Antibody production method

Abstract

The present disclosure relates to the production of antigen binding molecules. Animals, methods, nucleic acids, and cells for producing antigen binding molecules are disclosed. Also disclosed are animals comprising a sequence that induces a suppressed immune response.

Description

[0001] This application claims priority to US 63 / 513385, filed July 13, 2023; US 63 / 609417, filed December 13, 2023; and US63 / 645312, filed May 10, 2024, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0002] This disclosure relates to the fields of cell and molecular biology and immunology. More specifically, the present invention relates to the production of antigen-binding molecules, particularly in applications of therapy, prevention, diagnosis, imaging, and research.

[0003] background Animal immunization is widely used to develop monoclonal antibodies for both therapeutic and diagnostic purposes. Traditional mouse hybridoma technology involves fusing spleen cells collected from immunized mice with immortalized myeloma cells to generate antibody-secreting hybridoma cell lines. While mice are the most commonly used species for antibody production through animal immunization, other animal species, such as rabbits and goats, have also been used.

[0004] A key limitation of classic whole-protein target immunization approaches in antibody development is that they only produce antibodies against the target, with limited regulation of the binding site (i.e., the target epitope on the target protein), and the antibody response primarily targets the immunodominant epitope. Alternative strategies, such as immunization with small antigens (representing the desired binding region of the target protein), allow for greater regulation of the antibody binding site, but at the cost of an increased risk that the antibody will not bind to the protein in vivo (i.e., it will not recognize the protein in its "native" form), for example, because the antigen sequence is unavailable or presented differently in the natively folded protein. Therefore, extensive screening is required to find antibodies with high binding affinity for the target in vivo, but success is not guaranteed.

[0005] Existing methods for producing antibodies through animal immunization are not efficient and reliable for producing monoclonal antibodies with the desired functional properties associated with their target antigens.

[0006] Overview In a first aspect, the present invention provides an animal comprising an endogenous nucleotide sequence for inducing suppression of a primary humoral immune response.

[0007] In some embodiments, the animal contains an endogenous nucleotide sequence that induces the inhibition of the expression of one or more genes involved in generating a primary humoral immune response, or the activity of their products.

[0008] In some embodiments, the animal contains an endogenous nucleotide sequence that inducibly inhibits the expression of one or more genes involved in B cell maturation, or the activity of their products.

[0009] In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression of one or both genes selected from IGHM and IGHD, or the activity of their products. In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression of IGHM and / or IGHD, or the activity of their products.

[0010] In some embodiments, the animal contains an endogenous nucleotide sequence that, via recombinase-mediated disruption of the expression of one or more genes involved in generating a primary humoral immune response.

[0011] In some embodiments, the endogenous nucleotide sequence includes a target sequence of a recombinase located flanking all or part of the nucleotide sequence of a gene involved in generating a primary humoral immune response.

[0012] In some embodiments, the animal contains an endogenous nucleotide sequence that provides the induced expression or activity of the recombinase.

[0013] In some embodiments, an endogenous nucleotide sequence for inducible expression or activity of the recombinase is provided, encoding a conditional system for regulating the expression or activity of the recombinase.

[0014] In some embodiments, the target sequence of the recombinase is a loxP sequence, and the recombinase is a Cre recombinase.

[0015] In some embodiments, the animal contains an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

[0016] In some embodiments, the animal is a mouse, rat, or rabbit.

[0017] In some embodiments, the endogenous nucleotide sequence includes an endogenous nucleotide sequence encoding a recombinase target sequence located flanking one or more exons of IGHM and IGHD.

[0018] In some embodiments, the animal comprises an endogenous nucleotide sequence that comprises or consists of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:4.

[0019] In some embodiments, the animal contains an endogenous nucleotide sequence encoding a conditional system for regulating the expression and / or activity of Cre recombinase.

[0020] In some embodiments, the expression of the Cre recombinase is controlled by a promoter that drives expression in B-cell lineage cells.

[0021] In some embodiments, the animal contains an endogenous nucleotide sequence that comprises or consists of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO: 6.

[0022] The present invention also provides a method for producing antigen-binding molecules, comprising administering a peptide / polypeptide or a nucleic acid encoding a peptide / polypeptide to an animal of the present invention.

[0023] In some embodiments, the method includes: (i) administering to an animal a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) Treating animals to suppress their ability to initiate a primary immune response; and (iii) administering a second peptide / polypeptide or a nucleic acid encoding a second peptide / polypeptide to the animal, wherein the second peptide / polypeptide contains a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0024] In some embodiments, the method includes: (i) administering a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide to an animal, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) administering to the animal a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide, wherein the second peptide / polypeptide comprises a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence; and (iii) Treat the animal to suppress its ability to initiate a primary immune response.

[0025] In some implementations, treating an animal to suppress its ability to initiate a primary immune response includes administering a reagent that induces the expression or activity of a recombinase.

[0026] A method for producing antigen-binding molecules is also provided, wherein the method includes: (i) administering a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide to an animal, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) Treating the animal to suppress its ability to initiate a primary immune response; and (iii) administering a second peptide / polypeptide or a nucleic acid encoding a second peptide / polypeptide to the animal, wherein the second peptide / polypeptide contains a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0027] In some embodiments, the method includes treating the animal before, during, and / or after administration of a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide to suppress its ability to initiate a primary immune response.

[0028] In some embodiments, the method includes treating an animal to suppress its ability to initiate a primary immune response, including administering an agent that inhibits the expression of one or more genes involved in initiating a primary humoral immune response or the activity of their products. In some embodiments, treating an animal to suppress its ability to initiate a primary immune response includes administering an agent that reduces the number / proportion of naive B cells in the animal, reduces the number or proportion of cells expressing IgM and / or IgD in the animal, and / or inhibits B cell maturation. In some embodiments, the agent inhibits the expression of one or more genes selected from IGHM and IGHD or the activity of their products.

[0029] In some embodiments, the reagent inhibits the activity of IgM and / or IgD. In some embodiments, the reagent is selected from or includes antibodies, antigen-binding molecules, peptides, decoy receptors, aptamers, chelating agents, or small molecules.

[0030] In some embodiments, the reagent inhibits the expression of IGHM and / or IGHD. In some embodiments, the reagent is selected from or includes RNAi, siRNA, antisense nucleic acid, antisense oligonucleotide, or gene editing system.

[0031] In some embodiments, the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments. In some embodiments, the animal is a mouse, rat, or rabbit.

[0032] In some embodiments, the method further includes producing a hybridoma capable of generating an antigen-binding molecule that binds to the target protein / protein complex.

[0033] In some embodiments, the method further includes isolating one or more antigen-binding molecules capable of binding to proteins containing a target amino acid sequence.

[0034] In some embodiments, the method further includes formulating an antigen-binding molecule capable of binding to a protein containing a target amino acid sequence into a pharmaceutical composition.

[0035] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that induces inhibition of the primary humoral immune response.

[0036] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that induces the inhibition of the expression of one or more genes involved in initiating a primary humoral immune response, or the activity of their products.

[0037] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that inducibly inhibits the expression of one or more genes involved in B cell maturation, or the activity of their products.

[0038] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that induces the inhibition of the expression of one or both genes selected from IGHM and IGHD, or the activity of their products.

[0039] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that, via a recombinase-mediated disruption of the expression of one or more genes involved in initiating a primary humoral immune response.

[0040] The present invention also provides a nucleic acid or multiple nucleic acids comprising a nucleotide sequence, wherein the nucleotide sequence comprises all or part of a nucleotide sequence encoding a gene involved in initiating a primary humoral immune response, flanked by a target sequence of a recombinase.

[0041] In some embodiments, the nucleic acid / multiple nucleic acids further includes a nucleotide sequence encoding a conditional system for regulating recombinase expression or activity.

[0042] In some embodiments, the target sequence of the recombinase is a loxP sequence, and the recombinase is a Cre recombinase.

[0043] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that includes a target sequence of a recombinase located flanking one or more exons of IGHM and IGHD.

[0044] In some embodiments, the nucleic acid or multiple nucleic acids comprises a nucleotide sequence that contains, or is composed of, a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:4.

[0045] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that encodes a conditional system for regulating the expression and / or activity of Cre recombinase.

[0046] In some embodiments, the expression of the Cre recombinase is controlled by a promoter that drives expression in B-cell lineage cells.

[0047] In some embodiments, the nucleic acid or multiple nucleic acids comprise a nucleotide sequence that contains, or is composed of, a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:6.

[0048] This disclosure also provides one or more vectors containing nucleic acids or multiple nucleic acids according to this disclosure.

[0049] The present invention also provides a cell comprising one or more nucleic acids according to the present disclosure, or one or more vectors. In some embodiments, the cell comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

[0050] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an embryonic stem cell. In some embodiments, the cell is a mouse cell, rat cell, or rabbit cell.

[0051] In some embodiments, the animal is a hyperimmune mouse or a mouse with a hyperimmune phenotype.

[0052] In some embodiments, the animal is a humanized mouse.

[0053] illustrate This disclosure broadly relates to the production of antibodies (especially IgG antibodies, and ultimately preferably monoclonal IgG antibodies) having certain desired functional properties, said antibodies being products of binding to a target protein / protein complex region.

[0054] This can be achieved by: (i) immunizing the animal with a first peptide / peptide containing the target amino acid sequence (i.e., (a) the amino acid sequence of the target protein / protein complex, or (b) an amino acid sequence similar to (a)) to induce a primary immune response against the target amino acid sequence; (ii) inhibiting the animal’s ability to initiate a primary immune response; and subsequently (iii) immunizing the animal again with a second peptide / peptide containing the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0055] In some implementations, this can be achieved by: (i) immunizing an animal containing an endogenous nucleotide sequence (which can inducibly inhibit a primary humoral immune response) with a first peptide / peptide containing the target amino acid sequence (i.e., the amino acid sequence of (a) the target protein / protein complex, or (b) an amino acid sequence similar to (a) the amino acid sequence) to induce a primary immune response against the target amino acid sequence; (ii) inhibiting the animal's ability to initiate a primary immune response; and subsequently (iii) immunizing the animal with a second peptide / peptide containing the target amino acid sequence or a similar amino acid sequence.

[0056] Suppressing an animal's ability to initiate a primary immune response facilitates a secondary immune response targeting the amino acid sequence upon administration of a second peptide / peptide. The secondary immune response is associated with the production of antibodies, particularly IgG antibodies, which bind to their targets with high affinity.

[0057] Suppressing primary immune responses and / or promoting secondary immune responses Various aspects and embodiments of this disclosure relate to, for example, using reagents to suppress primary immune responses (e.g., primary humoral immune responses) in animals. In some embodiments, the animal comprises endogenous nucleotide sequences that induce suppression of primary immune responses.

[0058] The aim is to favor a secondary humoral immune response to the target amino acid sequence presented by the second peptide / peptide, rather than a primary humoral immune response to the second peptide / peptide region that is not present in the first peptide / peptide.

[0059] In other words, its purpose is to suppress the primary humoral immune response to regions of the second peptide / peptide other than the target amino acid sequence, and / or enhance the secondary humoral immune response to the target amino acid sequence present in the second peptide / peptide.

[0060] It is important to clarify that the purpose is not to suppress the primary humoral immune response to the first peptide / peptide. In particular, the purpose is not to prevent immunoglobulin isotype conversion induced by the administration of the first peptide / peptide, or to eliminate cells that are activated / stimulated and proliferate by the administration of the first peptide / peptide, or derivative cells of such cells (i.e., progeny of cells that are activated / stimulated and proliferate by the administration of the first peptide / peptide).

[0061] In some embodiments, the ability to inhibit the initiation of a primary humoral immune response does not inhibit the development of cells expressing IgG, IgA, and / or IgE in cells activated / stimulated by administration of a first peptide / peptide. In some embodiments, the development of cells activated / stimulated by administration of a first peptide / peptide into plasma B cells and / or memory B cells is not inhibited.

[0062] In some embodiments, suppressing the ability to initiate a primary humoral immune response does not deplete cells expressing IgG, IgA, and / or IgE (e.g., cells proliferating due to activation / stimulation by administration of a first peptide / peptide, which then undergo immunoglobulin isotype conversion to produce cells expressing IgG, IgA, and / or IgE). In some embodiments, plasma B cells and / or memory B cells (e.g., cells proliferating due to activation / stimulation by administration of a first peptide / peptide, which then undergo immunoglobulin isotype conversion to produce plasma B cells and / or memory B cells) are not depleted.

[0063] After the introduction of a second peptide / polypeptide, memory B cells that can recognize antigen-binding molecules of the target amino acid sequence tend to mature with affinity rather than generating a primary humoral immune response to regions outside the target amino acid sequence.

[0064] In the above embodiments, the reagent can suppress the primary immune response to a sequence present in the second peptide / polypeptide but not in the first peptide / polypeptide.

[0065] Primary and secondary immune responses are components of adaptive immune responses. Adaptive immune responses are described, for example, in Janeway's *Immunobiology*, 9th edition; Murphy et al., 2017 (Garland Science, Taylor & Francis), specifically in Part IV.

[0066] The primary immune response refers to the response of the adaptive immune system of a subject after initial exposure to an antigen.

[0067] The triggering of the primary immune response typically involves the uptake, processing, and presentation of MHC class II molecules by antigen-presenting cells (APCs), such as dendritic cells. In the presence of appropriate co-stimulation, naive T cells containing specific T cell receptors (TCRs) of MHC class II:peptide complexes are activated and stimulated to proliferate.

[0068] The antigen also binds to the homologous B cell receptor (BCR) expressed on the surface of naive B cells expressing IgM and IgD. The bound antigen is internalized, processed, and presented in the presence of the MHC class II: peptide complex on the B cell surface. Follicular T helper (TFH) effectors containing the peptide complex for MHC class II specific TCR: B cell presentation are stimulated to produce cytokines such as IL-4 and IL-21, which induce B cell proliferation and differentiation into plasma B cells and memory B cells.

[0069] The antibodies initially produced by the primary immune response are mainly IgM allotype antibodies, which bind to the target antigen with low affinity.

[0070] The secondary immune response refers to the response of the subject's adaptive immune system after exposure to the antigen, and the subject has already developed a primary immune response to the antigen.

[0071] In subsequent contact, the antigen binds to the BCR of memory B cells (i.e., memory B cells generated by the primary immune response upon initial exposure to the antigen), which are internalized, processed, and presented to memory TFH cells, thereby activating the memory TFH cells.

[0072] Following antigen binding, memory B cells undergo somatic hypermutation in the V region of the germinal center, resulting in B cell clones closely associated with BCRs possessing different affinities to the antigen. B cells with high-affinity BCRs recognize antigens presented via follicular dendritic cells and process and present them to TFH cells, thereby promoting B cell survival. Activated B cells maturing in the germinal center also undergo immunoglobulin type switching to produce IgG, IgA, or IgE antibodies. B cells expressing high-affinity BCRs differentiate into mature plasma B cells, which produce large amounts of high-affinity antibodies against the antigen.

[0073] As used in this article, "humoral immune response" refers to an immune response involving antibodies produced from B cells.

[0074] Primary humoral immune responses may be characterized by: activation of naive B cells (i.e., stimulation of naive B cells to proliferate, differentiate, and / or undergo immunoglobulin class switching), production of IL-4 and / or IL-21 by B cells, immunoglobulin class switching from cells expressing IgM and / or IgD to cells expressing IgG-, IgE-, or IgA, differentiation of naive B cells into plasma B cells, differentiation of naive B cells into memory B cells, and / or production of antibodies (such as IgM antibodies) that bind to their target antigens with low affinity.

[0075] B cell development is described, for example, in the article by Pieper et al. published in the *Journal of Clinical Immunology of Allergy* (Vol. 131, No. 4, pp. 959–71), the entire contents of which are incorporated herein by reference. Characteristic descriptions of B cells are also found, for example, in the paper by Carsetti et al. published in *Cytometry A*, Vol. 101, No. 2, 2022, pp. 131–139, the entire contents of which are incorporated herein by reference.

[0076] As used in this article, "naive" B cells refer to mature B cells whose BCRs have not yet been exposed to specific antigens. Naive B cells may also be referred to as mature naive B cells or mature B cells. Naive B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): MHC class II, IgM, and IgD. Naive B cells may also be characterized by the lack of CD27 expression (e.g., on the cell surface).

[0077] As used herein, "plasma" B cells refer to B cells that express a large number of soluble antibodies. Plasma B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): CD27, CD38, CD138, CD78, CD126, CXCR4, and BCMA. Plasma B cells may also be characterized by the lack of expression of CD20 and / or CD24 (e.g., on the cell surface).

[0078] As used in this article, “memory” B cells refer to B cells that form in germinal centers following a primary immune response. Memory B cells may be characterized by the expression of one or more of the following (e.g., on the cell surface): CD19, CD20, CD21, CD24, CD27, CD95, CD148, MHC class II, and TACI.

