Transgenic chicken that makes antibodies with long cdr-h3s stabilized by multiple disulfide bridges and diversified by gene conversion

Abstract

The present invention provides, among other things, a transgenic chicken. In some embodiments, the transgenic chicken comprises a B cell having an endogenous immunoglobulin heavy chain locus comprising: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain having a length of CDR3 in the range of 30 to 60 amino acids and containing at least two cysteine ​​residues; and (b) a plurality of pseudogenes operably linked to the functional immunoglobulin heavy chain gene and, by gene conversion, providing a nucleotide sequence to the nucleic acid encoding the heavy chain variable domain of (a), wherein the pseudogenes are located upstream or downstream of the functional immunoglobulin heavy chain gene.

Description

Technology Field

[0001] This application claims priority to U.S. provisional patent application No. 62 / 684,669 filed on June 13, 2018, and the entire specification is a reference to this application. Background Technology

[0002] The CDR3 region of the heavy chain of natural chicken antibodies can be considerably long, which appears to be primarily due to the frequent integration of two D segments into the heavy chain locus following VDJ recombination. The ability of the chicken antibody heavy chain to accommodate a long CDR3 makes chickens an attractive system for expressing modified antibodies with "long" CDRs—specifically, knobs or protuberances that can extend into cavities, such as the active site crevice of enzymes, ligand binding sites of GPCRs, or pockets of ion channels. One of the challenges in manipulating chickens to produce such "long CDR3" antibodies is how to diversify that region so that the chickens produce an antibody population with additional diversity within it.

[0003] A specific aspect of the present description relates to transgenic animals that produce antibodies having various extra-long cysteine-rich heavy chain CDR3 regions, e.g., heavy chain-only antibodies. The problem to be solved

[0004] The present invention provides, above all, a transgenic chicken comprising a B cell having an endogenous immunoglobulin heavy chain locus comprising: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain having a length of CDR3 in the range of 30 to 60 amino acids and containing at least two cysteine ​​residues; and (b) a plurality of pseudogenes operably linked to the functional immunoglobulin heavy chain gene and, by gene conversion, providing a nucleotide sequence to the nucleic acid encoding the heavy chain variable domain of (a), wherein the pseudogenes are located upstream or downstream of the functional immunoglobulin heavy chain gene.

[0005] In these animals, the nucleic acid encoding the heavy chain variable domain can be mutated by somatic hypermutation and, optionally, by gene conversion with a similar gene, resulting in a functional immunoglobulin heavy chain gene that expresses antibodies having various “knotty” CDR3s, wherein the “knotty” CDR3s are relatively long and form knobs or protrusions on the antibody binding surface. means of solving the problem

[0006] definition

[0007] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “assaying” are used interchangeably herein to refer to any form of measurement and include determining whether an element is present. These terms include both quantitative and / or qualitative determinations. Evaluations may be relative or absolute. “Determining the presence of” includes determining not only whether something is present but also determining the quantity of what is present.

[0008] The term “gene” above refers to a nucleic acid sequence consisting of a promoter region, a coding sequence, and a 3’UTR.

[0009] The terms “protein” and “polypeptide” are used interchangeably herein.

[0010] The term “nucleic acid” includes DNA, RNA, single-stranded or double-stranded DNA and chemical modifications thereof. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein.

[0011] The term “operably-linked” refers to the combination of nucleic acid sequences on a single nucleic acid fragment such that one function is influenced by another. For example, a promoter is operably linked to a coding sequence when it can influence the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Similarly, when an intron is operably linked to a coding sequence, the intron is spliced ​​from mRNA to provide for the expression of the coding sequence. “Unlinked” means that the related genetic elements are not closely related to each other and one function does not influence the other.

[0012] The term “homozygous” indicates that the same allele is present at the same locus on homologous chromosomes. In contrast, “heterozygous” indicates that different alleles are present at the same locus on homologous chromosomes. Transgenic animals may be homozygous for the transplanted gene, or hemizygous for the transplanted gene if there is no counterpart at the same locus on the same chromosome.

[0013] In relation to genes, the term “endogenous” indicates that a gene is unique to the cell; that is, it indicates that the gene exists at a specific locus in the genome of a non-modified cell. An endogenous gene may be a wild-type gene existing at a locus of a wild-type cell (as found in nature). An endogenous gene may be a modified endogenous gene if it exists at the same locus as a wild-type gene. An example of such a modified endogenous gene is a gene into which foreign nucleic acids have been inserted. Endogenous genes may exist in the nuclear genome, mitochondrial genome, etc.

[0014] The term “construct” above refers to recombinant nucleic acid, generally recombinant DNA, that is generated for the expression of specific nucleotide sequence(s) or used to construct other recombinant nucleotide sequences. The construct may exist in a vector or genome.

[0015] The term “recombination” above refers to a polynucleotide or polypeptide that does not occur naturally in a host cell. A recombinant molecule may contain two or more naturally occurring sequences linked together in a manner that does not occur naturally. A recombinant cell contains a recombinant polynucleotide or polypeptide. When a cell receives a recombinant nucleic acid, the nucleic acid is “exogenous” to the cell.

[0016] The above term “selectable marker” means a protein that can be expressed in a host that facilitates the selection of a host containing the introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, proteins that confer resistance to antimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol), proteins that confer metabolic benefits such as nutritional benefits to host cells, as well as proteins that confer functional or phenotypic benefits (e.g., cell division) to cells.

[0017] As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. This process includes both transcription and translation.

[0018] In the context of inserting a nucleic acid sequence into a cell, the term “introducing” includes “transfection” and “transformation” and all other methods of introducing a nucleic acid into a cell, wherein the nucleic acid sequence may be incorporated into the cell’s genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[0019] The term “coding sequence” above refers to a sequence of nucleic acids that, once transcribed and translated, produces a protein, for example, in vivo, when placed under the control of appropriate regulatory elements. As used herein, the coding sequence may have a continuous ORF or an ORF interrupted by the presence of an intron or a non-coding sequence. In this embodiment, the non-coding sequence is spliced ​​from pre-mRNA to produce mature mRNA.

[0020] In the context of replacing one gene locus with another, the term “replacing” refers to a single-step protocol or a multi-step protocol.

