain leaders and light chain leaders to a control leader containing osteonectin (SEQ ID NO: 117), which is often used for this purpose. This initial assay enabled the inventors to exclude suboptimal leaders and select leader candidates sufficient for expression in vitro. The expression of two exemplary antibodies was determined using corresponding combinations of the panel of heavy chain leader sequences and light chain leader sequences selected to drive the expression of each of the heavy chain and light chain. The results showed that some leaders support expression at very low levels, while most leaders support sufficient expression at or above the level of the osteonectin leader sequence. The benefit of selecting a common set of leaders with good function for expression in vitro is the strategic flexibility in the use of common leaders within recombinant vectors, and the direct use of the heavy chain and light chain sequences for amplification by polymerase chain reaction of transcriptional activity for direct screening of top antibody candidates from animals, thereby enabling the incorporation of a recovery strategy with universal sequences together with a functional screening strategy downstream.Benefits include potentially improved expression over osteonectin, moving from animals to in vitro situations that improve array recovery, simplification of molecular biology strategies, and the ability to scale up methods for automation.

[0104] Following initial functional screening by in vitro expression, the leader panel was further refined. These criteria included an assessment of the genomic sequence complexity and length to determine whether they would be suitable for manipulation across multiple variable genes for incorporation into transgenic constructs. Blast for the exome confirmed that the sequences used for primer design were unique across mouse transcripts and aided specificity. Consideration was also given to functional disadvantages in recombinant situations to further refine the criteria for high-value leaders. For example, leader sequences were also removed to avoid their use as cryptic translation start sites when they included downstream methionine residues, such as IGKV 1-9 (SEQ ID NO: 97), and IGKV 1-39 (SEQ ID NO: 104).

[0105] In addition, the sequences of potential amplification primers for use with leader peptide coding sequences were analyzed for undesired secondary structures and sequence disadvantages that would affect the ability to design high-speed PCR strategies. These included primer sequences immediately adjacent to the variable domain framework in the 3' region of the leader, which are strategic regions to support full-length variable domain sequencing as the more 5' region creates longer sequences elucidated by sequencing.

[0106] Based on the above considerations, a heavy-chain leader peptide coding sequence for IGHV 3-23 (SEQ ID NOs: 16 and 136), encoding SEQ ID NO: 86, was selected for the methods, constructs, and heavy chains in the mouse of the present invention, and a light-chain leader peptide coding sequence for IGKV 3-20 (SEQ ID NOs: 49 and 137), encoding SEQ ID NO: 112, was similarly selected. In a preferred embodiment, the leader peptide genomic coding sequences of IGHV 3-23 (SEQ ID NO: 136) and IGKV 3-20 (SEQ ID NO: 137) are used for the heavy-chain and light-chain V gene segments, respectively. These sequences were determined to be optimal based on the criteria presented herein, but the use of the same leader peptide coding sequences is essentially beneficial regardless of which specific sequences are selected, so other sequences can also be employed in the methods, constructs, and mice of the present invention. [Example 2]

[0107] Selection of V gene segments of heavy and light chains V gene segments for use in the methods and mice of the present invention were selected as follows. Briefly, V gene segments for both heavy and light chains were selected based on the prevalence in the overall human antibody repertoire, antibody therapeutics entering Phase I clinical trials, and approved antibody drugs. These were also evaluated for chemical disadvantages such as the presence of methionine (especially within the CDRs) or unpaired cysteines, as well as sequence disadvantages such as DG and NG, the presence of glycosylation sites, and immunogenicity {all of which were observed experimentally and predicted by computer [e.g., EPIVAX® immunogenicity assessment software, EpiVax Inc., Providence, R.I., USA]}.

[0108] After consideration and deliberation of the above factors, the following 19 V gene segments (hIGHV): 3-23; 5-51; 3-7; 1-2; 1-69-1; 3-48; 1-18; 1-46; 3-21; 3-30; 3-74; 4-39; 3-9; 2-5; 1-3; 4-4; 7-4-1; 3-66; and 1-24 were selected for the heavy chain variable domain locus. The following 17 V gene segments (hIGKV): 1-39; 3-11; 1-33; 3-20; 4-1; 1-27; 1-5; 1-16; 1-12; 2-30; 3-15; 2-28; 1D-13; 1-17; 6-21; 1-9; and 1D-43 were selected for the light chain variable domain locus.