[0079] The term "given gene or protein" as used in this specification includes isotypes, fragments, variants, or homologs of genes / proteins from any species. It should be understood that, in aspects and embodiments involving a particular animal, the gene or protein is a suitable homolog encoded by the animal's genome. For example, when the animal is a mouse, the gene / protein may be a mouse homolog of the relevant gene / protein. In a further example of a transgenic mouse encoding a human homolog of the relevant gene / protein, the gene may be a human homolog of the relevant gene / protein.

[0080] Secondary humoral immune responses may be characterized by: activation of memory B cells (i.e., stimulation of memory B cells to proliferate and / or differentiate), somatic hypermutation of memory B cells, differentiation of memory B cells into plasma B cells, and / or production of antibodies (such as IgG antibodies) that bind to their target antigens with high affinity.

[0081] Some aspects and embodiments of this disclosure relate to providing agents for inhibiting primary humoral immune responses in animals. Some aspects and embodiments of this disclosure relate to animals comprising endogenous nucleotide sequences in which said endogenous nucleotide sequences provide inducible inhibition of primary humoral immune responses. It should be understood that the inhibitory effect may predispose to / promote secondary humoral immune responses in the animal.

[0082] This disclosure also provides reagents for use in the methods disclosed herein, such as those described herein, for suppressing primary humoral immune responses in animals in the methods disclosed herein. Use of said reagents for suppressing primary humoral immune responses in animals is also provided.

[0083] In this article, "inhibition" may also be referred to as "antagonism". A reagent that can inhibit a reaction / expression / activity can be called an "inhibitor" or "antagonist" of the relevant reaction / expression or activity.

[0084] In some embodiments, the suppression of primary humoral immune response according to this disclosure includes one or more of the following: suppressing the expression of genes involved in initiating primary humoral immune response, suppressing the activity of gene products involved in initiating primary humoral immune response, reducing the number / proportion of naive B cells, and / or reducing the number / proportion of cells expressing IgM and / or IgD.

[0085] As used herein, “expression” can refer to gene or protein expression. Gene expression includes the transcription of DNA into RNA and can be analyzed using various methods known to those skilled in the art, such as measuring mRNA levels by quantitative real-time PCR (qRT-PCR) or reporter-based methods. Similarly, protein expression can be measured using various methods well known in the art, such as antibody-based methods, including Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, or reporter-based methods.

[0086] As used herein, a factor / activity / cell type “involved in” a given reaction / process (e.g., initiating an immune response, immunoglobulin isotype conversion, etc.) refers to a factor / activity / cell type involved in the relevant reaction / process. The factor / activity / cell type preferably has a positive effect on the relevant reaction / process (i.e., promotes, enhances), and may, for example, be essential to the relevant reaction / process, meaning that the reaction / process will not occur in the absence of the factor / activity or cell type.

[0087] Various aspects and embodiments of this disclosure relate to inhibiting the expression (i.e., gene or protein expression) of one or more genes involved in initiating a primary humoral immune response, and / or inhibiting the activity of products of one or more genes involved in initiating a primary humoral immune response.

[0088] In a preferred embodiment, the one or more genes are not involved in or are not required to initiate a secondary humoral immune response. In some embodiments, the one or more genes involved in initiating a primary humoral immune response are IGHM and / or IGHD.

[0089] Inhibition of gene or protein expression of a given gene may include, for example, inhibiting gene transcription, inhibiting post-transcriptional processing (e.g., splicing) of RNA transcribed from a gene, reducing the stability of RNA transcribed from a gene, promoting the degradation of RNA transcribed from a gene, inhibiting the translation of RNA transcribed from a gene into a protein, inhibiting post-translational processing of a polypeptide encoded by a gene, reducing the stability of a polypeptide encoded by a gene, or promoting the degradation of a polypeptide encoded by a gene.

[0090] Gene expression can be analyzed using methods well known to those skilled in the art. The RNA level encoding a given gene can be determined using techniques such as RT-qPCR. Protein expression can also be measured using methods well known to those skilled in the art. The level of a given protein / isoform can be determined using, for example, antibody-based methods, including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, ELISA, etc.

[0091] The suppression of gene or protein expression of a given gene may be less than 1-fold of the expression level observed in the unsuppressed state, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold. In some implementations, the degree of inhibition of gene or protein expression exceeds 5%, for example, the expression observed in the uninhibited state as ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0092] Suppression of gene or protein expression can be achieved, for example, by altering / destroying the nucleotide sequence of the gene, or by altering / destroying the nucleotide sequence required to express the gene (e.g., regulatory sequences that regulate gene expression). In some embodiments, suppressing gene or protein expression may include altering the nucleotide sequence, for example, by substituting, deleting, or inserting one or more nucleotides. For example, in certain aspects and embodiments, this disclosure contemplates suppressing gene or protein expression by deleting all or part of the nucleotide sequence of the relevant gene.

[0093] In some implementations, altering / degrading nucleotide sequences may include, for example, altering / removing regulatory sequences (e.g., promoters, enhancers) used for gene transcription, introducing premature stop codons into sequences transcribed from genes, altering nucleotide sequences to encode truncated and / or nonfunctional gene products, or altering nucleotide sequences to encode misfolded and / or degraded gene products.

[0094] Altering / disrupting nucleotide sequences to suppress / prevent gene or protein expression can be called gene "knockout".

[0095] Nucleotide sequences may be disrupted, for example, through homologous recombination, or by modifying the target nucleic acid using site-specific nucleases (SSNs, also referred to herein as “gene editing systems”).

[0096] Modifications involving homologous recombination may involve the exchange of nucleic acid sequences via crossover events guided by homologous sequences, as discussed by Mortensen in Chapter 4, Unit 4.29 of the Current Protocols for Neuroscience, 2007, and by Vasquez et al. in Proceedings of the National Academy of Sciences (PNAS), Volume 98, Issue 15, 2001, pp. 8403-8410, both of which are incorporated herein by reference in their entirety. Homologous sequences are located flanking all or part of the disrupted nucleotide sequence. Recombinases can catalyze recombination. For example, in certain aspects and embodiments, this disclosure contemplates the disruption of nucleotide sequences via homologous recombination between loxP sequences catalyzed by Cre recombinase.

[0097] Homologous recombination can disrupt nucleotide sequences in animals, as described above regarding the Cre-LoxP, Flp-FRT, and Dre-rox systems.

[0098] For example, Eid and Mahfouz reviewed in Experimental and Molecular Medicine, Vol. 48, No. 10, p. e265, 2016, which is incorporated herein by reference in its entirety. (2016) 48(10):e265, the entire contents of which are incorporated herein by reference. Enzymes capable of generating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into target nucleic acid sequences. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), in which the ends of the break are rejoined, usually accompanied by the insertion or deletion of nucleotides. Alternatively, DSBs can be repaired by highly homologous directed repair (HDR), in which a DNA template with ends homologous to the break site is provided and introduced into the DSB site. SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeats / CRISPR-related-9 (CRISPR / Cas9) systems.

[0099] The ZFN system is reviewed, for example, in *Nature Reviews Genetics*, Vol. 11, No. 9, 2010, pp. 636-646, the entire contents of which are incorporated herein by reference. ZFNs contain programmable zinc finger DNA-binding domains and DNA-cutting domains (e.g., FokI endonuclease domains). DNA-binding domains can be identified by screening zinc finger arrays capable of binding target nucleic acid sequences. The TALEN system is reviewed, for example, in *Journal of Plant Biotechnology*, Mahfouz et al., 2014, pp. 12(8): 1006-14, the entire contents of which are incorporated herein by reference. TALENs contain programmable DNA-binding TALE domains and DNA-cutting domains (e.g., FokI endonuclease domains). TALE contains a repeating domain consisting of repeats of 33–39 amino acids, which are identical except that the two residues at positions 12 and 13 of each repeat are repeat variable double residues (RVDs). Each RVD determines the binding of the repeat sequence to a nucleotide in the target DNA sequence according to the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501.). CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR-C2c2, and CRISPR / C22c3, have been reviewed, for example, in Bioengineering (2017) 8(3):265–273 by Nakade et al., the entire contents of which are incorporated herein by reference. These systems include endonucleases (such as Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. sgRNAs can be engineered to target nucleic acid sequences that enable endonuclease activity.

[0100] As used herein, the “product” of a gene can refer, for example, to nucleic acids transcribed from a gene, or peptides / polypeptides produced by translating nucleic acids transcribed from a gene (and, where appropriate, further post-translational processing). These peptides / polypeptides include those modified with chemical motifs, such as carbohydrate and / or lipid motifs.

[0101] Inhibiting the activity of a given gene product may include, for example, inhibiting the expression of the gene or protein (as described above), or inhibiting one or more activities of the gene product. The activity of the gene product may be, for example, catalytic activity, binding (e.g., protein-protein interactions, such as ligand-receptor binding, polymerization, etc.), signal transduction, transport, storage, structural support, etc.

[0102] Inhibition of the activity of a given gene product can be reduced to less than 1-fold the activity level observed in the uninhibited state, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≥0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold. In some implementations, the given gene product can be inhibited to an activity level exceeding 5% observed in the uninhibited state, for example ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0103] In some embodiments, the reagent is capable of reducing the expression of a target gene or protein (i.e., a gene or protein involved in initiating a primary immune response) and / or reducing the activity of the target gene or protein. In some cases, the reagent inhibits, degrades, silences, knocks out, reduces, or otherwise modifies the expression and / or activity of genes or proteins involved in initiating a primary immune response.

[0104] The reagent may have one or more of the following properties associated with the target gene or protein (i.e., the gene or protein involved in initiating the primary immune response): inhibiting gene and / or protein expression; interfering with gene transcription; interfering with the translation of protein-encoding mRNA; degrading protein-encoding mRNA; binding to proteins; isolating proteins; competing for protein binding; and / or blocking protein activity.

[0105] In some embodiments, the reagent is capable of inhibiting the expression of genes IGHM and / or IGHD or inhibiting the activity of their products.

[0106] Such reagents can be used to treat the animals described herein. In some embodiments, the animals contain endogenous nucleotide sequences, as described herein, that can induce suppression of the primary immune response. In some embodiments, the animals do not contain endogenous nucleotide sequences that provide inducible suppression of the primary immune response.

[0107] In some embodiments, the reagent is an antibody or antigen-binding molecule (both referred to herein as "antigen-binding molecules"), such as anti-IgM or anti-IgD antibodies. In some cases, the antigen-binding molecule is specific to proteins involved in initiating the primary immune response, such as those selected from IgM and / or IgD. In some cases, the antigen-binding molecule exhibits specific binding to proteins involved in initiating the primary immune response, such as those selected from IgM and / or IgD. In some cases, the antigen-binding molecule exhibits specific binding to IgM or IgD. In some cases, the antigen-binding molecule is an anti-IgM or anti-IgD antigen-binding molecule.

[0108] The antigen-binding molecule may be an antagonistic antigen-binding molecule that inhibits or reduces the biological activity of target proteins (such as IgM or IgD) involved in initiating the primary immune response.

[0109] The antigen-binding molecule can bind to a specific target region of a target protein involved in initiating a primary immune response, such as IgM or IgD. The antigen-binding region of the antigen-binding molecule can bind to a linear epitope of the target protein involved in initiating a primary immune response, such as IgM or IgD, and consists of a continuous amino acid sequence (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding region molecule can bind to a conformational epitope of the target protein involved in initiating a primary immune response, such as IgM or IgD, and consists of a discontinuous amino acid sequence.

[0110] The antigen-binding molecule may be a multispecific antigen-binding molecule. "Multispecific" means that the antigen-binding molecule exhibits specific binding to multiple single targets. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two distinct antigen-binding domains (i.e., at least two antigen-binding domains, for example, comprising different VH and VL domains). Multispecific antigen-binding molecules may be provided in any suitable form, such as those described by Brinkmann and Kontermann in Monoclonal Antibodies (MAbs) (2017) 9(2):182-212, the entire contents of which are incorporated herein by reference.

[0111] In some embodiments, the antigen-binding molecule binds to a target protein involved in initiating a primary immune response, such as IgM or IgD, and another target (e.g., an antigen other than the target protein), and is therefore at least bispecific. The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants.

[0112] The ability of a given polypeptide to specifically bind to a given molecule or another given peptide / peptide can be determined by analysis according to methods known in the art, such as ELISA, surface plasmon resonance (SPR; Hearty et al., *Methods in Molecular Biology*, 2012, Vol. 907, pp. 411-442), biolayer interference assay (see, for example, Lad et al., *Journal of Biomolecular Screening*, 2015, Vol. 20, No. 4, pp. 498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. In some embodiments, the binding may be a reaction detected in a given assay. Binding affinity can be expressed as a dissociation constant (KD).

[0113] Those skilled in the art can use various methods well-known in the art to determine the regions of antibody-bound peptides / peptides, including X-ray cocrystallation analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based “protective” methods. For example, this method is described in the article by Gershoni et al. published in BioDrugs, Volume 21, Issue 3, 2007, pp. 145-156, the entire contents of which are incorporated herein by reference.

[0114] In some embodiments, the antigen-binding molecule inhibits the interaction between two binding partners. The ability of the antigen-binding molecule to inhibit the interaction between two binding partners can be determined by analyzing the downstream functional effects of the interaction in appropriate assays, for example, by detecting protein production in the reaction system using ELISA, Western blotting, or electrophoresis.

[0115] Those skilled in the art will be able to produce suitable antigen-binding molecules using techniques such as those described herein or those known in the art, see, for example, the paper by Chiu and Gilliland published in Curr Opin Struct Biol, 2016, Vol. 38, pp. 163-173; the paper by Jakobovits A published in Curr Opin Biotechnol, October 1995, Vol. 6, No. 5, pp. 561-566; and the paper by Brüggemann M et al. published in Arch Immunol Ther Exp (Warsz), 2015, Vol. 63, No. 2, pp. 101-108. A suitable technique is phage display, see, for example, the paper by Hammers and Stanley published in the Journal of Dermatological Research, Volume 134, Issue 2, p. e17, 2014, and the paper by Bazan J et al. published in Human Vaccines and Immunother, Volume 8, Issue 12, pp. 1817-1828, 2012. Antigen-binding polypeptide chains can also be produced by techniques such as chemical synthesis (see Chandrudu et al., published in Molecules, Vol. 18, 2013, pp. 4373-4388), or by recombinant expression techniques (such as those described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2012, by Green and Sambrook, and in Nature Methods, Vol. 5, No. 2, 2008, pp. 135-146), all of which are incorporated herein by reference. Alternatively, cell-free protein synthesis (CFPS; see, for example, Zemella et al., Chemical Biochemistry (2015), 16(17): 2420-2431), all of which are incorporated herein by reference. The antigen-binding molecules can be monoclonal, i.e., a group of homologous antibodies that specifically target a single epitope on the antigen.Monoclonal antibodies against selected antigens can be prepared using known techniques, such as those described in H. Zola's *Monoclonal Antibodies: A Manual of Techniques* (CRC Press, 1988) and J.G.R. Hurrell's *Monoclonal Hybridoma Antibodies: Techniques and Applications* (CRC Press, 1982). Chimeric antibodies were discussed by Neuberger et al. (1988, 8th International Symposium on Biotechnology, Part 2, 792-799). Suitable polyclonal antibodies can also be prepared using methods well-known in the art.

[0116] The antigen-binding portion can be a part of the antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment [ScFv]). Antigen-binding fragments of the antibody, such as Fab and Fab2 fragments, can also be used / provided, and genetically engineered antibodies and antibody fragments can be used. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognized in early protease digestion experiments. The “humanization” of rodent antibodies further confirms this. Variable domains derived from rodents can be fused with constant domains derived from humans, resulting in antibodies that retain the antigen specificity of the rodent parent antibody (Morrison et al. (1984), Proceedings of the National Academy of Sciences, Vol. 81, pp. 6851-6855).

[0117] The antibody and antigen binding fragments disclosed herein include a complementarity-determining region (CDR) of an antibody capable of binding to an associated target molecule, namely one or more proteins involved in the primary humoral immune response as described herein.

[0118] The reagent may be nucleic acid-based or contain nucleic acid elements. The reagent may promote gene expression silencing through RNA-mediated interference (RNAi) or antisense degradation mechanisms (e.g., via RNase H).

[0119] In some embodiments, the reagent is or comprises an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid (e.g., DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid (e.g., mRNA that can be translated into a protein, such as FHR protein) and is capable of reducing transcription of the target nucleic acid (e.g., mRNA from DNA), reducing translation of the target nucleic acid (e.g., mRNA), or altering transcript splicing (e.g., via single-stranded morpholino oligomers). The antisense nucleic acid can be single-stranded, such as a spacer polymer, or double-stranded, such as siRNA. The antisense nucleic acid is capable of hybridizing with the target nucleic acid (e.g., target mRNA) via Watson-Crick base pairing (e.g., selective hybridization). In some cases, the antisense nucleic acid specifically binds to the target nucleic acid. In some cases, the antisense nucleic acid hybridizes with the target nucleic acid sequence (e.g., mRNA) under stringent hybridization conditions. In some cases, the antisense nucleic acid hybridizes with the target nucleic acid (e.g., mRNA) under reasonably stringent hybridization conditions.