[0021] With respect to the insertion of a nucleic acid sequence into a cell, the term “introduced” means “transfection,” “transformation,” or “transduction,” and includes the meaning of integrating the nucleic acid sequence into a eukaryotic or prokaryotic cell, wherein the nucleic acid sequence may exist temporarily in the cell or be integrated into the cell’s genome (e.g., chromosome, plasmid, chloroplast, or mitochondrial DNA) and converted into an autonomous replicon.

[0022] The above term “plurality” means at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000, or at least 10,000 or at least 50,000. In certain cases, the plurality includes at least 10 to 50. In other embodiments, the plurality may be at least 50 to 1,000.

[0023] As used herein, the term “isolated” refers to cells cultured in a test tube in relation to cells. Where an animal is described as containing isolated cells, such isolated cells were cultured in a test tube and then transplanted into the animal.

[0024] The terms “progeny” or “off-spring” above refer to all future generations that are descendants of a specific animal or cell. Therefore, offspring that are animals of any consecutive generations are included herein, and F1, F2, F3, generations, etc., are included in this definition.

[0025] The phrase “transgenic animal” above refers to an animal containing cells that possess foreign nucleic acids (i.e., recombinant nucleic acids not native to the animal). The foreign nucleic acids may be present in all cells of the animal or in some cells of the animal, but not all. The foreign nucleic acid molecule is referred to as a “transgene” and may contain one or more genes, cDNA, etc. By inserting a transgene into a modified oocyte or an early embryonic cell, the resulting transgenic animal can become fully transgenic and stably deliver the foreign nucleic acids in the germline. Alternatively, the foreign nucleic acids may be introduced to produce a partially transgenic animal, for example, by transplanting recombinant cells or tissues containing them into the animal. Alternatively, a transgenic animal may be produced by delivering a nucleus from a genetically modified somatic cell or by delivering genetically modified pluripotent cells, such as embryonic stem cells or primordial germ cells. A chimeric animal may possess cells donated by another animal in the germline, in which case the offspring of said animal may be heterozygous with respect to the chromosomes of the donated cells. If said donated cells contain exogenous nucleic acids (i.e., nucleic acids not endogenous to the cell), the offspring of said chimeric animal may be “transgenic,” wherein a “transgenic” animal is an animal composed of cells containing foreign nucleic acids (i.e., recombinant nucleic acids not endogenous to the animal). said foreign nucleic acid molecules may be referred to herein as “transgenes.”

[0026] The above terms, such as “hybrid animal” and “transgenic hybrid animal,” are used interchangeably herein to refer to an animal obtained from the mating of a first animal having specific characteristics and a second animal having specific characteristics. For example, the hybrid animal of the present invention may refer to a transgenic offspring obtained from the mating of a transgenic first animal that produces a common light chain and a second transgenic animal that produces a synthetic heavy chain. The hybrid animal may be immunized and may be used as a source for the production of antigen-specific antibodies.

[0027] The terms “antibody” and “immunoglobulin” are used interchangeably herein. These terms are well understood by those in the art and refer to a protein composed of one or more polypeptides that specifically bind to an antigen. One form of antibody consists of the basic structural unit of the antibody. This form is a tetramer and consists of two pairs of identical antibody chains, each pair having one light chain and one heavy chain. In each pair, the variable regions of the light and heavy chains together are responsible for binding to the antigen, while the constant region functions as an antibody effector.

[0028] The recognized immunoglobulin polypeptide comprises kappa and lambda light chains and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains or equivalents of other species. The full-length immunoglobulin “light chain” (about 25 kDa or about 214 amino acids) comprises a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. The full-length immunoglobulin “heavy chain” (about 50 kDa or about 446 amino acids), similarly comprises a variable region (about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (about 330 amino acids).

[0029] The terms “antibody” and “immunoglobulin” include any isotype of antibody or immunoglobulin that specifically binds to an antigen, and include, but are not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, etc. The antibody may be additionally conjugated to other moieties, such as members of specific binding pairs, such as biotin (a member of a biotin-avidin specific binding pair), for example. The antibody may also be conjugated to a solid support, including but not limited to polystyrene plates or beads. In addition, the above terms include Fab', Fv, F(ab') 2 and / or other antibody fragments and monoclonal antibodies that maintain specific binding to antigens.

[0030] Antibodies may exist in various other forms, including, for example, Fv, Fab, and (Fab')2, as well as dual-functional (i.e., dual-specific) hybrid antibodies (e.g., Lanzavecchia and Scheidegger, Eur. J. Immunol. 1987, 17(1):105-111) and single-stranded antibodies (e.g., Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85(16):5879-5883 and Bird et al., Science. 1988, 242(4877):423-426, which are incorporated herein by reference) (generally, see Hood et al., "Immunology", Benjamin, NY, 2nd ed. 1984, and Hunkapiller and Hood, Nature. 1986, 323(6083):15-16).

[0031] Chimeric antibodies are antibodies that are typically constructed by genetic engineering from variable and constant region genes of antibodies belonging to different species, with light and heavy chain genes. For example, the variable segment of a gene from a chicken or rabbit monoclonal antibody can be linked to a human constant segment, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain of a chicken or rabbit antibody and the constant or effector domain of a human antibody (e.g., the anti-Tac chimeric antibody produced by cells of ATCC deposit number CRL 9688), but other mammalian species may be used.

[0032] The term “pseudogene” is used to describe a non-transcribed nucleic acid region containing an open reading frame that may or may not contain start and / or stop codons. An amino acid sequence may be “encoded” by the pseudogene in that the nucleotide sequence of the open reading frame can be translated in silico to produce an amino acid sequence. In the context of the heavy and light chain immunoglobulin loci, the pseudogene does not contain a promoter region, a recombination signal sequence, or a leader sequence.

[0033] The above terms “upstream” and “downstream” are used in relation to the direction of transfer.

[0034] The above term “specific binding” refers to the ability of an antibody to preferentially bind to a specific analyte present in a homogeneous mixture of different analytes. In certain embodiments, the specific binding interaction will distinguish between desirable and undesirable analytes in a sample, and in some embodiments, will be about 10 to 100 times (e.g., about 1,000 times or more than 10,000 times).