[0109] Functional variable domain genes are two exons that are variable but separated by introns of moderate length. Observation that the germline leader terminates within the second exon led to efforts to engineer a common leader for the IGH variable gene and the IGK variable gene. This enabled a universal method for the design of transgenes for all variable genes by including the germline DNA sequence for the common leader from ATG in exon 1 to the end of the common leader in exon 2. The universal strategy ensures that it may be possible to design each variable domain gene without introducing functional disadvantages such as abnormal splicing across introns or negative effects on the expression of the variable domain gene.

[0110] The contents of all figures, as well as all references, patents, and patent application publications cited in this application are hereby expressly incorporated by reference into this specification.

[0111] Equivalents: One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed herein. Such equivalents are intended to be encompassed by the following claims.

Claims

**Claim 1** A method for producing a non-human transgenic animal having a human immunoglobulin variable region locus, comprising: (a) a plurality of human heavy chain leader / V gene segments, each of the plurality of human heavy chain leader / V gene segments comprising the same first leader peptide coding sequence, and / or (b) a plurality of human light chain leader / V gene segments, each of the human light chain leader / V gene segments comprising the same second leader peptide coding sequence, introducing into the genome of the non-human animal, wherein the non-human animal is a mouse, a rat, or a cow. **Claim 2** The method according to claim 1, wherein no additional human heavy chain leader / V gene segments other than the plurality of human heavy chain leader / V gene segments comprising the same first leader peptide coding sequence are introduced into or present in the genome of the non-human animal. **Claim 3** The method according to claim 1 or 2, wherein no additional human light chain leader / V gene segments other than the plurality of human light chain leader / V gene segments comprising the same second leader peptide coding sequence are introduced into or present in the genome of the non-human animal. **Claim 4** The method according to any one of claims 1 to 3, wherein the first leader peptide coding sequence is not the same as the second leader peptide coding sequence. **Claim 5** The method according to any one of claims 1 to 4, wherein the first leader peptide coding sequence and / or the second leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135. **Claim 6** A non-human transgenic animal, the genome of which contains: (a) a plurality of human heavy chain leader / V gene segments, each of the human heavy chain leader / V gene segments comprising the same first leader peptide coding sequence, and / or (b) a plurality of human light chain leader / V gene segments, each of the human light chain leader / V gene segments comprising the same second leader peptide coding sequence, wherein the non-human transgenic animal is a mouse, a rat, or a cow. **Claim 7** The non-human transgenic animal according to claim 6, wherein no additional human heavy chain leader / V gene segments other than the plurality of human heavy chain leader / V gene segments containing the same first leader peptide coding sequence are present in the genome of the non-human transgenic animal.

8. The non-human transgenic animal according to claim 6 or 7, wherein no additional human light chain leader / V gene segments other than the plurality of human light chain leader / V gene segments containing the same second leader peptide coding sequence are present in the genome of the non-human transgenic animal.

9. The non-human transgenic animal according to any one of claims 6 to 8, wherein the first leader peptide coding sequence is not the same as the second leader peptide coding sequence.

10. The non-human transgenic animal according to any one of claims 6 to 9, wherein the first leader peptide coding sequence and / or the second leader peptide coding sequence encodes a leader peptide selected from the group consisting of SEQ ID NOs: 71 to 133 and 135.

11. A method for producing a human antibody or an antigen-binding fragment thereof, comprising: (a) administering an antigen of interest to a non-human transgenic animal according to any one of claims 6 to 10; (b) obtaining, after step (a), nucleic acid sequences encoding the antigen-binding domains of the heavy and light chains of an antibody specific for the antigen, produced by the non-human transgenic animal; (c) expressing an antibody specific for the antigen, or an antigen-binding fragment thereof, from a gene construct comprising one or both of the nucleic acid sequences obtained in step (b); and (d) isolating or purifying the antibody or antigen-binding fragment expressed in step (c).

12. The method according to claim 11, wherein the non-human transgenic animal is a mouse.

13. A polynucleotide comprising two or more human heavy or light chain leader / V gene segments containing the same leader peptide coding sequence.

14. The polynucleotide according to claim 13, wherein two or more human heavy chain leader / V gene segments comprise two or more different naturally-occurring human V gene segments, and each leader / V gene segment further comprises the same first leader peptide coding sequence, and the first leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135.

15. The polynucleotide according to claim 13, wherein two or more human light chain leader / V gene segments comprise two or more different naturally-occurring human V gene segments, and each leader / V gene segment further comprises the same second leader peptide coding sequence, and the second leader peptide coding sequence encodes a leader peptide sequence selected from the group consisting of SEQ ID NOs: 71 to 133 and 135.

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