[0120] The nucleotide sequence of the antisense nucleic acid is fully complementary to that of the target nucleic acid, enabling it to bind or hybridize with the target nucleic acid. Therefore, if those skilled in the art are familiar with the sequence of the target nucleic acid, it is easy and conventional to design a suitable antisense nucleic acid that hybridizes with the target to achieve the desired effect.

[0121] The target RNA may be an mRNA encoding a protein involved in initiating a primary immune response, such as IgM and / or IgD.

[0122] In some cases, this reagent can promote RNA interference (RNAi). RNAi uses small double-stranded RNA molecules to induce the degradation of target mRNA. Non-limiting examples of antisense nucleic acids used as reagents according to the present invention include siRNA (including its derivatives or precursors, such as nucleotide analogs), short hairpin RNA (shRNA), microRNA (miRNA, including its long primary transcript (pri-miRNA) and partially processed 60-70 base pair hairpin transcripts (miRNA precursors)), saRNAs (small activating RNAs), and small nucleolar RNA (snoRNA), or certain derivatives or precursors thereof. Antisense nucleic acid molecules may stimulate RNA interference (RNAi).

[0123] siRNA nucleic acids are ~21-25 nucleotides in length, containing a guide strand that hybridizes to the target mRNA and a complementary passenger strand (e.g., each complementary sequence of a double-stranded siRNA is 21-25 nucleotides in length, and the length of the double-stranded siRNA is approximately 21-25 base pairs). They facilitate the degradation of the target mRNA via RISC. The structure and function of siRNAs are well known in the art and have been described, for example, in an article by Kim and Rossi published in the journal *Biotechniques*, April 2008, Volume 44, Issue 5 (pp. 613-616). Suitable siRNA molecules for use in the methods of this invention can be designed using methods well known in the art, for example, see Elbashir et al., *Nature*, 2001, Vol. 411, pp. 494-498; Amarzguioui et al., *Biochemical and Biophysical Research Communications*, 2004, Vol. 316, No. 4, pp. 1050-1058; and Reynolds et al., *Nature Biotechnology*, 2004, Vol. 22, No. 3, pp. 326-330. Detailed information on the preparation of siRNA molecules can be found on the websites of several commercial vendors such as Ambion, Dharmacon, GenScript, Invitrogen, and OligoEngine. The sequence of any potential siRNA candidate can typically be detected using a BLAST alignment procedure for any possible match with other nucleic acid sequences or nucleic acid sequence polymorphisms (see the National Library of Medicine website). Typically, a large number of siRNAs are generated and screened to obtain viable drug candidates, see U.S. Patent No. 7,078,196. siRNAs can be produced from vector expression and / or chemically or synthetically. Synthetic RNAi can be obtained from commercial sources, such as Invitrogen (Carlsbad, California). RNAi vectors are also available from commercial sources, such as Invitrogen.

[0124] microRNAs (miRNAs) also regulate gene expression via RISC. They are initially expressed as long primary transcripts (pri-miRNAs), which are processed in the nucleus into hairpins of 60-70 nucleotides (miRNA precursors), and further processed in the cytoplasm into small double-stranded nucleic acids that interact with RISC and target mRNAs. miRNAs contain a “seed sequence” that is crucial for binding to the target mRNA. The “seed sequence” typically consists of six nucleotides and is located at positions 2-7 of the 5' end of the miRNA.

[0125] In some embodiments, the reagent comprises a double-stranded nucleic acid molecule, one strand of which is fully or partially complementary to or hybridizes with an mRNA sequence encoding a protein involved in initiating a primary immune response (such as IgM or IgD, as described herein). In some embodiments, the reagent comprises a siRNA molecule containing a guide strand complementary to or hybridizing with a portion of an mRNA sequence encoding all or part of a protein involved in initiating a primary immune response, such as IgM or IgD, as described herein. In some embodiments, the reagent comprises a miRNA molecule (pri-, precursor, or mature miRNA) containing a seed sequence capable of hybridizing with a portion of an mRNA sequence encoding all or part of a protein involved in initiating a primary immune response, such as IgM or IgD, as described herein.

[0126] In some cases, the reagent is a single-stranded antisense oligonucleotide (ASO). ASOs alter the expression of the target RNA by modifying splicing or recruiting RNase H to degrade the target RNA. RNase H recognizes the DNA-RNA hybrid formed when an ASO binds to the target RNA. ASOs are typically 18-30 base pairs in length. Many ASOs are designed as chimeras, composed of a mixture of bases with different chemical compositions, or as spacer polymers consisting of a central DNA portion surrounded by “wings” of modified bases. ASOs are described, for example, in *Neurogenetics*, April 2019, Vol. 5, No. 2, p. e323, by Scoles et al.

[0127] Antisense nucleic acids can include naturally occurring nucleotides or modifications such as thiophosphate bonds, diamidine phosphate bonds, methoxyethyl nucleotide modifications such as 2-MOE, "locked" nucleic acids such as LNA, peptide nucleic acids (PNA), and / or 5'-methylcytosine modifications.

[0128] In some embodiments, the reagent includes an antisense oligonucleotide capable of hybridizing with a portion of an mRNA sequence encoding all or part of a protein involved in initiating a primary immune response, such as IgM or IgD, as described herein.

[0129] The antisense nucleic acid described herein may comprise or consist of a nucleotide sequence having complementarity with its target nucleic acid of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Complementarity may be calculated over the entire length of the antisense nucleic acid and / or over all or part of the target nucleic acid to which the antisense nucleic acid binds.

[0130] The nucleic acid molecule may be an aptamer. As used herein, the term "aptamer" refers to an oligonucleotide (e.g., a short oligonucleotide or deoxyribonucleotide) that binds to proteins, peptides, and small molecules (e.g., those with high affinity and specificity). Aptamers typically have well-defined secondary or tertiary structures because they tend to form complementary base pairs and are therefore often capable of folding into a variety of complex molecular structures. Three-dimensional structure is crucial for aptamer binding affinity and specificity; specific three-dimensional interactions drive the formation of the aptamer-target complex. Phylogenetic enrichment of ligands can be achieved through exponential enrichment (as described by Ellington and Szostak in *Nature*, Vol. 346, pp. 818-822, 1990; Tuerk C and Gold L. in *Nature*, Vol. 249, pp. 505-510, 1990), or through the development of SOMAmers (slow dissociation rate modified aptamers) (Gold L et al., in *PLoSONE*, Vol. 5, No. 12, p. e15004, 2010). SOMAMs are short, single-stranded deoxyoligonucleotides that possess protein-like properties due to functional groups mimicking amino acid side chains. The application of SELEX and SOMAmer techniques, including, for example, the addition of functional groups mimicking amino acid side chains, can expand the chemical diversity of aptamers. Therefore, high-affinity aptamers for targets can be enriched and identified.

[0131] Aptamers can be DNA or RNA molecules, and can be single-stranded or double-stranded. Aptamers can include chemically modified nucleic acids, such as sugars and / or phosphates and / or bases that are chemically modified. Such modifications can improve the stability of the aptamer or make it more resistant to degradation, and can include modifications at the 2' site of the ribose.

[0132] Aptamers can be synthesized using methods well known to those skilled in the art. For example, aptamers can be chemically synthesized, such as on a solid support. Solid-phase synthesis can be performed using a phosphorus amide chemistry method. Briefly, a solid-supported nucleotide is detrimethylated and then coupled with a suitably activated nucleoside phosphorus amide to form a phosphite trimer bond. End-capping may then occur, followed by oxidation of the phosphite trimer with an oxidizing agent (typically iodine). This cycle can then be repeated to assemble aptamers (see, for example, the paper by Sinha, ND et al., published in Nucleic Acids Research, Vol. 12, p. 4539, 1984; and the paper by Beaucage, SL and Iyer, RP, published in Tetrahedron, Vol. 48, No. 12, p. 2223, 1992).

[0133] Aptamers can be peptides that have been selected or modified to bind to specific target molecules. Reverdatto et al. reviewed peptide aptamers and their generation and identification methods in *Curr Top Med Chem* (2015, Vol. 15, No. 12, pp. 1082-1101). Peptide aptamers can optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Peptide aptamers can optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. Suitable peptide aptamers may optionally have a length of one of 2-30, 2-25, 2-20, 5-30, 5-25, or 5-20 amino acids.

[0134] The KD of the aptamer may be in the range of nM or pM, for example less than one of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, or 100 pM.

[0135] Aptamers or SOMAmers suitable for the purposes described herein can bind to proteins involved in initiating primary immune responses, such as IgM or IgD, as described herein. Aptamers or SOMAmers suitable for the purposes described herein can exhibit specific binding to proteins involved in initiating primary immune responses, such as IgM or IgD, as described herein. The aptamers can inhibit the function of proteins involved in initiating primary immune responses, such as IgM or IgD, for example, by blocking their binding to homologous binding partners or ligands.

[0136] The reagent may be a chelating agent, such as a protein involved in initiating a primary immune response as described herein. The reagent may be a protein molecule.

[0137] The reagent may be a small molecule. For example, as described herein, the small molecule can bind to proteins involved in initiating a primary immune response and prevent / reduce their normal function and / or prevent / reduce interactions with homologous binding partners. The small molecule may also prevent / reduce the proper folding of the target protein.

[0138] The reagent may be a decoy receptor. In some embodiments, a decoy receptor refers to a peptide or polypeptide capable of binding to proteins involved in initiating the primary immune response as described herein. The receptor may be a receptor for proteins involved in initiating the primary immune response as described herein, including fragments and derivatives thereof. A decoy receptor may be able to recognize and bind specific ligands, but may not be able to signal or activate a subsequent response. A decoy receptor may bind to proteins involved in initiating the primary immune response as described herein to form a complex. A decoy receptor can act as an inhibitor of proteins involved in initiating the primary immune response as described herein by blocking / reducing the ability or availability of a protein to bind to its receptor. A decoy receptor can also act as an inhibitor of proteins involved in initiating the primary immune response by binding to a binding chaperone of the protein (e.g., in a region where the binding chaperone would normally bind) and blocking the interaction between the protein and one or more binding chaperones.

[0139] Decoy receptors can be soluble (non-membrane-bound) or membrane-bound, such as those expressed on the cell surface. Decoy receptors can be presented and / or applied to the surface of nanocarriers, such as nanoparticles, liposomes, beads, polymers, metal particles, dendritic macromolecules, nanotubes, or micron-sized silica rods, see, for example, the literature by Wilczewska AZ et al., published in Pharmacology Reports, 2012, Vol. 64, No. 5, pp. 1020-1037.

[0140] Methods for detecting whether a decoy receptor competitively binds to a target protein may include, for example, SPR (see, for example, Hearty et al., Methods in Molecular Biology, Vol. 907, pp. 411-442, 2012), competitive ELISA assays, or solid-binding assays. Other suitable methods are known in the art.

[0141] Agents that reduce the number of proteins involved in initiating the primary immune response and / or reduce the expression of genes encoding proteins involved in initiating the primary immune response may fall into one of the above categories. For example, antigen-binding molecules or decoy receptors can also be chelating agents.

[0142] Any of the reagents described herein can optionally be separated and / or substantially purified.

[0143] Reduce the number / proportion of immature B cells or cells expressing IgM and / or IgD. Various aspects and embodiments of this disclosure relate to reducing the number / proportion of naive B cells or reducing the number / proportion of IgM and / or IgD-expressing cells in animals. It should be understood that naive B cells express IgM and / or IgD.

[0144] The aim is to deplete / remove a cell population that would otherwise be available to initiate a primary humoral immune response following a subsequent immunization of the animal. In other words, reducing the number / proportion of naive B cells or cells expressing IgM and / or IgD reduces / prevents the animal's ability to initiate a primary humoral immune response to an antigen upon subsequent challenge.

[0145] Its purpose is not to deplete / remove cells that have been activated / stimulated to proliferate by the application of first peptides / polypeptides, or cells derived from these cells (i.e., the progeny of cells that have been activated / stimulated by the application of first peptides / polypeptides to proliferate).

[0146] In some embodiments, after a period of time sufficient for cells (or cells derived from these cells) to proliferate through application of a first peptide / peptide activation / stimulation to undergo immunoglobulin isotype conversion (i.e., cells expressing IgG-, IgE-, or IgA), the animal is treated to reduce the number / proportion of naive B cells or cells expressing IgM and / or IgD. In some embodiments, after a period of time sufficient for the cells (or cells derived from these cells) to proliferate through application of a first peptide / peptide activation / stimulation to differentiate into plasma B cells and / or memory B cells, the animal is treated to reduce the number / proportion of naive B cells or cells expressing IgM and / or IgD.

[0147] In some embodiments, after a period of time sufficient for cells (or cells derived from these cells) to proliferate upon application of a first peptide / peptide activation / stimulation to undergo immunoglobulin isotype conversion (i.e., conversion to cells expressing IgG-, IgE-, or IgA), the animal is treated to induce a reduction in the number / proportion of naive B cells or cells expressing IgM and / or IgD. In some embodiments, after a period of time sufficient for cells (or cells derived from these cells) to differentiate into plasma B cells and / or memory B cells upon application of a first peptide / peptide activation / stimulation to undergo differentiation, the animal is treated to induce a reduction in the number / proportion of naive B cells or cells expressing IgM and / or IgD.

[0148] In some embodiments, reducing the number / proportion of naive B cells or reducing the number / proportion of cells expressing IgM and / or IgD does not include reducing the number / proportion of cells expressing IgG-, IgE-, or IgA. In some embodiments, reducing the number / proportion of naive B cells or reducing the number / proportion of cells expressing IgM and / or IgD does not include reducing the number / proportion of plasma B cells and / or memory B cells.

[0149] By inhibiting the development of such cells, the number / proportion of naive B cells or the number / proportion of cells expressing IgM and / or IgD can be reduced. Inhibiting the development of naive B cells or cells expressing IgM and / or IgD can be achieved by inhibiting the expression or activity of one or more factors involved in precursor cell development / maturation into relevant cell types. Inhibiting the development of naive B cells or cells expressing IgM and / or IgD may include inhibiting the maturation of precursor cells into naive B cells, IgM and / or IgD-expressing cells.

[0150] As used herein, "precursor cells of naive B cells" refers to a cell type upstream of naive B cells during B cell development. In some embodiments, the precursor cells of naive B cells may be selected from: stem cells, pre-B cells, early pre-B cells, late pre-B cells, precursor B cells, large precursor B cells, small precursor B cells, or immature B cells.

[0151] Stem cells can be hematopoietic stem cells and can be characterized, for example, by the expression of CD34 (e.g., surface expression) and / or the absence of CD10 expression (e.g., surface expression). Early pre-B cells can be characterized by the expression of CD10, CD43, CD45, and / or MHC class II (e.g., surface expression). Late pre-B cells can be characterized by the expression of CD19, CD43, CD45, and / or MHC class II (e.g., surface expression). Large pre-B cells can be characterized by the expression of precursor BCR, CD19, CD43, CD45, and / or MHC class II (e.g., surface expression). Small pre-B cells can be characterized by the expression of precursor BCR, CD19, CD45, and / or MHC class II (e.g., surface expression). Immature B cells can be characterized by the expression of CD10, CD19, CD20, CD24, CD38, CD45, IgM, and / or MHC class II (e.g., surface expression), and / or the absence of CD27 expression (e.g., surface expression).

[0152] The number / proportion of naive B cells or the number / proportion of cells expressing IgM and / or IgD can also be reduced by inhibiting the expression of one or more factors expressed by naive B cells. For example, by inhibiting the expression of IGHM and / or IGHD, the number / proportion of naive B cells or cells expressing IgM and / or IgD can be reduced.

[0153] By inhibiting the activity of one or more factors expressed by naive B cells, it is possible to reduce the number / proportion of naive B cells or the number / proportion of cells expressing IgM and / or IgD. For example, by inhibiting the activity of IgM and / or IgD, the number / proportion of naive B cells or cells expressing IgM and / or IgD can be reduced.

[0154] Inhibit immunoglobulin isotype conversion The aspects and embodiments of this disclosure relate to inhibiting immunoglobulin isotype switching. A general objective is to eliminate, after subsequent immunization, the ability of an animal to generate an IgG response to a region of the second peptide / peptide that differs from the first peptide / peptide.

[0155] Some aspects and embodiments of this disclosure include inhibiting the expression (gene or protein expression) of genes involved in immunoglobulin isotype conversion and / or inhibiting the activity of gene products involved in immunoglobulin isotype conversion.

[0156] Immunoglobulin type switching, also known as "isotype switching" and "type switching recombination," is discussed in the article by Stavnezer and Schrader in the Journal of Immunology, Vol. 193, No. 11, 2014, pp. 5370–5378, the entire contents of which are incorporated herein by reference.