[0035] In certain embodiments, the affinity between the antibody and the analyte when specifically bound to the antibody / analyte complex is 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, or about 10 -12 It is characterized by a KD (dissociation constant) of less than M.

[0036] The "variable region" of a heavy or light chain antibody is the N-terminal maturation domain of the chain containing CDR1, CDR2, CD3, and the framework region (where CDR stands for "complementarity determining region"). Both the heavy and light chains of the antibody contain the variable region. All domain, CDR, and residue numbers are assigned based on sequence alignment and structural knowledge. The identification and numbering of framework and CDR residues are described by Chothia et al. (Chotia et al., J. Mol. Biol. 1998, 278 (2): 457-479).

[0037] VH is the variable domain of the antibody heavy chain. VL is the variable domain of the antibody light chain.

[0038] The terms “gene” and “locus” are used interchangeably herein. Neither term implies that the gene is actively transcribed or intact. Both terms include inactive genes.

[0039] As used herein, a “chimeric” chicken is a chicken containing a significant number of genetically distinct cells from at least two sources. A chimeric animal can be created by transplanting cells from one animal into the embryo of another animal, or by transplanting cultured cells (e.g., having a modified genome) into an embryo. The transplanted cells may be harvested from the culture before being inserted into the host embryo. The embryo develops into an animal, and the resulting animal may contain the host cells as well as the transplanted cells. If the donated cells contain exogenous nucleic acids (i.e., nucleic acids that are not endogenous to the cell), the offspring of the chimeric animal may be “transgenic,” wherein a “transgenic” animal is an animal composed of cells containing foreign nucleic acids (i.e., recombinant nucleic acids that are not endogenous to the animal). The foreign nucleic acid molecules may be referred to herein as “transgenes.”

[0040] The term “inactivated” above is intended to denote a non-expressed gene, meaning that the protein encoded by the gene is not expressed. A gene can be inactivated by removing part of the gene’s coding sequence and / or regulator sequence. A destroyed gene, e.g., a “knockout,” is a type of inactivated gene. A locus containing a once-expressed endogenous sequence that has been replaced by a human immunoglobulin sequence that is subsequently expressed contains an inactivated endogenous gene. As such, a locus containing an expressed human immunoglobulin sequence may have an inactivated endogenous immunoglobulin gene when the endogenous immunoglobulin gene is replaced by a human immunoglobulin sequence. In many cases, this can be accomplished by knocking out the endogenous sequence (e.g., by deleting at least part of the sequence) and then inserting the human immunoglobulin sequence into the position that the endogenous sequence once occupied.

[0041] The term “recombination” above refers to a polynucleotide or polypeptide that does not occur naturally in a host cell. A recombinant molecule may contain two or more naturally occurring sequences linked together in a manner that does not occur naturally. A recombinant cell contains a recombinant polynucleotide or polypeptide. When a cell receives a recombinant nucleic acid, the nucleic acid is “exogenous” to the cell.

[0042] The term “genetically linked” refers to two genetic elements located on the same chromosome that tend to be inherited together during meiosis (i.e., the elements have a recombination frequency of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%). Two closely linked genetic elements are less likely to separate into different chromosomes during chromosomal crossover events (or “recombination”). The probability of two genetically linked elements separating during recombination depends on the amount of sequence between the two elements and can be calculated as a percentage of probability called the “recombination frequency.”

[0043] The above term “autonomous heavy chain variable domain” means a heavy chain variable domain capable of folding and binding to epitopes autonomously, that is, without aggregating and / or associated light chains. “Heavy chain-only” or “HCO” antibodies, shark antibodies, VHH antibodies, camilids, and single-domain antibodies are all examples of antibodies containing an autonomous heavy chain variable domain. Several strategies for producing such antibodies have been reviewed, for example, in Janssens et al (Proc. Natl. Acad. Sci. 2006 103:15130-5), Bruggemann et al (Crit. Rev. Immunol. 2006 26:377-90), Zou et al (J. Immunol. 2005 175:3769-79), and Nguyen et al (Immunology 2003 109: 93-101). Autonomous heavy chain variable domains can be created by introducing camelizing substitutions into the variable domain of VH antibodies. Brief explanation of the drawing

[0044] A skilled technician will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present invention in any way. Figure 1 shows the crystal structure of BLV1H12 Fab. This antibody contains a very long two-beta-stranded stalk with a disulfide-rich “knob”. FIG. 2 illustrates how a heavy chain having a knotty CDR3 can be diversified in B cells of a transgenic chicken. In this embodiment, the endogenous immunoglobulin heavy chain gene locus comprises: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain (“knotty functional variable domain” or “kfV”) having a length of 30-60 amino acids and containing at least two cysteine ​​residues; and (b) a plurality of pseudo-genes (P1-P4) operably linked to the functional immunoglobulin light chain gene and, by gene conversion, providing nucleotide sequences to the nucleic acid encoding the heavy chain variable domain of (a), wherein the pseudo-genes are located upstream or downstream of the functional immunoglobulin light chain gene. In this example, the pseudo-gene that diversifies the coding sequence for the CDR3 region of the heavy chain variable domain by gene conversion also has a length in the range of 30-60 amino acids and encodes an amino acid sequence containing at least two cysteine ​​residues (indicated separately). Specific details for implementing the invention

[0045] Description of exemplary embodiments

[0046] Before the present invention is further described, it should be understood that the invention is not limited to the specific embodiments described and, of course, may be modified. Furthermore, since the scope of the invention is limited only by the appended claims, it should be understood that the terms used herein are merely for describing specific embodiments and are not intended to be limiting.

[0047] Where a range of values ​​is provided, unless otherwise specified in the context, each intermediate value is understood to be up to one-tenth of the lower limit unit, and between the upper and lower limits of the range and any other mentioned or described value of the mentioned range is included in the present invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Any method and material similar or equivalent to that described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference to describe and explain the methods and / or materials in which the publications are cited.

[0049] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “and,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes multiple cells, and a reference to “a candidate agent” includes one or more candidate agents and equivalents thereof known to those skilled in the art. It should be noted that the claims may be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a precedence basis for the use of exclusive terms such as “solely,” “only,” etc., in connection with the citation of claim elements or the use of “negative” limitations.