[0157] Mature naive B cells express both IgM and IgD. Activation of antigen binding leads to cell proliferation, and if they encounter a suitable factor (such as IL-4), they are triggered to undergo type conversion reorganization, switching from expressing IgM and IgD to expressing IgG, IgE, or IgA. During type conversion, the constant regions of the immunoglobulin heavy chain change, but the variable regions, and therefore antigen specificity, remain unchanged.

[0158] Immunoglobulin type conversion involves replacing the constant heavy chain (CH) regions of expressed Ig (μ and δ) with CH regions of γ, ε, or α, followed by recombination via deletions between two distinct transition (S) regions. Type conversion recombination (CSR) is initiated by activation-induced cytidine deaminase (AICDA), which converts cytosine in the S region to uracil. The uracil is subsequently removed via two DNA repair pathways, resulting in the mutations, single-strand DNA breaks, and double-strand breaks required for CSR.

[0159] Various aspects and embodiments of this disclosure relate to inhibiting the immunoglobulin type conversion of B cells expressing IgM and / or IgD to cells expressing IgG, IgE, and / or IgA. In a particular embodiment, the present invention relates to inhibiting the immunoglobulin type conversion of B cells expressing IgM and / or IgD to B cells expressing IgG.

[0160] Its purpose is not to inhibit immunoglobulin isotype conversion in cells that have been activated / stimulated by the administration of a first peptide / polypeptide or in cells derived from such cells (i.e., the progeny of cells that have been activated / stimulated by the administration of a first peptide).

[0161] In some embodiments, after a period of time sufficient for cells (or cells derived from these cells) to proliferate upon application of a first peptide / peptide to undergo immunoglobulin isotype conversion (i.e., cells expressing IgG-, IgE-, or IgA), the animal is treated to inhibit, for example, by inducing an inhibition of immunoglobulin isotype conversion. In some embodiments, after a period of time sufficient for cells (or cells derived from these cells) to proliferate upon application of a first peptide / peptide to differentiate into plasma B cells and / or memory B cells, the animal is treated to inhibit, for example, by inducing an inhibition of immunoglobulin isotype conversion.

[0162] In some implementations, inhibiting immunoglobulin isotype conversion does not include inhibiting immunoglobulin isotype conversion of cells (or cells derived from these cells) that are activated / stimulated by the administration of a first peptide / polypeptide to proliferate.

[0163] IgM and IgD are involved in immunoglobulin class switching. Dual depletion of IgM+ and IgD+ cells has been shown to prevent the production of IgG after immunization (Chentoufi et al., Cellular Immunology (2000, Vol. 205, pp. 40-51). Therefore, inhibiting the expression of IGHM and / or IGHD will impair immunoglobulin class switching.

[0164] In some embodiments, inhibiting immunoglobulin type switching includes inhibiting the expression or activity of one or more factors involved in immunoglobulin type switching. In some embodiments, inhibiting immunoglobulin type switching includes inhibiting the expression or activity of one or both of IGHM and / or IGHD.

[0165] It should be understood that inhibition of immunoglobulin type switching can also be achieved, for example, by reducing the number / proportion of immature B cells, or by reducing the number / proportion of IgM and / or IgD expressing cells, as described above. In other words, immunoglobulin type switching can be inhibited by depleting / removing the cell population that would otherwise undergo immunoglobulin type switching.

[0166] Various aspects and embodiments of this disclosure include treating animals to suppress primary immune responses (e.g., primary humoral immune responses) and / or promote secondary immune responses (e.g., secondary humoral immune responses).

[0167] Various aspects and embodiments of this disclosure include treating animals containing endogenous nucleotide sequences that induce suppression of primary immune responses (e.g., primary humoral immune responses) and / or promotion of secondary immune responses (e.g., secondary humoral immune responses).

[0168] In some embodiments, the methods of this disclosure include administering, for example, a reagent as described above, to animals, said reagent inducing suppression of primary immune responses and / or promoting secondary immune responses. In some embodiments, said reagent induces suppression of primary immune responses in animals.

[0169] In some embodiments, the reagent effectively inhibits primary immune responses and / or promotes secondary immune responses in animals. In some embodiments, the reagent inhibits the expression of genes involved in initiating primary humoral immune responses, inhibits the activity of gene products involved in initiating primary humoral immune responses, reduces the number / proportion of naive B cells, reduces the number / proportion of cells expressing IgM and / or IgD, inhibits immunoglobulin isotype switching, inhibits the expression of genes involved in immunoglobulin xenotype switching, and / or inhibits the activity of gene products involved in immunohistoprotein isotype switching.

[0170] In some embodiments, the reagent induces the expression or activity of one or more factors, thereby inhibiting primary immune responses and / or promoting secondary immune responses.

[0171] Various aspects and embodiments of this disclosure relate to the use of such reagents in methods for preparing antigen-binding molecules, such as the methods for preparing antigen-binding molecules described herein.

[0172] In some embodiments, the reagent is capable of inhibiting the expression (gene or protein expression) of genes involved in initiating the primary humoral immune response. In some embodiments, the reagent is capable of inhibiting the expression (gene or protein expression) of genes involved in immunoglobulin isotype switching.

[0173] In some embodiments, the reagent can alter / destroy the nucleotide sequence of a target gene (i.e., a gene involved in initiating a primary immune response) or alter / destroy the nucleotide sequence required for the expression of a target gene (e.g., a regulatory sequence that regulates the expression of a target gene or transcription factor).

[0174] In some embodiments, the reagent can induce alteration / destruction of the nucleotide sequence of a target gene (i.e., a gene involved in initiating a primary immune response) through homologous recombination. In some embodiments, the reagent can increase the expression or activity of a recombinase, which can alter / destruct the nucleotide sequence of the target gene (i.e., a gene involved in initiating a primary immune response).

[0175] In some embodiments, the animal contains an endogenous nucleotide sequence encoding a tamoxifen / 4-hydroxytamoxifen regulatory system that regulates the activity of the recombinase.

[0176] In various aspects and embodiments of employing such a system, the agent capable of inhibiting the primary immune response and / or promoting the secondary immune response may be tamoxifen / 4-hydroxytamoxifen. Similarly, methods relating to such a system in this disclosure may include administering tamoxifen / 4-hydroxytamoxifen to an animal to inhibit the animal's ability to initiate a primary immune response.

[0177] nucleotide sequence Various aspects and embodiments of this disclosure relate to animals comprising endogenous nucleotide sequences that provide inducible inhibition of primary humoral immune responses.

[0178] As used herein, nucleotide sequences that “provide” inductive inhibition of the primary humoral immune response may encode one or more factors that are involved in (e.g., are necessary for or permitted inductive inhibition of the primary humoral immune response).

[0179] In some embodiments, the nucleotide sequence mediates the inhibition of expression of one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) involved in initiating the primary humoral immune response via site-specific recombinase.

[0180] In some embodiments, the nucleotide sequence inhibits the expression of one or both of IGHM and / or IGHD via site-specific recombinase-mediated inhibition.

[0181] Site-specific recombinase (SSR) systems for suppressing gene expression are well known in the art. Such SSR systems include, for example, the Cre-LoxP, Flp-FRT, and Dre-rox systems, and are described, for example, in the article by Branda and Dymecki published in *Developmental Cell*, Vol. 6, No. 1, 2004, pp. 7–28, and in the article by Kim et al. published in *Lab Anim Res*, Vol. 34, No. 4, 2018, pp. 147–159, the entire contents of which are incorporated herein by reference. Such SSR systems and other variants of SSR systems are also well known in the art and can be similarly used to suppress the expression of target genes.

[0182] Targeted disruption of nucleotide sequences can be achieved by providing a target sequence, or the nucleotide sequence required for gene expression, to a recombinase on either side (i.e., upstream / 5' and downstream / 3') of all or part of the target nucleotide sequence of a gene. In the presence of the corresponding recombinase, homologous recombination occurs between the target sequences, disrupting the nucleotide sequence of the gene or the nucleotide sequence required for gene expression.

[0183] In the Cre-loxP system, Cre recombinases bind to inverted repeats of loxP target sequences and promote recombination and excision of nucleotide sequences between loxP target sequences. As used herein, “Cre recombinase” refers to any peptide / peptide having the catalytic activity of a Cre recombinant protease. Cre recombinases may comprise the amino acid sequence of UniProtKB Q71TG5-1,v1, or an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of UniProtKB Q21TG5-11,v1. Cre recombinases include, for example, fusion proteins of Cre recombinant proteases, including, for example, CreERT and CreERT2 as described below.

[0184] While the examples above describe the use of the SSR system for targeted excision of nucleotide sequences, the SSR system can also be used for targeted inversion, insertion, and translocation of nucleotide sequences.

[0185] SSR-mediated gene knockout, particularly knockout using the Cre-loxP system, is exemplified by the work of Kim et al., published in Lab Anim Res, 2018, Vol. 34, No. 4, pp. 147–159, the full text of which is incorporated herein by reference.

[0186] It should be understood that the SSR system according to this disclosure is used to alter / disrupt the expression of a gene or protein by inhibiting / preventing gene expression. In some embodiments, the SSR system is used to alter / disrupt the production of a product encoded by an unaltered nucleotide sequence of the gene.

[0187] In some implementations, the SSR system is used to alter / disrupt the transcription of genes by introducing an early stop codon into the sequence transcribed from the gene, altering the nucleotide sequence to encode a truncated and / or nonfunctional gene product, or altering the nucleotide sequence to encode a misfolded and / or degraded gene product.

[0188] In some embodiments, the nucleotide sequence encoding factors of this disclosure provide site-specific recombinase-mediated interference with the expression of one or more genes that initiate a primary humoral immune response.

[0189] In some embodiments, the nucleotide sequence includes a recombinase target sequence flanking all or part of the nucleotide sequence of a gene involved in initiating a primary humoral immune response. In some embodiments, the nucleotide sequence includes a recombinase target sequence flanking all or part of the nucleotide sequence required for gene expression.

[0190] In some embodiments, the target sequence of the recombinase is loxP The target sequence of the recombinase is an FRT sequence, and the recombinase is an Flp recombinase. In some embodiments, the target sequence of the recombinase is a rox sequence, and the recombinase is a Dre recombinase.

[0191] In some embodiments, the recombinase target sequence is located flanking the nucleotide sequence required for gene product expression. In some embodiments, the recombinase target sequence is located flanking all or part of the nucleotide sequence encoding one or more exons of a gene. In some embodiments, the recombinase target sequence is located flanking all or part of the nucleotide sequence encoding a regulatory sequence that regulates gene expression (e.g., a promoter or enhancer).

[0192] It should be understood that the recombinase target sequences are located on either side of a given nucleotide sequence, provided as "flanks". That is, in the context of the endogenous nucleotide sequences of this disclosure, they are provided at the 5' and 3' of a given nucleotide sequence. For illustration, the endogenous nucleotide sequences of this disclosure may include arrangements of the following nucleotide sequences: 5'-[Target sequence of recombinase]-[All or part of the nucleotide sequence of the target gene / nucleotide sequence required for target gene expression]-[Target sequence of recombinase]-3' In some embodiments, the target sequence of the recombinase may be within about 5, 10, 50, 100, 250, 500, or 1000 bases of the first and / or last nucleotide sequence of the target gene or the nucleotide sequence required for expression. That is, in some embodiments, the last base of the target sequence of the recombinase may be provided within about 5, 10, 50, 100, 250, 500, or 1000 bases of the nucleotide sequence required for target gene expression / the first base of the target gene's nucleotide sequence, and / or the first base of the target sequence of the recombinase may be provided within about 5, 10, 50, 100, or 1000 bases of the nucleotide sequence required for target gene expression / the last base of the target gene's nucleotide sequence.

[0193] In some aspects and embodiments, the endogenous nucleotide sequence of this disclosure further includes a nucleotide sequence encoding a recombinase, i.e., a recombinase corresponding to an associated target sequence of the recombinase. For example, in an embodiment where the target sequence of the recombinase is a loxP sequence, the endogenous nucleotide sequence may further include a nucleotide sequence encoding a Cre recombinase.

[0194] The endogenous nucleotide sequence may also include a nucleotide sequence encoding a regulatory nucleotide sequence (e.g., a promoter and / or enhancer) for recombinase expression. The regulatory nucleotide sequence may be operatively linked to the nucleotide sequence encoding the recombinase. In some embodiments, the endogenous nucleotide sequence encodes an expression cassette for the recombinase.

[0195] In various aspects and embodiments of this disclosure, the endogenous nucleotide sequence provides an inducible inhibition of the primary humoral immune response. This inhibition may be inducible, for example, by responding to a given chemical or physical treatment.

[0196] In implementations employing the SSR system, SSR-mediated gene expression repression can be induced by increasing the level or activity of the relevant recombinase (e.g., by administering the recombinase and / or increasing its expression).

[0197] In embodiments employing endogenous nucleotide sequences, the expression inhibition of related genes can be induced by administering recombinases to animals, administering nucleic acids (e.g., vectors) encoding recombinases to animals, and / or treating animals to increase the expression or activity of recombinases. The endogenous nucleotide sequence contains a target sequence of the recombinase located flanking all or part of the nucleotide sequence of a gene involved in initiating a primary humoral immune response, or flanking all or part of the nucleotide sequence required for gene expression.

[0198] It should be understood that increasing the level and / or activity of recombinases in cells containing endogenous nucleotide sequences enhances recombination and thereby disrupts the target gene and subsequently inhibits its expression, wherein the endogenous nucleotide sequence contains the target sequence of the recombinase located flanking the target nucleotide sequence.

[0199] In some embodiments, the upregulation of the expression / activity of the recombinase is chemically induced. Chemically induced SSR-mediated gene knockout is described, for example, in an article by Kim et al. published in *Lab Anim Res.* (2018, Vol. 34, No. 4, pp. 147-159), which is incorporated herein by reference.

[0200] In some embodiments, the endogenous nucleotide sequence encodes a conditional system for regulating the expression or activity of the recombinase.

[0201] In various aspects and embodiments employing the above-described conditional system, the reagent capable of inhibiting the primary immune response and / or promoting the secondary immune response may be a reagent that induces the expression or activity of recombinases.

[0202] In some implementations, the endogenous nucleotide sequence includes a nucleotide sequence encoding a conditional expression system for regulating recombinase expression.

[0203] "Conditional expression," also referred to as "inducible expression" in this paper, refers to gene / protein expression that depends on certain conditions, such as the presence of a specific reagent. Conditional expression systems are well-known in the field, for example in the article reviewed by Ryding et al. in the Journal of Endocrinology (2001, Vol. 171, pp. 1-14), the entire contents of which are incorporated herein by reference.

[0204] Conditional expression systems include those that employ tetracycline-regulated transcriptional activation, such as the Tet-On and Tet-Off systems.

[0205] The Tet-On system employs a nucleic acid encoding a reverse tetracycline transactivator (rtTA) protein, a fusion of the tetracycline repressor (TetR) protein with mutations at four amino acid sites to reverse the response to tetracycline / doxycycline, and an activation domain of VP16. In the absence of tetracycline (or its derivatives, such as doxycycline), rtTA does not bind to the TetO operon sequence, and the polypeptide is not expressed. In the presence of tetracycline / doxycycline, rtTA binds to the TetO sequence in the TRE and activates transcription of downstream nucleic acids of the promoter. The Tet-On system is described in an article by Das et al. published in *Curr Gene Ther.* (2016, Vol. 16, No. 3, pp. 156-67), the entire contents of which are incorporated herein by reference, and includes systems employing optimized rtTA variants, such as the upgraded Tet-On system (which uses the rtTA variant protein rtTA2). s -M2) and Tet-On 3G system.

[0206] The Tet-On Advanced system is also described in the article by Urlinger et al. published in the Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 14, pp. 7963–8) (the entire contents of which are incorporated herein by reference), and the Tet-On 3G system is described in the article by Zhou et al. published in Gene Therapy (Vol. 13, No. 19, pp. 1382–1390) (the entire contents of which are incorporated herein by reference).

[0207] The Tet-Off system employs a nucleic acid encoding a tetracycline transactivator (tTA) protein, a fusion of a tetracycline repressor (TetR) protein and an activation domain HSV protein VP16. In the absence of tetracycline (or its derivatives, such as doxycycline), tTA does not bind to the TetO operon sequence, forming a tetracycline response element (TRE) upstream of a minimal promoter (such as a CMV promoter). Binding of tTA to the TetO sequence in the TRE activates transcription of downstream nucleic acids. In the presence of tetracycline / doxycycline, tTA cannot bind to the TetO sequence in the TRE, and transcription of downstream nucleic acids is repressed. The Tet-Off system is described in the article by Bujard et al. published in the Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA, 1992, Vol. 89, No. 12, pp. 5547-51), the full text of which is incorporated herein by reference.