[0050] The publications discussed herein are provided solely for disclosure prior to the filing date of this application. Nothing in this application shall be construed as an acknowledgment that the present invention is not qualified to precede such publications by prior art. Additionally, the provided publication dates may differ from the actual publication dates, which require individual verification.

[0051] All publications and patents cited herein are incorporated by reference as specifically and individually indicated where each individual publication or patent is incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials cited therein. All citations of publications are intended for disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention is not qualified to precede such publications due to prior art. Additionally, the provided publication dates may differ from the actual publication dates requiring individual verification.

[0052] As will be apparent to those skilled in the art upon reading this description, each individual embodiment described and illustrated herein has distinct components and features that can be easily separated from or combined with the features of some other embodiments without departing from the scope or spirit of the invention. All mentioned methods may be performed in the order of the mentioned events or any other logically possible order.

[0053] As mentioned above, a transgenic animal is provided. In certain embodiments, the animal may be any non-human animal having a relatively small number of light chain genes, or an animal that uses genetic modification to develop a primary antigen repertoire, and thus the animal may be one of various other animals. In one embodiment, the animal may be a bird, a member of the Galliformes order such as a chicken or turkey, or a member of the Anseriformes order such as a duck or goose, or a mammal, a member of the Lagomorpha order such as a rabbit, or a domestic animal such as a cow, sheep, pig, or goat.

[0054] Part of the present invention relates to transgenic chickens containing one or more transgenic genes. Since the nucleotide sequences of immunoglobulin loci of many animals and methods for modifying the genomes of such animals are known, the general concepts described below can be easily applied to any suitable animal, particularly to animals that use genetic modification to develop a primary antigen repertoire. The generation of antibody diversity by gene conversion between operantly linked (upstream) analogs containing variable regions different from the variable regions of transcribed immunoglobulin heavy or light chain genes is as follows, for example, various such as Butler (Rev. Sci. Tech. 1998 17: 43-70), Bucchini (Nature 1987 326: 409-11), Knight (Adv. Immunol. 1994 56: 179-218), Langman (Res. Immunol. 1993 144: 422-46), Masteller (Int. Rev. Immunol. 1997 15: 185-206), Reynaud (Cell 1989 59: 171-83), and Ratcliffe (Dev. Comp. Immunol. 2006 30: 101-118). It is explained in the publication.

[0055] As mentioned above, a transgenic chicken is provided. In some embodiments, the transgenic chicken comprises a B cell having an endogenous immunoglobulin heavy chain locus comprising: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain having a length of CDR3 in the range of 30 to 60 amino acids and containing at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues; and (b) a plurality of pseudogenes operably linked to the functional immunoglobulin light chain gene and, by gene conversion, provide a nucleotide sequence to a nucleic acid encoding a heavy chain variable domain of (a), wherein the pseudogenes are located upstream or downstream of the functional immunoglobulin heavy chain gene. In some embodiments, the chicken non-B cell comprises an endogenous immunoglobulin heavy chain gene locus comprising a VH segment, a D cluster, a J segment, and a plurality of upstream pseudogenes, wherein each D segment in the D cluster encodes a different sequence in the range of 30-60 amino acids in length and comprises at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues.In these embodiments, the functional gene of (a) is generated from VDJ recombination between a VH segment, a D cluster, and a J segment in B cells, wherein each D segment of the D cluster encodes a different sequence ranging in length from 30 to 60 amino acids and independently contains at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues. In other words, the D segments of the D cluster are all different from one another, and each D segment encodes a CDR3 sequence that is expected to be long and heavily folded as a result of disulfide bonds within that region. Since the D segment selected during VDJ recombination is largely random, the heavy chain CDR3 sequence must be diversified in this animal, as different B cells will contain different D segments after VDJ recombination. For example, in some embodiments, if the D cluster has 50 D segments, the B cell must contain 50 heavy chain CDR3 coding sequences before somatic hypermutation and / or gene conversion creates additional mutations. The heavy chain CDR3 must have a "cysteine ​​knot" or "knottin" motif present in some mammalian growth factors, cytokines, and some toxins. Classical notin has three disulfide bridges. Natural chicken antibodies also feature disulfides in H3, but generally have only one bridge, sometimes two, in the short H3. Antibodies that kneadb knots or knobs are described, for example, in Wang et al. Cell 153: 1379-1393. Bovine antibodies generally have a "stalk and knob" structure that can be maintained in this system (e.g., see Haakenson Front. Immunol. 2019 9: 1262 and Wang et al. supra).The crystal structures of several small antibody Fab fragments having very long CDR H3 regions have been reported (e.g., see Wang, supra and Stanfield et al, Sci Immunol 2016: aaf7962). The CDR H3 regions of these antibodies were found to adopt the same general structure, consisting of a β-ribbon stalk supporting a knob region containing one type I β-turn, one conserved disulfide bond, and three antiparallel β strands. The supported knob region extends far from the traditional antigen-binding surface generated by other CDR I antibodies, thus making it possible to bind to the antigen with a concave epitope.

[0056] In another embodiment, chicken non-B cells contain the functional immunoglobulin heavy chain gene of (a) and the analogue gene of (b). In these embodiments, the functional immunoglobulin heavy chain may be identical in all chicken cells (although it is expressed only in B cells). In these embodiments, the heavy chain CDR3 region may be diversified by somatic hypermutation and / or gene conversion.

[0057] The above-mentioned pseudogene may or may not diversify the heavy chain CDR3 coding sequence of the functional gene. Accordingly, in some embodiments, the above-mentioned pseudogene does not include a sequence corresponding to the CDR3 region of the heavy chain variable domain of (a). In these embodiments, the CDR3 coding sequence may be diversified by somatic hypermutation alone. In other embodiments, the above-mentioned pseudogene may include a sequence that diversifies the coding sequence for the CDR3 region of the heavy chain variable domain of (a) by gene transformation. In these embodiments, the sequence of the pseudogene that diversifies the coding sequence for the CDR3 region of the heavy chain variable domain of (a) by gene transformation encodes an amino acid sequence with a length in the range of 30 to 60 amino acids and includes at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues. In other words, the sequence donated from the above-mentioned pseudogene to the functional gene may also include a long cysteine-rich coding sequence.

[0058] In any embodiment, the CDR3 encoded by the functional heavy chain gene may be in the range of 30 to 60 amino acids in length and contain 3 to 10 cysteine ​​residues. Similarly, the pseudogene may encode an amino acid sequence in the range of 30 to 60 amino acids in length and containing at least 3 to 10 cysteine ​​residues. In these embodiments, the functional V region and the HC CDR3 of the pseudogene may be designed to have a "knob and stalk" structure as shown in FIG. 1, wherein the stalk sequence is predicted using a secondary structure prediction algorithm, and the knob sequence may differ for each pseudogene, and the sequence encoding the knob in each pseudogene varies and includes multiple cys codons to promote knob formation. In many embodiments, the sequence may have 4, 6, 8, or 10 cystes. In these embodiments, the CDR3 region encoded by the functional V is the Wang, supra It may be any sequence shown in Figure S1, and the CDR3 region encoded by the similar gene is Wang in Figure S1, supra It may be another CDR3 sequence shown in.

[0059] In some embodiments, the heavy chain variable domain of (a) may be an autonomous heavy chain (AHC) variable domain. As mentioned above, there are various ways to implement the autonomous heavy chain (AHC) variable domain. One method is, for example, the camelization of a human antibody.

[0060] Typical “natural” antibodies from humans and mice consist of heavy and light chains. In the absence of the light chain, the heavy chain of these antibodies does not fold properly and aggregates. “Camelization” refers to the process in which the VH domain (a term describing the binding domain type of human and mouse IgG) is modified so that it can bind to an epitope on its own (i.e., without a light chain) without aggregating. The term “camelization” was coined for this process because camels (e.g., camels, llamas, etc.) produce functional antibodies without a light chain in which a single N-terminal domain (VHH) can fully bind to an antigen. These autonomous heavy-chain antibodies possess high stability and solubility. Sharks and other cartilaginous fish also produce VHH antibodies.

[0061] Similar to conventional VH domains, autonomous heavy-chain antibodies consist of four framework regions (FRs) that form the core structure of the immunoglobulin domain and three complementarity determining regions (CDRs) involved in antigen binding. For example, see Conrath et al., Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH, J Mol Biol, 2005, 350: 112-25. Sequence comparison of human and camel variable domains induced a camelizing process involving the transfer of several hallmark residues from the agglutination-resistant VHH domain to the human VH domain. For example, see Davies et al., 'Camelising' human antibody fragments: NMR studies on VH domains, FEBS Lett, 1994, 339: 285-90. For example, a characteristic feature of VHH is the presence of amino acid substitutions at four FR2 positions (positions 37, 44, 45, and 47; Kabat numbering) that are conserved in the existing VH domain and involved in the formation of a hydrophobic interface with the VL domain. Specifically, the sequences of camel FR and the human VH3 family were remarkably similar, except for three residues of FR2 (44, 45, and 47), which are generally highly conserved in the VH domain. See, for example, Conrath et al., 2005. These solvent-exposed residues (mostly hydrophilic in camels) are located at the preceding light chain interface and interfere with binding to the VL domain. Non-specific binding of VH by interface to the above light chain variable domain (VL) was prevented through amino acid mutations in frameworks 2 and 4 (Val37F, G44E, L45R, W47G, and W103R).Da Silva et al, Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity J Mol Biol. 2004Jul9;340(3):525-42 참조. 낙타화를 위한 다른 전략은 Tanha et al Protein Eng Des Sel. 2006 Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach Nov;19(11):503-9 및 Davies et al, Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng. 1996 Jun;9(6):531-7에 설명되어 있다.

[0062] Similar mutation approaches to humanize VHH have also been attempted. For example, see Conrath et al., 2005. Also, see Vincke et al., General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold, J Biol Chem, 2009, 284: 3273-84. Although the increased hydrophilicity of VHH depends primarily on the aforementioned changes in the prior VL interface, some amino acids at positions forming a slightly hydrophobic patch in the existing VH domain in contact with the CH1 domain are also changed to hydrophilic residues in VHH. For example, see Lesk et al., Elbow motion in the immunoglobulins involves a molecular ball-and-socket joint, Nature, 1988 Sep 8, 335 (6186): 188-90. See also Muyldermans et al., Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains, Protein Eng, 1994 Sep, 7(9): 1129-35.

[0063] As such, in some embodiments, any VH antibody may be camelized by performing camel amino acid substitution of one or more of positions 37, 44, 45 and 47 in FR2, and optionally any hydrophobic residue at position 103 (Kabat number) of FR4, which is a non-hydrophobic residue. The camelization amino acid substitution may include substitution of hydrophobic residues such as glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp) at one or more positions where non-hydrophobic residues are present.

[0064] Heavy chain-only antibodies possess an autonomous heavy chain variable domain, also known as a heavy chain variable domain, which can fold and bind to an epitope autonomously—that is, without aggregation and / or associated light chains. Strategies for producing antibodies containing autonomous heavy chain variable domains are, for example, Janssens et al (Proc. Natl. Acad. Sci. 2006 103:15130-5), Br It was reviewed in ggemann et al (Crit. Rev. Immunol. 2006 26:377-90), Zou et al (J. Immunol. 2005 175:3769-79) and Nguyen et al (Immunology 2003 109: 93-101).

[0065] In some embodiments, the autonomous heavy chain variable domain of (a) is a camelized human heavy chain variable domain having a CDR3 of length 30 to 60 amino acids and containing at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues. This variable domain may be, for example, a camelized human monoclonal antibody, many of which are known in the art. In other embodiments, the autonomous heavy chain variable domain of (a) is encoded by a human germline heavy chain V segment, and the D segment encodes an amino acid sequence of length in the range of 30 to 60 amino acids and contains at least two (e.g., up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) cysteine ​​residues and a human J segment, wherein the variable domain contains up to 10 camelized amino acid substitutions.

[0066] In some embodiments, the pseudogene of (b) encodes a human germline antibody sequence (excluding any D segment). In some embodiments, the pseudogene is less than 600 nucleotides in length, for example, 300 to 600 nucleotides in length.