[0208] Other tetracycline regulatory systems include the T-REx conditional expression system, described in the article by Yao et al. published in *Human Gene Therapy* (1998, Vol. 9, No. 13, pp. 1939-1950) (the entire contents of which are incorporated herein by reference). In the T-REx system, TetR is expressed under the regulation of the CMV promoter, and in the absence of tetracycline / doxycycline, TetR binds to two Tet operator 2 (TetO2) sequences upstream of the target region, repressing transcription in the target region. When tetracycline / doxycycline is added to the system, it binds to TetR and releases it from the TetO2 sequence, thereby protecting the target region from transcriptional repression.

[0209] In some embodiments, the endogenous nucleotide sequences of this disclosure include nucleotide sequences encoding elements of a system for providing conditional expression of the recombinase. In some embodiments, the endogenous nucleotide sequences encode a tetracycline / doxycycline-regulated transcriptional activation system for regulating the expression of the recombinase.

[0210] In various aspects and embodiments of employing the above-described system, the agent capable of inhibiting the primary immune response and / or promoting the secondary immune response may be tetracycline / doxycycline. Similarly, methods of this disclosure involving the above-described system may include administering tetracycline / doxycycline to an animal to inhibit the animal's ability to initiate a primary immune response.

[0211] In some embodiments, the endogenous nucleotide sequence encodes a conditional system for regulating recombinase activity.

[0212] In some embodiments, the recombinase encoded by an endogenous nucleotide sequence includes a portion that inducibly regulates recombinase activity. Regulation of recombinase activity can be achieved, for example, by influencing the subcellular localization of the recombinase. That is, regulation of recombinase activity can be achieved by modulating contact with the recombinase target sequence.

[0213] The active system of inducible recombinases is described, for example, in an article by Kim et al. published in *Lab Anim Res.* (2018, Vol. 34, No. 4, pp. 147-159) (incorporated above by reference). The tamoxifen-induced Cre system employs a fusion protein called CreER recombinase, which comprises a Cre recombinase fused to an estrogen receptor containing a mutant ligand-binding domain (ER-LBD). CreER is normally localized in the cytoplasm of cells expressing the fusion protein in a form bound to HSP90. However, binding to synthetic steroids (such as tamoxifen or 4-hydroxytamoxifen) disrupts the interaction between HSP90 and CreER, and CreERT (i.e., CreER-tamoxifen) translocates to the nucleus, where it binds to a loxP target sequence and exerts recombinase activity. CreERT2 is a variant of CreER that is approximately 10-fold more sensitive to 4-OHT in vivo.

[0214] In some embodiments, the nuclear translocation of the recombinase is inducible. In some embodiments, the recombinase comprises a portion of an estrogen receptor having a mutant ligand-binding domain (ER-LBD). In some embodiments, the recombinase is a Cre recombinase. In some embodiments, the Cre recombinase is CreERT, CreERT2, or a variant thereof. In a preferred embodiment, the Cre recombinase is CreERT2.

[0215] In some embodiments, the endogenous nucleotide sequence encodes a system regulated by tamoxifen or 4-hydroxytamoxifen, which is used to regulate the activity of the recombinase.

[0216] In various aspects and embodiments of employing the above-described system, the agent capable of inhibiting the primary immune response and / or promoting the secondary immune response may be tamoxifen / 4-hydroxytamoxifen. Similarly, methods of this disclosure involving such systems may include administering tamoxifen / 4-hydroxytamoxifen to an animal to inhibit the animal's ability to initiate a primary immune response.

[0217] In hematopoietic cells, the expression of the recombinase may be driven by regulatory sequences, such as those in B cell lineages. Therefore, in some embodiments, in hematopoietic cells, the endogenous nucleotide sequence encodes the recombinase under the regulation of a driving regulatory sequence (e.g., a promoter). In some embodiments, in B cell lineage cells, the endogenous nucleotide sequence encodes the recombinase under the regulation of a driving regulatory sequence (e.g., a promoter).

[0218] In some embodiments, the expression of the recombinase may be regulated by cell type or tissue-specific regulatory sequences. For example, the expression of the recombinase may be regulated by cell type or tissue-specific promoters or enhancers. In this way, the expression of the recombinase, and SSR-mediated gene knockout, may be restricted to target cells or tissues. Therefore, in some embodiments, the endogenous nucleotide sequence encodes the recombinase under the regulation of cell type or tissue-specific regulatory sequences, such as cell type or structure-specific promoters.

[0219] In some embodiments, the endogenous nucleotide sequence encodes a recombinase under the regulation of a hematopoietic cell or tissue-specific regulatory sequence (e.g., a hematopoietic cell or structure-specific promoter). In some embodiments, the endogenous nucleotide sequence encodes a recombinase under the regulation of a B-cell lineage-specific regulatory sequence (e.g., a B-cell lineage-specific promoter).

[0220] In some embodiments, the endogenous nucleotide sequence encodes a recombinase (e.g., Cre recombinase, such as CreERT2) under the regulation of the CD79A promoter.

[0221] In some embodiments, the endogenous nucleotide sequence encodes a target sequence of a recombinase (e.g., a loxP sequence) flanking one or more exons of one or more genes involved in initiating the primary humoral immune response. In some embodiments, the one or more genes involved in initiating the primary humoral immune response are IGHM and / or IGHD.

[0222] In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes flanking exons of the associated gene. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the entire coding sequence of the associated gene. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the promoter of transcription of the associated gene. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes / destroys one or more splice donor and / or acceptor sites encoded by the associated gene. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the translation start codon for RNA translation encoded by the associated gene. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence introduces a frameshift in the nucleotide sequence of the associated gene. In some embodiments, the result of recombinase-mediated excision of the flanking region of the recombinase target sequence is that the locus encodes a truncated and / or non-functional form of the protein encoded by the associated gene. In some implementations, recombinase-mediated excision of flanking regions of the recombinase target sequence leads to nonsense-mediated degradation of RNA transcribed from the locus.

[0223] In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence (e.g., a loxP sequence) flanking one or more exons of IGHM. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking one or more of exons 1, 2, 3, 4, 5, and 6 of IGHM. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking exons 1 through 6 of IGHM. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the entire coding sequence of IGHM. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the promoter of IGHM transcription.

[0224] In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence (e.g., a loxP sequence) flanking one or more exons of IGHD. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking one or more of exons 1, 2, and 3 of IGHD. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking exons 1 to 3 of IGHD. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the promoter of IGHD transcription.

[0225] In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence (e.g., a loxP sequence) flanking one or more exons of IGHM and IGHD. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking one or more of exons 1, 2, 3, 4, 5, and 6 of IGHM and one or more of exons 1, 2, and 3 of IGHD. In some embodiments, the endogenous nucleotide sequence encodes a recombinase target sequence flanking exons 1 to 6 of IGHM and exons 1 to 3 of IGHD. In some embodiments, recombinase-mediated excision of the flanking regions of the recombinase target sequence removes the promoters of IGHM and IGHD transcription. In some embodiments, recombinase-mediated excision of the flanking regions of the recombinase target sequence removes the promoter and all exons of IGHM. In some embodiments, recombinase-mediated excision of the flanking regions of the recombinase target sequence removes the promoter and one or more exons (e.g., exons 1 to 3) of IGHD.

[0226] In some embodiments, the endogenous nucleotide sequence encodes a target sequence flanking the IGHM / IGHD locus region shown in SEQ ID NO:3.

[0227] In some embodiments, the endogenous nucleotide sequence comprises or consists of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:4, for example, nucleotide sequence identity with SEQ ID NO:4 ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%. Or 100% of one of them. In some embodiments, the endogenous nucleotide sequence comprises or consists of the nucleotide sequence of SEQ ID NO:4.

[0228] In some embodiments, after recombinase-mediated excision, the IGHM / IGHD locus contains a nucleotide sequence with 60% or more nucleotide sequence identity to SEQ ID NO:5, for example, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%. ≥99% or 100%. In some embodiments, the IGHM / IGHD locus contains the nucleotide sequence of SEQ ID NO:5 after recombinase-mediated excision.

[0229] In some embodiments, the endogenous nucleotide sequence encodes a recombinase, such as a Cre recombinase. In a preferred embodiment, the Cre recombinase is CreERT2.

[0230] In some embodiments, the endogenous nucleotide sequence encodes a recombinase (e.g., a Cre recombinase, such as CreERT2) under the regulation of a conditional system for regulating the expression and / or activity of the recombinase. In some embodiments, the endogenous nucleotide sequence encodes a tamoxifen / 4-hydroxytamoxifen-regulated system for regulating the activity of the recombinase.

[0231] In some embodiments, in hematopoietic cells, the expression of the recombinase is regulated by a regulatory sequence (e.g., a promoter) driving its expression. In some embodiments, in B-cell lineage cells, the expression of the recombinase is regulated by a regulatory sequence (e.g., a promoter) driving its expression. In some embodiments, the expression of the recombinase is regulated by the CD79A promoter.

[0232] In some embodiments, the endogenous nucleotide sequence comprises a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:6, for example, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% nucleotide sequence identity relative to SEQ ID NO:6. In some embodiments, the endogenous nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:6.

[0233] animal This invention relates to animals for generating antigen-binding molecules that can suppress primary immune responses, for example, using the methods described herein.

[0234] Various aspects and embodiments of this disclosure relate to animals containing endogenous nucleotide sequences that induce suppression of primary immune responses. It should be understood that, in animals, endogenous nucleotide sequences induce suppression of primary humoral immune responses.

[0235] In various aspects and embodiments of this disclosure, according to the embodiments described herein, animals may contain endogenous nucleotide sequences that induce suppression of primary humoral immune responses.

[0236] The animal described in this disclosure can be any kind of animal individual / subject. In a preferred embodiment, the animal is a non-human animal.

[0237] The animal is preferably an individual / subject of a species commonly used to generate antibodies through immunization. For example, in some embodiments, the animal may be a mouse, rat, hamster, camel, guinea pig, rabbit, goat, chicken, primate (e.g., non-human primates, such as monkeys), sheep, donkey, cow, cat, dog, pig, or horse. In some embodiments, the animal is a mammal (e.g., a non-human mammal).

[0238] In some embodiments, the animal is a rodent species or a lagomorph (e.g., rabbit). Leporidae Individuals / subjects of species in the family (e.g., mice) Mus ) genus, rat ( Rattus ) genus or guinea pig ( Cavia(Individuals / subjects of a species of the genus ) In some embodiments, the animal is a mouse, rat, or rabbit.

[0239] In some embodiments, the animal is a mouse (that is, in some embodiments the animal is a mouse). Mus Individuals / subjects of species of the genus ); for example, house mice ( Mus musculus (Individuals or subjects of the species).

[0240] In some embodiments, the animal is a rat (e.g., a rat ( Rattus Individuals / subjects of the genus ); for example, brown rats ( Rattus norvegicus ) or black rat ( Rattus Rattus (Individuals / subjects of the species).

[0241] In some embodiments, the animal is a rabbit (e.g., a European rabbit). Oryctolagus Individuals / subjects of the genus ); for example, the European domestic rabbit ( Orychtolagus cuniculus (Individuals or subjects of the species).

[0242] Animals according to this disclosure may have a genome containing nucleotide sequences that induce suppression of the primary humoral immune response. The nucleotide sequences that induce suppression of the primary humoral immune response are preferably contained in the genomic DNA of the animal. That is, the nucleotide sequences may be integrated into or be part of the genomic DNA of the animal cells.

[0243] Animals according to this disclosure may have a genome or may contain genomic DNA containing nucleotide sequences that induce suppression of primary humoral immune responses, including, to say the endogenous nucleotide sequences that induce suppression of primary humoral immune responses.

[0244] Animals according to this disclosure may contain one or more (e.g., one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) endogenous nucleotide sequences that induce suppression of primary humoral immune responses.

[0245] Animals according to this disclosure may contain a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) endogenous nucleotide sequences, each endogenous nucleotide sequence conforming to one embodiment of the endogenous nucleotide sequences described herein. In the above embodiments, the plurality of endogenous nucleotide sequences may inducibly inhibit the expression or activity of the different genes / products. For example, animals according to this disclosure may include: endogenous nucleotide sequences that inducibly inhibit the expression or activity of IGHM products; and / or endogenous nucleotide sequences that inducibly inhibit the expression or activity of IGHD products.

[0246] In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression or product activity of one or more genes involved in initiating a primary humoral immune response. In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression or product activity of one or both of IGHM and / or IGHD.

[0247] In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly knocks out one or more genes involved in initiating the primary humoral immune response. In some embodiments, the animal comprises an endogenous nucleotide sequence that inducibly knocks out one or both of IGHM and / or IGHD.

[0248] As used herein, “inducible knockout” refers to gene knockout that is induced, for example, by responding to a given chemical or physical treatment. Inducible knockout can also be called “conditional knockout.” Examples of induced gene knockout techniques can be found in the literature by Kim et al., published in *Lab Anim Res.*, 2018, Vol. 34, No. 4, pp. 147-159. Knockout can be induced by treatment, leading to an increase in the expression or activity levels of the factor mediating gene knockout. For example, knockout can be mediated by site-specific recombinase (SSR) systems and can be induced by treatments that lead to an increase in the expression or activity levels of the relevant recombinase, which can be achieved by binding to the target sequence of the recombinase located flanking all or part of the nucleotide sequence of the target gene. Knockout can be restricted to a target cell type or tissue, for example, by placing the expression of the relevant recombinase under the control of regulatory sequences such as promoters or enhancers to regulate its expression in the target cell type or tissue.

[0249] In some embodiments, the animal according to this disclosure includes an endogenous nucleotide sequence (e.g., a loxP sequence) encoding a recombinase target sequence flanking one or more exons of one or more genes involved in initiating the primary humoral immune response. In some embodiments, the one or more genes involved in initiating the primary humoral immune response are IGHM and / or IGHD.

[0250] In some embodiments, the animal contains an endogenous nucleotide sequence (e.g., a loxP sequence) encoding a recombinase target sequence, located at... IGHMThe animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking one or more exons 1, 2, 3, 4, 5, and 6 of the IGHM. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking exons 1 through 6 of the IGHM. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the entire coding sequence of the IGHM. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the promoter of IGHM transcription.

[0251] In some embodiments, the animal contains an endogenous nucleotide sequence (e.g., a loxP sequence) encoding a recombinase target sequence, located at... IGHD The animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking one or more exons 1, 2, and 3 of IGHD. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking exons 1 through 3 of IGHD. In some embodiments, recombinase-mediated excision of the flanking region of the recombinase target sequence removes the promoter of IGHD transcription.

[0252] In some embodiments, the animal contains an endogenous nucleotide sequence (e.g., a loxP sequence) encoding a recombinase target sequence flanking one or more exons of IGHM and IGHD. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking one or more exons 1, 2, 3, 4, 5, and 6 of IGHM and one or more exons 1, 2, and 3 of IGHD. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a recombinase target sequence flanking exons 1 to 6 of IGHM and exons 1 to 3 of IGHD. In some embodiments, recombinase-mediated excision of the flanking regions of the recombinase target sequence removes the promoters for IGHM and IGHD transcription.

[0253] In some embodiments, the animal contains an endogenous nucleotide sequence encoding a target sequence flanking the IGHM / IGHD gene locus region shown in SEQ ID NO:3. In some embodiments, the animal contains an endogenous nucleotide sequence for excising the IGHM / IGHD gene locus region shown in SEQ ID NO:3.

[0254] In some embodiments, the animal comprises an endogenous nucleotide sequence that includes or consists of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:4, for example, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% nucleotide sequence identity relative to SEQ ID NO:4. In some embodiments, the animal comprises an endogenous nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:4.

[0255] In some embodiments, after recombinase-mediated excision, the animal comprises a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:5, for example, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% nucleotide sequence identity relative to SEQ ID NO:5. In some embodiments, after recombinase-mediated excision, the animal comprises the nucleotide sequence of SEQ ID NO:5.

[0256] In some embodiments, the animal according to this disclosure may contain more than one endogenous nucleotide sequence that induces suppression of the primary immune response according to this disclosure. In some embodiments, the animal may contain one of 1, 2, 3, 4, 5, 6, 7, 9, or 10 endogenous nucleotide sequences according to this disclosure. In the above embodiments, the plurality of endogenous nucleotide sequences may each independently conform to any embodiment of the endogenous nucleotide sequences described herein. In some embodiments, wherein in an animal according to this disclosure containing a plurality of endogenous nucleotide sequences, a single endogenous nucleotide sequence may induce suppression of a different gene (e.g., selected from IGHM and IGHD) involved in initiating the primary humoral immune response.

[0257] In some embodiments, the animal according to this disclosure contains an endogenous nucleotide sequence encoding a recombinase (e.g., Cre recombinase). In a preferred embodiment, the Cre recombinase is CreERT2.

[0258] In some embodiments, under the regulation of a conditional system for regulating the expression and / or activity of the recombinase, the animal according to this disclosure contains an endogenous nucleotide sequence encoding a recombinase (e.g., a Cre recombinase, such as CreERT2). In some embodiments, the animal disclosed includes an endogenous nucleotide sequence encoding an endogenous nucleotide sequence encoding a tamoxifen / 4-hydroxytamoxifen regulatory system for regulating the activity of the recombinase.