[0067] In some embodiments, the sequence encoding FR1, FR2, and FR3, and optionally FR4, when present in the pseudogene of (b), is combined and is at least 90% identical to the combined sequence encoding the corresponding FR1, FR2, and FR3, and optionally FR4 in the variable domain of (a). For each individual FR of the pseudogene, the level of sequence identification may be at least 80%, for example, at least 90% in many cases, or at least 95%. In some embodiments, the sequence encoding the CDR in the pseudogene of (b) is 90% or less identical to the sequence encoding the corresponding CDR in the variable domain of (a). In other words, in some embodiments, there may be more diversity in the CDR encoded by the pseudogene compared to the FW sequence encoded by the pseudogene.

[0068] In any embodiment, the endogenous immunoglobulin heavy chain locus may encode a heavy chain containing a CH1 deletion. In these embodiments, the genome of the animal may also contain a knockout light chain immunoglobulin gene. Alternatively, the genome of the animal may contain a light chain immunoglobulin gene encoding a truncated light chain containing an invariant region rather than a variable region.

[0069] In any embodiment, the transgenic chicken may be homozygous or heterozygous with respect to the gene locus.

[0070] Additionally, a method is provided comprising the steps of: (a) immunizing a transgenic animal with an antigen; and (b) obtaining an antibody from the animal that specifically binds to the antigen. The antibody may be polyclonal or monoclonal. In these embodiments, the method may further comprise the steps of: (c) creating a hybridoma using B cells of a transgenic animal; and (d) screening the hybridoma to identify the hybridoma that produces an antibody that specifically binds to the antigen. Alternatively, the method may comprise the step of (c) screening B cells without creating a hybridoma to identify B cells that produce an antibody that specifically binds to the antigen. In any screening method, the method may comprise the step of amplifying at least a heavy chain variable region-encoding nucleic acid from B cells of a transgenic animal using PCR, and expressing a recombinant antibody using said amplified nucleic acid.

[0071] Monoclonal or polyclonal antibodies produced by transgenic animals are also provided, wherein the antibodies have various toxin-like HC CDR3s and, in some embodiments, may be autonomous heavy chain (AHC) variable domain antibodies.

[0072] In addition, B cells isolated from animals are provided.

[0073] The above-mentioned transgenic animal contains a functional immunoglobulin heavy chain expressed to produce an antibody heavy chain (i.e., transcribed to produce mRNA that is subsequently translated), and is operably linked to a functional heavy chain gene, a plurality of different pseudo-gene heavy chain variable regions (in this case, chickens and many species are immediately upstream), wherein the pseudo-gene variable region operably links the functional immunoglobulin heavy chain by altering the sequence of the functional immunoglobulin heavy chain gene by gene conversion (i.e., by replacing the sequence of the functional immunoglobulin heavy chain variable region with the sequence of the pseudo-gene). In the above-mentioned transgenic animal, gene conversion between the functional immunoglobulin heavy chain gene variable region and the pseudo-gene variable region alters the sequence of the functional immunoglobulin heavy chain gene variable region by a single codon less than the entire length of the variable region. In certain cases, the pseudo-gene variable region may donate the sequence of at least one CDR (e.g., CDR1, CDR2, or CDR3) from the pseudo-gene variable region to the variable region of the functional gene. Therefore, the heavy chains of antibodies produced by transgenic animals are encoded by all sequences donated from the pseudogenetic variable region to the variable region of the functional heavy chain gene. Since different sequences are donated from different cells of the animal, the animal's antibody repertoire is determined by which sequences are donated from the pseudogenetic variable region to the variable region of the functional gene.

[0074] In some embodiments, the framework segments of the human functional gene and the similar gene may be identical or nearly identical to each other, and the CDR segments of the functional gene and the similar gene may be different, while a gene transition may occur between the CDR segment of the similar gene and the germline sequence. Additionally, the length of the CDR may vary. In certain embodiments, an antibody having a CDR of a length outside these ranges is conceived. The heavy chain CDR1 may have a length in the range of 6 to 12 amino acid residues, the heavy chain CDR2 may have a length in the range of 4 to 12 amino acid residues, and the heavy chain CDR3 may have a length in the range of 30 to 60 amino acid residues.

[0075] In some embodiments, the nucleotide sequence and / or amino acid sequence of the introduced transcriptional variable region may be human, that is, may contain the nucleotide and / or amino acid sequence of a human antibody or germline sequence. In these embodiments, the CDR and the framework may both be human. In other embodiments, the nucleotide sequence and / or amino acid sequence of the introduced transcriptional variable region may not be human, but instead may be at least 80% identical, at least 90% identical, at least 95% identical, or more identical to a human sequence. For example, the introduced transcriptional variable region may include one or more nucleotide or amino acid substitutions relative to a human sequence. Any germline human VH segment may be selected from the following: VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81. For a description of different germline sequences, see PCT / WO 2005 / 005604.

[0076] In some embodiments, a portion of the heavy chain locus comprising the invariant region, a portion of the intron region, and the 3'UTR of the functional gene may be endogenous to the animal and the remainder of the heavy chain locus, and the functional gene, the remainder of the intron, and the pseudogene may be exogenous to the animal, that is, recombinantly produced in such a way that the functional gene is generated and the pseudogene can donate sequences to the functional gene by genetic transformation and is introduced into an animal adjacent to the invariant domain, the portion of the intron, and the 3'UTR. In certain cases, the heavy chain locus of the animal is in operable linkage: an intron region, an invariant domain-encoding region, and a 3' non-translation region; said intron region, invariant domain-encoding region, and 3' non-translation region are endogenous to the genome of the transgenic animal and a plurality of pseudogene heavy chain variable regions, wherein the plurality of pseudogene heavy chain variable regions are exogenous to the genome of the transgenic animal.

[0077] In certain embodiments, antibodies produced by individual transgenic animals may contain an endogenous constant domain and a variable domain that is exogenous to said animal. Because the endogenous constant domain may be used in these embodiments, said antibodies may still undergo class switching and affinity maturation, which allows said animal to undergo normal immune system development and generate a normal immune response. In certain embodiments, transgenic chickens have three endogenous constant domains at heavy chain loci encoding IgM, IgY, and IgA. In the early stages of B cell development, B cells express IgM. As affinity maturation progresses, class switching converts the constant domain to IgY or IgA. IgY provides humoral immunity to both adults and newborn chicks, receiving approximately 200 mg of IgY through reserves accumulated in the yolk. IgA is found primarily in lymphoid tissues (e.g., spleen, Peyer's patch, and Harderian glands) and oviducts.