[0259] In some embodiments, the expression of the recombinase in animals is regulated by a regulatory sequence (e.g., a promoter) that drives expression in hematopoietic cells. In some embodiments, the expression of the recombinase in animals is regulated by a regulatory sequence (e.g., a promoter) that drives expression in B-cell lineage cells. In some embodiments, the expression of the recombinase in animals is regulated by the CD79A promoter.

[0260] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:6, for example, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, ≥70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% nucleotide sequence identity relative to SEQ ID NO:6. In some embodiments, the animal comprises an endogenous nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:6.

[0261] Animals containing endogenous nucleotide sequences that induce suppression of the primary humoral immune response may contain the aforementioned endogenous nucleic acid sequences through genetic engineering. Therefore, in some embodiments, the animal is a genetically engineered animal containing endogenous nucleotide sequences that induce suppression of the primary humoral immune response. Genetically engineered animals may also be referred to as transgenic animals.

[0262] Methods of genetically engineering animals to include target nucleotide sequences are well known to those skilled in the art, as described, for example, in the articles by Huijbers in Methods Mol Biol (2017, Vol. 1642: 1-19), Sumiyama et al. in PLoS One (2018, Vol. 13, No. 9: e0203056), and Asfaw et al. in Cogent Food & Agriculture (2019, Vol. 5, No. 1: 1686802), the entire contents of which are incorporated herein by reference. The aforementioned methods include, for example, pronuclear microinjection, which is described, for example, in the article by Pu et al. published in Methods MolBiol (2019, Vol. 1874: 17–41) (the entire contents of which are incorporated herein by reference), and, for example, SSN-mediated genetic modification of germ cells, fertilized eggs, or embryos, which is described, for example, in the article by Lee et al. published in Drug Discovery Today: Disease Models (2016, Vol. 20: 13–20) (the entire contents of which are incorporated herein by reference).

[0263] Methods for producing genetically engineered animals include, for example, methods for transfecting embryonic stem cells with nucleic acid sequences to integrate the genome via homologous recombination, selecting cells in which nucleic acid sequences have been integrated into their genomic DNA, introducing genetically modified embryonic stem cells into blastocysts, and implanting blastocysts containing genetically modified embryonic stem cells into the uterus for pregnancy.

[0264] The target nucleotide sequence can be prepared using recombinant DNA technology and introduced into cells / embryos via viral transduction vectors, or through microinjection, electroporation, etc. Cells / embryos / animals containing the target nucleotide sequence can be identified through appropriate screening, such as by Southern blotting, PCR, etc.

[0265] A major obstacle to the use of monoclonal antibodies derived from immunized animals in humans is their xenogeneic origin. The host initiates an immune response to non-host antibodies, leading to elimination and potentially adverse side effects. Various approaches have been taken to reduce or eliminate their immunogenicity, such as producing chimeric antibodies containing the human Fc region, and humanized chimeric antibodies in which the variable domains of the antibody are engineered to be similar to the sequence of human antibodies.

[0266] Recently, transgenic technology has been employed, in which endogenous immunoglobulin gene loci in animals are replaced by their human homologs. Monoclonal antibodies produced from these mice using conventional hybridoma technology are fully human. Human Ig transgenic mouse strains include xenogeneic mice (Abgenix; Green et al., *Nature Genetics*, 1994, Vol. 7: 13–21; Green, *Journal of Immunology Methods*, 1999, Vol. 231, pp. 1–2: 11–23) and UltiMAb (Mederex; Lonberg and Huszar, *International Review of Immunology*, 1995, Vol. 13: 65–93); Lonberg, *Nature Biotechnology*, etc. Biotechnol (2005, Vol. 23: 1117–1125) and Velocimmune (Regeneron; Murphy, Proceedings of the National Academy of Sciences (PNAS, 2014, Vol. 111, No. 14: 5153–5158)) have produced a number of human monoclonal antibodies, all of which have been approved for marketing and have acceptable safety and efficacy. Several human monoclonal antibodies have been produced and approved for acceptable safety and efficacy. Using the aforementioned antibody discovery platform, a major hurdle has been overcome by reducing the immunogenicity of the generated antibodies while preserving the practicality of mouse immunization. These mice have been engineered to maintain a strong B-cell response and generate a strong secondary immune response through repeated immunization with human antigens, enabling the development of multiple monoclonal antibody libraries.

[0267] In various aspects and embodiments of this disclosure, the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments. In some embodiments, the animal comprises genomic DNA encoding one or more human immunoglobulin genes or gene segments.

[0268] In some embodiments, the animal's genome encodes sequences of the VH and / or VL regions of human immunoglobulin. In some embodiments, the animal's genome encodes sequences of the Fc region of human immunoglobulin. In some embodiments, the animal's genome encodes sequences of the VH, VL, and / or Fc regions of human immunoglobulin.

[0269] Animals containing endogenous human immunoglobulin genes or gene fragments can be used to produce antibodies containing fully human Fv (i.e., VH and VL regions). Transgenic mice encoding human immunoglobulin genes / fragments have been described in articles such as those by Lu et al. published in *J Biomed Sci.* (2020, Vol. 27: 1) and Brüggemann et al. published in *Archives of Experimental Immunology and Therapeutics* (Warsz, 2015, Vol. 63, No. 2: 101–108), and also include Xenomouse (Abgenix; Green et al., *Nature Genetics* (1994, Vol. 7: 13–21); Green published in *J Immunol Methods* (1999, Vol. 231, No. 1–2: 11–23)) and UltiMAb (Mederex; Lonberg and Huszar published in *International Review of Immunology*). Immunol, 1995, Vol. 13: 65–93; Lonberg published in Nature Biotechnol (2005, Vol. 23: 1117–1125), TransChromo™ mice (Ishida et al. published in Cloning Stem Cells (2002, Vol. 4: 91–102)), and Velocimmune® (Regeneron; Murphy published in Proceedings of the National Academy of Sciences (PNAS, 2014, Vol. 111, No. 14: 5153–5158)).

[0270] In some embodiments, the animals of this disclosure contain endogenous nucleotide sequences encoding human immunoglobulin V, D and / or J genes or fragments thereof.

[0271] In some embodiments, the animals of this disclosure contain endogenous nucleotide sequences encoding human immunoglobulin genes or gene fragments, which have been published by Lu et al. (J Biomed Sci., 2020, Vol. 27: 1), Brüggemann et al. (Arch Immunol Ther Exp (Warsz), 2015, Vol. 63, No. 2: 101–108), Green et al. (Nature Genetics, Nat. Genet., 1994, Vol. 7: 13–21), Green (J Immunol Methods, 1999, Vol. 231, No. 1–2: 11–23), Lonberg and Huszar (International Review of Immunology, Int Rev Immunol, 1995, Vol. 13: 65–93), and Lonberg (Nature Biotechnology, Nat. Genet., 2015, Vol. 63, No. 2: 101–108), respectively. The entire contents of the animal genomes described in Biotechnol, 2005, Vol. 23: 1117–1125, Ishida et al. in Cloning Stem Cells, 2002, Vol. 4: 91–102, or Murphy in Proceedings of the National Academy of Sciences of the United States of America, PNAS, 2014, Vol. 111, No. 14: 5153–5158, US 7135287 B1, US 7105348 B2, or US 2006 / 059575 A1 are incorporated herein by reference.

[0272] In some embodiments, antibodies generated by the animal's immune system according to the present disclosure comprise fully human VH and / or VL region sequences. In some embodiments, antibodies generated by the animal's immune system according to the present disclosure comprise fully human Fc region sequences. In some embodiments, antibodies generated by the animal's immune system according to the present disclosure comprise fully human VH, VL, and / or Fc region sequences. In some embodiments, antibodies generated by the animal's immune system according to the present disclosure comprise fully human amino acid sequences.

[0273] In some embodiments, the animals according to this disclosure have been immunized with a first peptide / peptide containing the target amino acid sequence (i.e., (a) the amino acid sequence of the target protein / protein complex, or (b) an amino acid sequence similar to (a)) to elicit a primary immune response against the target amino acid sequence.

[0274] In some embodiments, the animal according to this disclosure is an individual / subject of a specific strain of mouse. In some embodiments, the animal is a C57BL / 6 mouse, aBALB / c mouse, a / J mouse, CD1 mouse, ICR mouse, 129S2 / SvPas mouse, or FVB / N mouse. The mouse strains cited above are described, for example, in *The Jackson Laboratory Handbook on Genetically Standardized Mice* (6th edition, October 2009, published by Jackson Laboratory, edited by Kevin Flurkey and Joanne M. Currer).

[0275] In some embodiments, the animal is a genetically engineered mouse (i.e., a transgenic mouse).

[0276] In some embodiments, the animal is a genetically engineered mouse in which the endogenous immunoglobulin locus is replaced by its human homolog. In some embodiments, the animal is a xenogeneic mouse, an UltiMAb mouse, a TransChromo mouse, or a Velocimune mouse. In some embodiments, the animals are Lu et al. (J Biomed Sci., 2020, Vol. 27: 1), Brüggemann et al. (Arch Immunol Ther Exp (Warsz), 2015, Vol. 63, No. 2: 101–108), Green et al. (Nature Genetics, Nat. Genet., 1994, Vol. 7: 13–21), Green (J Immunol Methods, 1999, Vol. 231, No. 1–2: 11–23), Lonberg and Huszar (International Review of Immunology, Int RevImmunol, 1995, Vol. 13: 65–93), Lonberg (Nature Biotechnology, Nat. Genet., 2015, Vol. 63, No. 2: 101–108), Green et al. (Nature Genetics, Nat. Genet., 1994, Vol. 7: 13–21), Green (J Immunol Methods, 1999, Vol. 231, No. 1–2: 11–23), Lonberg and Huszar (International Review of Immunology, Int RevImmunol, 1995, Vol. 13: 65–93), Lonberg (Nature Biotechnology, Nat. Genet. Genet., 2015, Vol. 63, No. 2: 101–108), Green et al. (Nature Biotechnology, Nat. Genet. Genet., 1994, Vol. 7: 13–21), Green (J Immunol Methods, 1999, Vol. 231, No. 1–2: 11–23), Lonberg and Huszar (Nature Biotechnology, Nat. Genet. Genet. Genet., 1995, Vol. 1 The mice described in Biotechnol, 2005, Vol. 23: 1117–1125, Ishida et al. (Cloning Stem Cells, 2002, Vol. 4: 91–102), or Murphy (Proceedings of the National Academy of Sciences, PNAS, 2014, Vol. 111, No. 14: 5153–5158), and U.S. Patents 7,135,287 B1, 7,105,348 B2, or U.S. Publication 2006 / 059575 A1.

[0277] In some embodiments, the animal is a genetically engineered mouse with extended lifespan (i.e., compared to an equivalent mouse lacking the aforementioned genetic modifications). Ladiges et al., in their article published in *Aging Cell* (2009, Vol. 8, No. 4: 346-352), described genetically modified mice with extended lifespans (the full text of which is incorporated herein by reference), and see, in particular, Table 1. In some embodiments, the animal is the mouse described in Table 1 of Ladiges et al. (*Aging Cell*, 2009, Vol. 8, No. 4: 346-352). In some embodiments, the animal is an AmesDwarf mouse, an αMUPA Tg mouse, or a p66-shc mouse. - / - Mice, GHr / BP - / - Mice, Ghrhr lit / lit Mice, Snell Dwarf mice, Igf1 + / - Mice, FIRKO mice, Klotho Tg mice, Mit CAT Tg mice, MT Tg mice, Tg heart-specific mice, UCP2 Tg brain-specific mice, PappA - / - Mice, AC5 - / - Mice, Surf1 - / - Mice, PEPCK muscle-specific expression mice, Irs1 - / - Mice, Irs2 + / - Mice, Irs2 + / - Brain-specific mice or IGF-1 Tg heart-specific mice.

[0278] In some embodiments, the animal is a mouse with a low incidence of spontaneous tumors, such as a genetically engineered mouse with a low incidence of spontaneous tumors (i.e., compared to an equivalent mouse lacking the aforementioned genetic modifications). In some embodiments, the animal is the mouse described by Rithidech et al. in Blood Cells, Molecules and Diseases (1999, Vol. 25, No. 1: 38-45).

[0279] In some embodiments, the animal is a mouse with a deficiency in immune cell tolerance, such as a genetically engineered mouse with a deficiency in immune cell tolerance. In some embodiments, the animal is the mouse described by Khattri et al. in the Journal of Immunology (2001, Vol. 167, No. 11: 6312-6320).

[0280] In some embodiments, the animal is a mouse with chronically activated immunity, such as a genetically engineered mouse with chronically activated immunity. In some embodiments, the animal is the mouse described by Subramanian et al. in the Proceedings of the National Academy of Sciences of the United States of America (2006, Vol. 103, No. 26: 9970-9975).

[0281] In some embodiments, the animal is a mouse with an autoimmune or hyperimmune dysregulation phenotype, such as a genetically engineered mouse. In some embodiments, the animal is a mouse containing genetic variations that spontaneously generate an autoimmune or hyperimmune dysregulation phenotype. In some embodiments, the animal is a mouse in which an autoimmune or hyperimmune dysregulation phenotype has been induced by treatment with chemicals or peptides / peptides. In some embodiments, the mouse is a NOD mouse, NZB / WF1 mouse, MRL mouse, BXSB mouse, LPR mouse, GLD mouse, motheaten mouse, Scurfy mouse, BaffTg mouse, etc. Bcl2 Tg mice, Bim - / - mice, C1qa - / - mice, C4 - / - mice, Cd19 Tg mice, Cd19 Cre- Traf3 fl / fl mice, Cd22 - / - Mice, Cd40l Tg mice, Cd45E613R KI mice Ctla4 - / - Mice, Dnase1 - / - mice, FoxP3 - / - mice, G2a - / - mice, Gadd45a - / - mice, Gadd45a - / - p21cip1 / waf - / - mice Gadd45b - / - Gadd45g - / - Mice, Il4 Tg mice, LatY136F KI mice Lck Cre- Pten fl / - mice Lyn - / - mice 、 Man2a1 - / - mice Mark2 - / - mice Mfge8 - / - mice 0x40 Cre- Pten fl / fl mice p65P13K Tg mice 、 Pd1 - / - mice 、Pkbα Tg mice 、Prkcd - / - Mice, Pten + / - mice Taci - / - Mouse or Tyro3 - / - Axl - / - Mertk - / - Mice.

[0282] In some embodiments, the animal is a mouse with autoimmune encephalitis, such as a genetically engineered mouse with autoimmune encephalomyelitis. In some embodiments, the animal is a mouse with autoimmune encephalitis in an SJL background, as described by Rajan et al. in the *Journal of Immunology* (1996, Vol. 157, No. 2: 941-949). In some embodiments, the animal is a mouse with collagen-induced arthritis in a DBA / 1 background, as described by Courtenay et al. in *Nature* (1980) 283(5748): 666-8. In some embodiments, the animal is a mouse with imiquimod-induced psoriasis in a BALB / c or C57BL / 6 background, as described by Van der Fits et al. in the *Journal of Immunology* (2009) 182(9): 5836-45.

[0283] In some embodiments, the animal is a hyperimmune mouse, such as a genetically engineered hyperimmune mouse. Genetically engineered hyperimmune mice produce a stronger immune response to the antigen than equivalent mice lacking the aforementioned genetic modifications. Genetically engineered hyperimmune mice include DiversimAb mice (Abveris) and DivergimAb mice (Ab). In some embodiments, the animal is a DiversimAb mouse or a DivigimAb mouse.

[0284] The inventors have advantageously discovered that the animals of this invention, produced using hyperimmune mice, can be used to generate antibodies with high affinity and titers.

[0285] Therefore, in some embodiments, the animal is a hyperimmune mouse. As used herein, a “hyperimmune mouse” (also referred to as an “autoimmune mouse”) can mean a mouse with a background or phenotype of hyperimmunity, dysregulated autoimmunity, or high immune response and / or a mouse with a hyperimmune, dysregulated autoimmune, or high immune-reactive strain.

[0286] In some embodiments, the hyperimmune mouse is a genetically engineered mouse.

[0287] In some embodiments, the hyperimmune mouse may have one or more of the following characteristics: - Compared to mice without hyperimmune, dysregulated autoimmune, or hyperimmune background or phenotype, they exhibit enhanced immune responses (or strong or overactive adaptive responses), for example, characterized by an enlarged pool of naive B cells, enhanced activation of primary or secondary immune responses after immunization with antigens, or enhanced activation of B cells after immunization with antigens.

[0288] - Compared to mice without hyperimmune response, with dysregulated autoimmune or hyperimmune background or phenotype, tolerance to antigens (self or foreign) is altered, for example, characterized by increased retention of self-reactive B cells, unrestrained stimulation and proliferation of self-reactive B cells, or loss of negative selection for somatic hypermutation-induced self-reactivity.

[0289] - B and T cell development is normal or near normal.

[0290] -Lifetime matches the length of time antibody discovery is active.