[0078] The number of introduced pseudogenetic variable regions present at light chain and / or heavy chain gene loci may vary and, in certain embodiments, may be in the range of 1 to 50, e.g., 2 to 50 or 10 to 25. In certain embodiments, at least one of the plurality of pseudogenetic light chain variable regions (e.g., at least 2, at least 3, at least 5, at least 10) may be reversed with respect to the transcribed light chain variable region. Likewise, in certain embodiments, at least one of the plurality of pseudogenetic heavy chain variable regions (e.g., at least 2, at least 3, at least 5, at least 10) may be reversed with respect to the heavy chain transcribed variable region. In certain embodiments, the plurality of pseudogenetic variable regions may not be alternately oriented and may contain a series of at least 5 or at least 10 adjacent pseudogenetic regions that are opposite to the transcribed variable region in certain cases. In one embodiment, the closest similar gene region from the transcribed variable region is in the same direction as the transcribed variable region, and the similar gene region between the closest region and the transcribed variable region is in the opposite direction with respect to the transcribed variable region.

[0079] A pseudogene typically comprises a sequence of at least 50, at least 100, at least 200, or at least 300 adjacent nucleotides that are at least 80% identical to the sequence of the transcribed region, e.g., at least 85% identical, at least 90% identical, or at least 95% identical. In some embodiments, the framework sequence of the pseudogene is at least 90% identical to the framework sequence corresponding to the functional gene, and the CDR has less sequence identity.

[0080] The aforementioned transgenic animals can be created by recombinantly modifying the animal genome. Methods for producing transgenic animals, e.g., mice and chickens, are known, and methods for modifying the genomes of animals, particularly using gene conversion, are also known (e.g., for birds, see Sayegh, Vet. Immunol. Immunopathol. 1999 72:31-7 and Kamihira, Adv. Biochem. Eng. Biotechnol. 2004 91: 171-89; for rabbits, see Bosze, Transgenic Res. 2003 12:541-53 and Fan, Pathol. Int. 1999 49: 583-94; for cattle, see Salamone J. Biotechnol. 2006 124: 469-72), as well as the structures and / or sequences of germline immunoglobulin heavy and light chain loci of many species (e.g., Butler Rev Sci Tech 1998 17:43-70 and Ratcliffe Dev Comp Immunol 2006 30: 101-118), the animals described above can be produced by the conventional methods given herein. Methods for producing transgenic chickens are known. For example, see 8,592,644, US8,889,662, Collarini et al (Poult Sci. 2015 94: 799-803), van de Lavoir (Nature. 2006 441: 766-9) and Schusser et al (Proc Natl Acad Sci US A. 2013 110: 20170-5).

[0081] A method for producing antibodies containing an autonomous heavy chain (AHC) variable domain is also provided. In some embodiments, the method may include the step of immunizing a transgenic animal as described above with an antigen, and if the antibody is polyclonal, the method may include the step of isolating the antibody from the animal's hemorrhage. If the animal is homozygous for a common light chain sequence, all antibodies in the polyclonal antisera must be autonomous heavy chain (AHC) variable domain antibodies. If a monoclonal antibody is required, the method comprises the steps of: b) creating a hybridoma using cells of the immunized transgenic animal; c) screening the hybridoma to identify an antigen-specific hybridoma; and d) isolating an antigen-specific antibody from the antigen-specific hybridoma. Alternatively, B cells may be screened.

[0082] In certain embodiments, animals may be immunized to the following: GD2, EGF-R, CEA, CD52, CD20, Lym-1, CD6, complement activating receptor (CAR), EGP40, VEGF, tumor-associated glycoprotein TAG-72 AFP (alpha-fetoprotein), BLyS (TNF and APOL-related ligand), CA125 (carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2 (T-cell surface antigen), CD3 (heteromultimer associated with TCR), CD4, CD11a (integrin alpha-L), CD14 (monocyte differentiation antigen), CD20, CD22 (B-cell Receptor), CD23 (low-affinity IgE receptor), CD25 (IL-2 receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), CD44v6 (leukocyte-like mediator), CD52 (CAMPATH-1), CD80 (costimulator for CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin (cytokine A11), HER2 / neu, HER3, HLA-DR, HLA-DR10, HLA Class II, IgE, GPiib / iiia (integrin), integrin aVβ3, integrins a4β1 and a4β7, integrin β2, IFN-gamma,Therapeutic antibodies can be produced through IL-1β, IL-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15, KDR (VEGFR-2), Lewis, mesothelin, MUC1, MUC18, NCAM (neural cell adhesion molecule), oncofetal fibronectin, PDGFβR (beta platelet-derived growth factor receptor), PMSA, renal carcinoma antigen G250, RSV, E-Selectin, TGFbeta1, TGFbeta2, TNFα, DR4, DR5, DR6, VAP-1 (vascular adhesion protein 1), or VEGF, etc.

[0083] The above antigen may be administered to a transgenic host animal in any convenient manner, with or without an adjuvant, and may be administered according to a predetermined schedule.

[0084] After immunization, the serum or milk of the immunized transgenic animal may be fractionated for the purification of pharmaceutical-grade polyclonal antibodies specific to the antigen. In the case of transgenic birds, the egg yolk may be fractionated to produce antibodies. The concentrated and purified immunoglobulin fraction may be obtained by chromatography (affinity, ion exchange, gel filtration, etc.), selective precipitation using salts such as ammonium sulfate, organic solvents such as ethanol, or polymers such as polyethylene glycol.

[0085] To produce monoclonal antibodies, antibody-producing cells, e.g., splenocytes, may be isolated from the immunized transgenic animal and used for cell fusion with a cell line transformed for hybridoma production, or cDNA encoding the antibody may be cloned by standard molecular biology techniques and expressed in transfected cells. Procedures for producing monoclonal antibodies are well established in the art. For example, see European Patent Application 0 583 980 A1, US Patent No. 4,977,081, WO 97 / 16537, and EP 0 491 057 B1, the disclosures of which are incorporated herein by reference. In vitro production of monoclonal antibodies from cloned cDNA molecules is described by Andris-Widhopf et al., J Immunol Methods 242:159 (2000), and by Burton, Immunotechnology 1:87 (1995), the descriptions of which are incorporated herein by reference.