[0291] - A strong T-dependent antibody response.

[0292] - Enlarged germinal centers and B cell populations - Capable of producing autoantibodies that can switch classes.

[0293] - When immunized with self-epitopes, high serum antibody titers can be generated, such as epitopes or antigens that have high homology (>80%) with the self.

[0294] - When immunized with antigens that have poor immunogenicity, high serum antibody titers can be produced, meaning that a strong immune response will not be induced in mice without hyperimmune response, dysregulated autoimmunity, or hyperimmune response background or phenotype.

[0295] Therefore, in some implementations, the animals (e.g., hyperimmunized mice) may be able to produce strong antibody titers when immunized with self-epitopes or antigens with poor immunogenicity.

[0296] The production of hyperimmunized mice can be achieved using one or more of the following methods: - Mutations that affect B cell activation, proliferation, and survival, such as alterations in BCR and co-receptor signaling or loss of Fas-FasL-dependent apoptosis (as described by Miyamoto et al. in Nature (2002, Vol. 416, pp. 865-869) and Groom et al. in the Journal of Clinical Research (2002, Vol. 109, No. 1, pp. 59-68) in mice.

[0297] - Alterations in antigen presentation, such as the use of negative regulation of cytokine signaling for APC recruitment and migration (e.g., CCL2) or mutations in antigen-processing genes (e.g., TAP1 or LMP2).

[0298] - Mutations that affect the activation and function of Treg and TFH, such as those that alter TCR signaling or lead to FOXP3-mediated loss of differentiation (as described by Zahorsky Reeves and Wilkinson in the European Journal of Immunology (2001, Vol. 31, No. 1, pp. 196-204)).

[0299] - Spontaneously acquired gene mutations lead to an autoimmune phenotype, such as in NZB / W F1 mice.

[0300] In some embodiments, the hyperimmune mice are NOD mice, NZB / W F1 mice, MRL mice, BXSB mice, lpr mice, gld mice, motheaten mice, Scurfy mice, Baff Tg mice, Bcl2 Tg mice, Bim - / - mice, C1qa - / - mice, C4 - / - mice, Cd19 Tg mice, Cd19 Cre- Traf3 fl / fl mice, Cd22 - / - Mice, Cd40l Tg mice, Cd45E613R KI mice Ctla4 - / - Mice, Dnase1 - / - mice, FoxP3 - / - mice, G2a - / - mice, Gadd45a - / - mice, Gadd45a - / - p21cip1 / waf - / - mice Gadd45b - / - Gadd45g - / - mice ,Il4 Tg mice, LatY136F KI mice Lck Cre- Pten fl / - mice Lyn - / - mice, Man2a1 - / - mice Mark2 - / - mice, Mfge8 - / - mice 0x40 Cre- Pten fl / fl mice p65P13K Tg mice Pd1 - / - mice 、Pkbα Tg mice, Prkcd - / - Mice, Pten + / - mice Taci - / - Mouse or Tyro3 - / - Axl - / - Mertk - / - Mice.

[0301] In some embodiments, the hyperimmune mouse is an NZB / W F1 mouse. The NZB / W F1 mouse (also referred to herein as “NZBWF1” ​​or “NZBWF1 / J” mouse) is a New Zealand Black (NZB) x New Zealand White (NZW) F1 mouse, as described by Dubois et al. in JAMA (1966, Vol. 195, No. 4, pp. 285-289) and Bagavan et al. in Autoimmune Reviews (2020, Vol. 19, No. 2, Article No. 102686).

[0302] In some embodiments, the hyperimmune mouse comprises an endogenous nucleotide sequence that inducibly knocks out one or both of IGHM and / or IGHD. In some embodiments, the mouse is an NZBWF1 / J mouse. In some embodiments, the mouse is a highly immune... IghdIghm flox / flox ; Cd79a + / CreERT2 Mice. In some embodiments, the mice are NZBWF1 / J. IghdIghm flox / flox ; Cd79a + / CreERT2 Mice.

[0303] In some embodiments, the animal is a humanized mouse. The humanized mouse may have been implanted with cells or tissues derived from humans. The humanized mouse may also be a transgenic mouse.

[0304] In some embodiments, the humanized mice are as described by Chen and Murawsky in *Frontiers in Immunology* (2018, Vol. 9) and Ma B. and Osborn M. in *Transgenic Animals for Human Antibody Generation* (in *Introduction to Antibody Engineering*, a textbook in biological sciences, edited by Rüker F. and Wozniak-Knopp G., Springer, Chalmers, 2021, DOI:10.1007 / 978-3-030-54630-4_5). Transgenic animals used to produce human antibodies. In: Rüker, F., Wozniak-Knopp, G. (eds.) *Introduction to Antibody Engineering*. Biological Science Learning Materials. Chalmers Springer. https: / / doi.org / 10.1007 / 978-3-030-54630-4_5. In some embodiments, the animal is a humanized hyperimmune mouse.

[0305] Generate antigen-binding molecules Various aspects of this disclosure relate to methods for inducing the generation of antigen-binding molecules capable of binding to target proteins.

[0306] Various aspects of this disclosure relate to the generation of antigen-binding molecules, for example, for subsequent purification. Aspects of this disclosure also relate to the generation of cell populations that produce antigen-binding molecules.

[0307] As described herein, within the context of various aspects and embodiments of this disclosure, an "antigen-binding molecule" refers to a molecule capable of binding to a target antigen, such as an antibody / immunoglobulin. In some embodiments, the antibody / immunoglobulin according to this disclosure is IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In a preferred embodiment, the antibody / immunoglobulin is IgG.

[0308] Cells capable of producing antigen-binding molecules / antigen-binding molecules for downstream use, such as in therapeutic, research, imaging, and / or diagnostic applications. The methods disclosed herein can be used to prepare antigen-binding molecules having specific properties relevant to targets in therapeutic, research, imaging, and / or diagnostic applications.

[0309] In short, the method involves introducing a substance recognized as foreign (i.e., non-host) by the animal's immune system, causing the animal to selectively produce antibodies capable of binding to that substance. The substance may include, or may be processed into, an antigen. The immune system's ability to produce antibodies capable of specifically binding to antigens can be used to generate antibodies for the detection of target molecules in a variety of research, diagnostic, imaging, therapeutic, and preventative applications.

[0310] Methods for preparing antibodies are well known in the art, for example in *Antibodies: A Laboratory Manual (Second Edition, 2014)* (by Edward A. Greenfield, Cold Spring Harbor Laboratory Press), the full text of which is incorporated herein by reference. In particular, Chapter 6 describes in detail the methods for immunizing animals to produce antibodies.

[0311] In particular, aspects of the methods disclosed herein relate to the production of monoclonal antibodies. The methods may include isolating cells from a subject that generate antigen-binding molecules. The methods may also include generating monoclonal hybridomas from cells isolated from the subject, wherein the hybridomas produce a single type (i.e., monoclonal antibodies). one Specific antibodies.

[0312] Methods for antibody production involve introducing an antigen into an animal to induce antibody production, which can then be recovered from the animal.

[0313] Various aspects and implementations of the method disclosed herein include: (i) administering to an animal a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) Treating animals to suppress their ability to initiate a primary immune response; and (iii) administering a second peptide / polypeptide or nucleic acid encoding a second peptide / polypeptide to an animal, wherein the second peptide / polypeptide contains a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0314] In some embodiments, after a period of time sufficient for the animal to be treated to suppress its ability to initiate a primary immune response, cells (or cells derived from such cells) that have proliferated by administration of a first peptide / peptide activation / stimulation undergo immunoglobulin isotype conversion (i.e., cells expressing IgG-, IgE-, or IgA). In some embodiments, after a period of time sufficient for the cells (or cells derived from such cells) that have proliferated by administration of a first peptide / peptide activation / stimulation to differentiate into plasma B cells and / or memory B cells, the animal is treated to suppress its ability to initiate a primary immune response.

[0315] In some embodiments, the method for generating antigen-binding molecules includes one or more of the following: The preparation / formulation requires the introduction of animal peptides / polypeptides / nucleic acids / cells; Introducing peptides / polypeptides / nucleic acids / cells into animals; Detection and / or monitoring of antigen-binding molecules produced by animals; Detect and / or monitor the production of cells in animals that express / contain antigen-binding molecules; Collect antigen-binding molecules produced by the animals; Isolate / purify antigen-binding molecules produced by the animals; Cells that produce antigen-binding molecules are collected from the animal. Isolate / purify cells that produce antigen-binding molecules from the animals described above; The formation of hybridomas that produce antigen-binding molecules; Culture cells that produce antigen-binding molecules; and Isolate / purify antigen-binding molecules produced by cultured cells.

[0316] In some embodiments, the method includes isolating cells that produce antigen-binding molecules from an animal. In some embodiments, cells that produce antigen-binding molecules are obtained from the animal's blood (e.g., PBMCs obtained from animal blood) or from an animal's organ (e.g., spleen). In some embodiments, the isolated antigen-binding molecules-producing cells are cultured in vitro. In some embodiments, the method includes culturing cells isolated from a subject in vitro.

[0317] In some embodiments, the method includes isolating antigen-binding molecules capable of binding to a target protein. In some embodiments, the antigen-binding molecules are isolated from an animal. The antigen-binding molecules can be recovered from, for example, animal blood, plasma, serum, or ascites fluid.

[0318] In some embodiments, the antigen-binding molecule can be isolated from cells obtained from an animal. In some embodiments, the cell is a B cell. In some embodiments, the antigen-binding molecule can be isolated from the cell culture supernatant of B cells cultured in vitro.

[0319] In some embodiments, the antigen-binding molecule is obtained from a hybridoma that produces an antigen-binding molecule capable of binding to a target protein. In some embodiments, the method includes isolating an antigen-binding molecule capable of binding to a target protein from a hybridoma culture produced according to the present disclosure. In some embodiments, the antigen-binding molecule is obtained from the cell culture supernatant of a hybridoma culture. In some embodiments, the antigen-binding molecule is obtained from the blood, plasma, serum, or ascites fluid of an animal immunized with a hybridoma.

[0320] The methods for isolating (i.e. purifying) antigen-binding molecules from samples containing antigen-binding molecules (such as cells, cell extracts, cell culture media, blood, plasma, serum, ascites fluid) are well known to those skilled in the art and are described in detail in *Antigen-Binding Molecules: A Laboratory Manual (Second Edition, 2014)* (edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, cited above and incorporated herein by reference), particularly in Chapter 10. These methods include, for example, ion-exchange chromatography, purification based on protein A or protein G, gel electrophoresis, dialysis, and affinity purification based on target binding.

[0321] As used herein, isolated or purified antigen-binding molecules refer to compositions containing antibodies, wherein at least 80%, 90%, 95%, 99%, or 100% (by weight, or by weight of the protein component of the composition) is an antigen-binding small molecule component of the composition.

[0322] In some embodiments, the methods of this disclosure employ single B-cell clones. Methods for monoclonal antibody production using single B-cell cloning technology are described, for example, in articles by Carbonetti et al. in *Journal of Immunological Methods* (2017, Vol. 448, pp. 66-73) and Lei et al. in *Frontiers in Microbiology* (2019, Vol. 10, Article No. 672), both of which are incorporated herein by reference in their entirety. The aforementioned methods generally involve culturing B cells obtained from animals as monoclonal clones in vitro, for example in the presence of factors that promote B-cell proliferation and / or antibody production from B cells. B cells can be obtained from animal blood (e.g., PBMCs derived from animal blood) or from animal organs (e.g., spleen). B cells can be sorted into single-cell cultures using FACS or another cell sorting technique. B cells expressing antibodies with the target characteristics can be sequenced to determine the amino acid sequence of the antibody and / or the nucleic acid sequence encoding the antibody.

[0323] Methods for forming hybridomas are well known to those skilled in the art, for example, as described in detail in *Antibodies: A Laboratory Manual (Second Edition, 2014)* (edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, cited above and incorporated herein), particularly in Chapter 7. In short, according to the invention, an animal is immunized to stimulate an adaptive immune response, B lymphocytes are isolated from said animal, and these are fused with a suitable myeloma cell line to generate a hybridoma.

[0324] Prior to hybridoma formation, the antigen-binding molecules produced by the subject (e.g., antigen-binding molecule titer) can be determined as described herein. If the titer is too low, one or more enhancement steps can be performed as described herein, and the production of antigen-binding molecules can be monitored (e.g., by repeated blood sampling) until a sufficiently high titer is reached. In some embodiments, the method includes selecting subjects for hybridoma production based on the production or titer of antigen-binding molecules. The production or titer of antigen-binding molecules can be determined, for example, in blood, plasma, serum, or ascites samples obtained from animals.

[0325] In some implementations, antigen-binding molecules can be generated based on the detection of antigen-binding molecules capable of binding to target proteins, and subjects and / or cells can be selected.

[0326] In some embodiments, collecting cells that produce antigen-binding molecules includes obtaining the spleen and / or lymph nodes of the subject. In some embodiments, generating a hybridoma includes fusing cells capable of producing antigen-binding molecules obtained from the subject (e.g., B cells) with myeloma cells. In some embodiments, fusing cells capable of producing antigen-binding molecules obtained from the subject (e.g., B cells) with myeloma cells included in polyethylene glycol (PEG) co-centrifugation. In some embodiments, generating a hybridoma includes selection by culturing cells in a selective medium (e.g., a medium containing hypoxanthine-aminopterin-thymidine (HAT)).

[0327] As described herein, the production of antibodies by hybridoma populations capable of binding target proteins and / or other peptides and polypeptides can be tested, for example, by immunoprecipitation, immunoblotting, or in vitro binding assays (such as flow cytometry, ELISA, etc.). In some embodiments, antibody production or antibody titers can be determined in the culture supernatant of in vitro cultured hybridoma cells.

[0328] The present invention also provides an antigen-binding molecule capable of binding to a target protein, wherein the antigen-binding molecule is obtained by a method for generating antigen-binding molecules as described herein.

[0329] In some embodiments, the method further includes formulating the antigen-binding molecule into a composition, such as a pharmaceutical composition. In some embodiments, the method includes mixing the antigen-binding molecule with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The pharmaceutical composition may be formulated as a liquid (including gel) or a solid (e.g., piece Liquid formulations can be formulated for administration to selected areas of the human or animal body by injection or via catheter. This disclosure also provides a pharmaceutical composition formed by the method according to this disclosure.

[0330] The antigen-binding molecules produced by the methods disclosed herein can be mass-produced using methods well known to those skilled in the art.

[0331] Hybridomas can be cultured using standard cells. square The hybridoma can be propagated in vitro or in vivo, for example, as ascites in a host animal. In some embodiments, the method of this disclosure includes propagating the hybridoma by in vitro cell culture. In some embodiments, the method includes propagating the hybridoma in vivo by injecting the hybridoma into a host animal.

[0332] In some embodiments, antigen-binding molecules can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, the polynucleotide encoding the antibody can be derived from antibody-producing B cells or hybridoma cells. ，For example, reverse transcription PCR (RT-PCR) can be performed using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of antibodies, and the sequences of the polynucleotides can be determined. When the isolated polynucleotides encoding the heavy and light chains are transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, they can be cloned into suitable expression vectors that produce monoclonal antibodies, which would otherwise not produce immunoglobulins.

[0333] Target amino acid sequence The term “target amino acid sequence” as used in this article refers to (i) the amino acid sequence of the target protein / protein complex, or (ii) an amino acid sequence similar to the amino acid sequence of the target protein / protein complex (i.e., an amino acid sequence similar to the amino acid sequence of (i)).

[0334] As used herein, an amino acid sequence “similar” to a reference amino acid sequence refers to an amino acid sequence that shares some common characteristics with the reference amino acid sequence. In some embodiments, an amino acid sequence “similar” to a reference amino acid sequence is an amino acid sequence of a homolog, variant, or homologue of a protein that contains the reference amino acid sequence. In some embodiments, an amino acid sequence “similar” to a reference amino acid sequence (e.g., the amino acid sequence of a target protein) contains at least one of the following sequence identity: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, an amino acid sequence “similar” to a reference amino acid sequence differs from the reference amino acid sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0335] Conversely, an amino acid sequence "different from" the reference amino acid sequence refers to an amino acid sequence having less than 100% sequence identity with the reference amino acid sequence, such as less than 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%. In some embodiments, the amino acid sequence "different from" the reference amino acid sequence differs from the reference amino acid sequence by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0336] By aligning sequences and comparing phases answerThe amino acid position allows for comparison of a given amino acid sequence (i.e., the query sequence) with a reference amino acid sequence. In some embodiments, the sequence comparison is performed on regions where the query and reference sequences are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15 to 20, 20 to 25, 25 to 30, 30 to 60, 40 to 80, or 80 to 100 amino acids in length.

[0337] As used herein, “protein” includes peptides and polypeptides. In some embodiments, a target protein may be a target peptide or a target polypeptide. A “peptide” is a chain of two or more amino acid monomers linked by peptide bonds. The length of a peptide is typically in the range of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. The length of a polypeptide is typically greater than about 50 amino acids.