[0086] If the above antibody does not yet contain a human framework domain, the method may further include a step of humanizing the antibody, and this method may include a step of preparing a chimeric antibody by swapping the constant domain of the antibody with a human constant domain, as well as, in specific cases, a step of humanizing the variable domain of the antibody by, for example, CDR transplantation or repackaging. Humanization is described as follows: Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), US Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; It may be carried out in accordance with the methods of 4,816,567, PCT / :US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755; WO90 / 14443, WO90 / 14424, WO90 / 14430, EP 229246, each of which is incorporated herein in its entirety, including the references cited herein.

[0087] Thus, a method is also provided comprising, in addition to a transgenic animal, the steps of immunizing said transgenic animal with an antigen and obtaining an antibody from the transgenic animal that specifically binds to the antigen. The method comprises the steps of: creating a hybridoma using cells of the transgenic animal; and screening said hybridoma to identify a hybridoma that produces an antibody that specifically binds to the antigen. Alternatively, B cells may be screened without creating a hybridoma.

[0088] The heavy chain variable domain of the above antibody is “naturally” produced by the animal’s immune system. In certain cases, such antibodies may be post-translated modified (e.g., glycosylated) by host cells and may have glycosylation patterns and compositions characteristic of transgenic animal species.

[0089] The sequence of the antigen-specific binding domain of the antibody produced by the transgenic animal described above can be obtained relatively simply because, if desired, all or any coding sequence for a diversified population of heavy chain variable domains can be amplified from cDNA using a pair of PCR primers. Since the specificity and affinity of each antibody must be determined solely by the amino acid sequence of the heavy chain variable domain, there is no need to identify or sequence the cognate light chain. Therefore, the amino acid sequence for the antigen-specific heavy chain variable domain should be relatively easy to obtain. As mentioned above, in some cases, B cells or hybridomas can be functionally screened to select cells expressing antigen-specific heavy chains. The heavy chain variable domain coding sequences are obtained simultaneously from enriched or non-enriched B cell populations (e.g., PBMCs) en masse) can be amplified. When sequences are amplified in a non-enriched B cell population, sequences encoding antigen-specific variable domains must be identifiable because they are expressed more than non-antigen-specific sequences (due to B cell activation) and potentially belong to more diverse clades. Furthermore, since these heavy chains do not require specific light chains for binding, there is no need to determine which light chains pair with which heavy chains before performing subsequent operations.

Claims

Claim 1 A transgenic chicken comprising a B cell having an endogenous immunoglobulin heavy chain locus including the following: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain having a length of 30 to 60 amino acids and containing at least two cysteine ​​residues; and (b) a plurality of pseudogenes operably linked to the functional immunoglobulin light chain gene and providing nucleotide sequences to the nucleic acid encoding the heavy chain variable domain of (a) by gene conversion, wherein the pseudogenes are located upstream or downstream of the functional immunoglobulin heavy chain gene. Claim 2 A transgenic chicken according to claim 1, wherein the non-B cells of the chicken comprise an endogenous immunoglobulin heavy chain locus comprising a VH segment, a D cluster, a J segment, and a plurality of upstream similar genes, and wherein each D segment in the D cluster encodes a different sequence in the range of 30-60 amino acids in length and comprises at least two cysteine ​​residues. Claim 3 A transgenic chicken according to claim 1, characterized in that the non-B cells of the chicken include the functional immunoglobulin heavy chain gene of (a) and the similar gene of (b). Claim 4 A transgenic chicken according to claim 1, characterized in that the similar gene does not include a sequence corresponding to the CDR3 region of the heavy chain variable domain of (a). Claim 5 A transgenic chicken according to claim 1, characterized in that the similar gene comprises a sequence that diversifies the coding sequence for the CDR3 region of the heavy chain variable domain of (a) by gene conversion. Claim 6 A transgenic chicken according to claim 5, characterized in that the pseudo-gene sequence that diversifies the coding sequence for the CDR3 region of the heavy chain variable domain of (a) by gene conversion is 30-60 amino acids long and encodes an amino acid sequence containing at least two cysteine ​​residues. Claim 7 A transgenic chicken according to claim 1, characterized in that the heavy chain variable domain of (a) is an autonomous heavy chain (AHC) variable domain. Claim 8 A transgenic chicken according to claim 7, wherein the AHC variable domain of (a) is a camelized human heavy chain variable domain having a CDR3 of 30-60 amino acid length and containing at least two cysteine ​​residues, the AHC variable domain of (a) is a camelized human monoclonal antibody, the AHC variable domain of (a) is encoded by a human germline heavy chain V segment, a D segment encoding an amino acid sequence in the range of 30-60 amino acids and containing at least two cysteine ​​residues, and the AHC variable domain comprises up to 10 camelized amino acid substituents. Claim 9 A transgenic chicken according to claim 1, characterized in that the similar gene of (b) above encodes a human germline antibody sequence. Claim 10 A transgenic chicken according to claim 9, characterized in that the similar gene has a length of less than 600 nucleotides. Claim 11 A transgenic chicken according to claim 1, characterized in that the endogenous immunoglobulin heavy chain locus encodes a heavy chain containing a CH1 deletion, or the chicken genome contains a light chain immunoglobulin gene that has been knocked out. Claim 12 A transgenic chicken according to claim 1, characterized in that the genome of the animal comprises a light chain immunoglobulin gene encoding a truncated light chain that includes an invariant region rather than a variable region. Claim 13 (a) a step of immunizing a transgenic animal of any one of claims 1 to 12 with an antigen; (b) a step of obtaining an antibody from said animal that specifically binds to said antigen. Claim 14 A monoclonal or polyclonal antibody produced by a transgenic animal of any one of claims 1 to 12, wherein the monoclonal or polyclonal antibody is an autonomous heavy chain (AHC) variable domain antibody. Claim 15 B cells isolated from an animal according to any one of paragraphs 1 to 12.

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