[0338] The peptides and polypeptides mentioned in this article also include complexes containing the aforementioned peptides / polypeptides, which may be homologous or heterologous multimeric complexes (e.g., formed through non-covalent interactions) containing two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) peptides / polypeptides.

[0339] The target protein can be any protein. For example, a target protein can be a diagnostic, prognostic, imaging, or therapeutically relevant protein. In some embodiments, the target protein can be a candidate therapeutic target of an antigen-binding molecule. As used herein, "target protein" means "one or more target proteins." That is, an antibody capable of binding to a protein may bind to more than one target protein. The term "target protein" as used herein also includes target protein complexes, which can be homologous or heterologous multimeric complexes (e.g., formed through non-covalent interactions) comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) polypeptides.

[0340] In some embodiments, the target protein is a protein whose expression / activity or upregulated expression / activity is positively correlated with a disease or condition (e.g., cancer, infectious disease, or autoimmune disease). In some embodiments, the target protein is expressed by a pathogen / infecting agent, cell, or cell of a tissue intended to disrupt or remove it. In some embodiments, the target protein is expressed by a pathogen / infecting agent, cell, or cell of a tissue intended to guide a humoral immune response. In some embodiments, the target protein is associated with cancer, an infectious disease, or an autoimmune disease. Pathogens include prokaryotes (bacteria), eukaryotes (e.g., protozoa, worms, fungi), and viral pathogens. In some embodiments, the target protein is expressed by cancer cells, an infectious agent, cells infected by an infectious agent, or autoimmune effector cells (i.e., effector cells of autoimmune pathologies). In some embodiments, the target protein is a disease / disease-associated (e.g., cancer-related and / or autoimmune disease) variant of the protein.

[0341] In some embodiments, the antigen-binding molecules generated according to the method of this disclosure are capable of recognizing proteins associated with the target protein.

[0342] "A protein associated with the target protein" refers to a protein that shares some characteristics with the target reference protein. For example, the methods disclosed herein can be used to trigger antigen-binding molecules capable of recognizing protein variants of pathogens / infectious agents. That is, the methods can be used to trigger broadly neutralizing antigen-binding molecules. In some embodiments, the protein associated with the target protein includes the target amino acid sequence. In some embodiments, the protein associated with the target protein includes the amino acid sequence of the target protein.

[0343] Related proteins include isotypes and fragments of the target protein. 、 Variants or homologs (such as paralogs or orthologs) include proteins belonging to the same protein family. A given target protein isotype, fragment, variant, or homolog may optionally be characterized by having an amino acid sequence that is at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the reference protein.

[0344] As used in this article, “sequence identity” refers to the percentage of nucleotide / amino acid residues in a subject sequence that are identical to those in a reference sequence after comparing sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity between sequences. Pairwise and multiple sequence alignments used to determine the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J., Bioinformatics, Vol. 21, pp. 951-960, 2005), T-coffee (Notredame et al., Journal of Molecular Biology, Vol. 302, pp. 205-217, 2000), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics, Vol. 6, No. 298, 2005), and MAFFT (Katoh and Standley, Molecular Biology & Evolution, Vol. 30, No. 4, pp. 772-780, 2013). When using the above software, it is preferable to use default parameters, such as space penalty and expansion penalty.

[0345] In some embodiments, the target amino acid sequence may be, for example, a shared sequence or a majority sequence of one or more related proteins in the corresponding region. In some embodiments, the target amino acid sequence may be a shared sequence in the corresponding region of two or more isotypes, homologs, or variants of the target protein.

[0346] In some embodiments, the target amino acid sequence is an antigenic amino acid sequence. As used herein, the term "antigenic" refers to the ability to stimulate an immune response, particularly an adaptive immune response (e.g., B cell and / or T cell-mediated immune responses). In some embodiments, the target amino acid sequence is capable of stimulating a B cell-mediated immune response. In some embodiments, the target amino acid sequence is an amino acid sequence that forms or is predicted to form a B cell epitope. In some embodiments, the target amino acid sequence is capable of stimulating the production of antigen-binding molecules.

[0347] In some embodiments, the target amino acid sequence is a continuous sequence of amino acids of the target protein, or a similar sequence. In some embodiments, such as in embodiments where the target amino acid sequence folds to form a discontinuous epitope, the target amino sequence is a discontinuous sequence of amino acids of the target protein, or a similar sequence.

[0348] In some embodiments, the target amino acid sequence is a continuous amino acid sequence that provides a linear epitope. In some embodiments, the target amino acid sequence is a continuous amino acid sequence that folds to provide a discontinuous epitope. In some embodiments, the target amino acid sequence is a discontinuous sequence of amino acids that together form a discontinuous epitope.

[0349] In some embodiments, the target amino acid sequence is a continuous or similar sequence of amino acids of the target protein. In some embodiments, the target amino acid sequence is a discontinuous or similar sequence of amino acids of the target protein. In some embodiments, the target amino acid sequence is a discontinuous or similar sequence of amino acids of the target protein complex. In some embodiments, the target amino acid sequence is a discontinuous amino acid sequence formed by the amino acid sequences or similar sequences of two or more polypeptides of the target protein complex.

[0350] In some embodiments, the target amino acid sequence is one of the following lengths: 5 to 100, 5 to 50, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, or 5 to 10 amino acids. In some embodiments, the target amino acid sequence is one of the following lengths: 10 to 100, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, or 10 to 12 amino acids. In some embodiments, the target amino acid sequence is one of the following lengths: 15 to 100, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 15 to 19, 15 to 18, or 15 to 17 amino acids. In some embodiments, the target amino acid sequence is one of the following lengths: 20 to 100, 20 to 50, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 amino acids. In some embodiments, the target amino acid sequence has a length of 5 to 30 amino acids.

[0351] In cases where the target amino acid sequence is provided or predicted to provide discontinuous epitopes, the target nucleic acid sequence can refer to either a continuous amino acid sequence folded to provide discontinuous epitopes or a discontinuous amino acid sequence forming discontinuous epitopes.

[0352] When the target amino acid sequence provides discontinuous epitopes, the total length of the discontinuous epitopes can be approximately 5 to 30 amino acids. Discontinuous epitopes can include, for example, 2, 3, 4, 5, 6, or 7 discontinuous amino acid sequences. Each of these discontinuous sequences can each contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.， However, it is preferable to have fewer than, for example, 30, 25, 20 or 15 amino acids.

[0353] Those skilled in the art can determine the antigenic sequence of a target protein using methods well-known in the art. For some proteins, known or predicted amino acid sequences of the antigens are available in databases such as BciPep, AntiJen, AntigenDB, SPTR, FIMM, HPTAA, IEDB, Epitome, MHCBN, MHCPEP, MPID-T2, and Protegen. The antigenic amino acid sequence of a protein can also be determined through literature searches, such as using the internet.

[0354] The amino acid sequence that forms a B-cell epitope and thus may effectively generate antibodies can be predicted from a query sequence using a variety of methods. Those skilled in the art can predict whether an amino acid sequence is antigenic by comparison with known or predicted antigenic sequences (e.g., for other proteins) and / or based on the properties of the amino acid sequence. For example, see the article by El-Manzalawy and Honavavar published in *Immunome Research*, Volume 6 (Supplement 2): S2, 2010, the full text of which is incorporated herein by reference. The methods described above take into account, for example, the hydrophilicity, flexibility, accessibility, turning, exposed surface, polarity, and antigenicity of the amino acid sequence. Whether a peptide / polypeptide contains a T-cell epitope can be determined, for example, using the predictive methods described by Desai et al. in *Methods of Molecular Biology*, Volume 1184, pp. 333-364, 2014.

[0355] Some of these methods take into account three-dimensional structures and can be used to predict conformational epitopes. Software that can be used to identify / predict amino acid antigen sequences includes EMBOSS:antigenic, BepiRed, IEDB Analysis Resource, SVMTriP and SCRATCH, ElliPro, COBEPro, BEPro, PEPITO, and DiscoTope.

[0356] The antigenic sequence of a target protein is determined by experimentally analyzing whether an amino acid or amino acid sequence possesses antigenicity. For example, those skilled in the art can determine whether a given amino acid sequence is antigenic, for instance, by using the amino acid... base The peptide sequence of the target protein is immunized with an acid sequence and the resulting peptide is used to determine whether an adaptive immune response is elicited. In the methods disclosed herein, one or more of the methods described above may be used alone or in combination to determine the antigenic sequence of the amino acids of the target protein.

[0357] When assessing the similarity of an amino acid sequence to a reference amino acid sequence of a target protein that provides or predicts to provide a discontinuous epitope, the amino acid sequence can be compared with a continuous amino acid sequence folded to form a discontinuous epitope or a discontinuous amino acid sequence that co-forms a discontinuous epitope.

[0358] First peptide / polypeptide The methods disclosed herein include administering a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide to an animal, wherein the first peptide / polypeptide comprises a target amino acid sequence. In some embodiments, the first peptide / polypeptide consists of or is substantially composed of the target amino acid sequence.

[0359] It should be understood that the first peptide / polypeptide includes peptides / polypeptides that comprise or consist of: (i) the amino acid sequence of the target protein, or (ii) an amino acid sequence similar to the amino acid sequence of the target protein (i.e., amino acids similar to those in (i)). sequence (Similar amino acid sequences).

[0360] In some embodiments, the first peptide / peptide comprises the target amino acid sequence and additionally comprises other amino acids. In some embodiments, the first peptide / peptide comprises the target amino sequence and one of 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, or 1-50 additional amino acids at one or both ends (i.e., the N or C terminus) of the target amino acid sequence.

[0361] In some embodiments, in the context of a protein derived from the target amino acid sequence, the additional amino acid corresponds to an amino acid provided at those sites relative to the target amino acid sequence. For example, in the case where the target amino acid sequence corresponds to amino acid positions 20 to 30 of the amino acid sequence of the target protein, and wherein the first peptide / polypeptide comprises the target amino acid sequence and five additional amino acids located at the N-terminus, these five additional amino acids may correspond to positions 15 to 19 of the amino acid sequence of the target protein.

[0362] The first peptide / polypeptide may comprise more than one peptide / polypeptide chain. For example, the first peptide / polypeptide may be a complex of peptides / polypeptides, such as comprising 2, 3, 4, 5, or 6 or more peptides / polypeptides. The peptides / polypeptides may be coupled to each other or administered as a composition (e.g., a mixture) of two or more unbound peptides / polypeptides.

[0363] In some embodiments, the first peptide / peptide is provided as a conjugate to a carrier protein. As used herein, a "carrier protein" refers to a protein that can be used to induce an immune response to the peptide / peptide conjugated thereto (i.e., act as a "carrier"). Due to their size and complexity, carrier proteins induce immune responses, including immune responses to conjugated peptides / peptides. Many proteins can be used as carriers and are selected based on the immunogenicity, solubility, and availability of available functional groups through which conjugation to the target peptide / peptide is achieved. Carrier proteins are well known in the field of immunology, for example, as described in Thermo Scientific Antibody Production and Purification Technology Manual (2nd Edition, 2010; Thermo Scientific, Inc., Document No. 1601975 09 / 10), the entire contents of which are incorporated herein by reference.

[0364] In some embodiments, the carrier protein is selected from keyhole hemocyanin (KLH), spirocyanin (CCH, also known as blue carrier protein), bovine serum albumin (BSA), cationic bovine serum albumin (cBSA), hepatitis B virus core antigen (HBc), thyroglobulin, and ovalbumin (OVA). In some embodiments, the carrier protein is selected from KLH, BSA, HBc, and OVA.

[0365] Peptides / peptides can be coupled to carrier proteins using methods well known to those skilled in the art, including, for example, amine-thiol crosslinking (e.g., using succinimide-6-[(β-maleimide propionamido)hexanoate] (SMPH)), EDC coupling (carboxyl-amino crosslinking), maleimide coupling (thiol crosslinking), and glutaraldehyde coupling (amine-amine crosslinking) (see Thermo Scientific Antibody Production and Purification Technical Manual, 2nd Edition (2010), Thermo Scientific, Inc., USA, 1601975 09 / 10).

[0366] As used herein, “first peptide / polypeptide” can refer to at least one peptide / polypeptide (i.e., one or more peptides / polypeptides). Similarly, “nucleic acid encoding the first peptide / polypeptide” can refer to a nucleic acid encoding at least one peptide / polypeptide, or a nucleic acid encoding one or more peptides / polypeptides. For example, “first peptide / polypeptide” can refer to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, such as a mixture or coupled to each other. “Nucleic acid encoding the first peptide / polypeptide” can refer to a nucleic acid encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, or a nucleic acid encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides. “First peptide / polypeptide” or “nucleic acid encoding the first peptide / polypeptide” can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 peptides / polypeptides or a mixture of nucleic acids encoding peptides / polypeptides.

[0367] As described in this article, two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially.

[0368] Second peptide / polypeptide The method disclosed herein includes administering a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide to an animal, wherein the second peptide or polypeptide contains a target amino acid sequence or an amino acid sequence similar to a target nucleic acid sequence.

[0369] In some embodiments, the second peptide / polypeptide is the same as the first peptide / polypeptide. In some embodiments, the second peptide / polypeptide is not the same as the first peptide / polypeptide.

[0370] It should be understood that a peptide / peptide "not identical" to a reference peptide / peptide includes an amino acid sequence having less than 100% sequence identity with the reference peptide or peptide. In some embodiments, the second peptide / peptide has an amino acid sequence having less than 100%, for example less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the first peptide / peptide. In some embodiments, the second peptide / peptide has an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the first peptide / peptide.

[0371] It should be understood that the second peptide / polypeptide includes peptides / polypeptides that comprise: (a) a target amino acid sequence contained in the first peptide / polypeptide; or (b) an amino acid sequence that is similar to the target amino acid sequence in the first peptide / polypeptide.

[0372] Conversely, it can be understood (as explained above) that the target amino acid sequence contained in the first peptide / polypeptide can be (i) the amino acid sequence of the target protein, or (ii) an amino acid sequence similar to the amino acid sequence of the target protein (i.e., similar to the amino acid sequence of (i)). base Amino acid sequences similar to acid sequences.

[0373] In some embodiments, the second peptide / peptide comprises the target amino acid sequence and additionally comprises other amino acids. In some embodiments, the second peptide / peptide comprises at least 5, 10, 15, 20, 40, 50, 80, 100, 200, or 300 additional amino acids at one or both ends (i.e., the N- or C-terminus) of the target amino acid sequence. In some embodiments, the second peptide / peptide comprises at least one of the following: 1-5, 1-10, 1-15, 1-20, 1-40, 1-50, 1-80, 1-100, 1-200, or 1-300 additional amino acids at one or both ends (i.e., the N- or C-terminus) of the target amino acid sequence.

[0374] The second peptide / polypeptide may comprise more than one peptide / polypeptide chain. For example, the second peptide / polypeptide may be a peptide / polypeptide complex, such as comprising 2, 3, 4, 5, or 6 or more peptides / polypeptides. The peptides / polypeptides may be coupled to each other or administered as a composition (e.g., a mixture) of two or more unbound peptides / polypeptides.

[0375] The peptides / polypeptides and nucleic acids described herein can be administered in the form of cells containing / expressing the peptides / polypeptides / nucleic acids or in the form of synthetic reagents containing the peptides / polypeptides / nucleic acids.

[0376] In some embodiments, the second peptide / polypeptide may further contain one or more amino acids at either end or both ends. In some embodiments, the second peptide / polypeptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids at one end or both ends, or 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4 or 1-3 amino acids.

[0377] In some embodiments, the second peptide / polypeptide can induce the generation of antigen-binding molecules capable of binding the target protein and its isotypes, variants, or homologs.

[0378] As used herein, “second peptide / polypeptide” can refer to at least one peptide / polypeptide (i.e., one or more peptides / polypeptides). Similarly, “nucleic acid encoding a second peptide / polypeptide” can refer to a nucleic acid encoding at least one peptide / polypeptide, or a nucleic acid encoding one or more peptides / polypeptides. For example, “second peptide / polypeptide” can refer to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, for example, in mixtures or coupled to each other. “Nucleic acid encoding a second peptide / polypeptide” can refer to a nucleic acid encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, or a nucleic acid encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides. “Second peptide / polypeptide” or “nucleic acid encoding a second peptide or polypeptide” can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 peptides / polypeptides or a mixture of nucleic acids encoding peptides / polypeptides.

[0379] As described in this article, two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially.

[0380] Nucleic acids encoding peptides / polypeptides As described herein, the peptides / polypeptides of this disclosure can be expressed by nucleic acids encoding the peptides / polypeptides. The nucleic acid may be a vector, or may be contained within a vector. In some embodiments, the nucleic acid may be DNA encoding the peptides / polypeptides described herein.

[0381] Nucleic acids / vectors can be administered to subjects to express the peptides / peptides described herein. The nucleic acids / vectors can be present in cells and can be administered to animals. The nucleic acids / vectors can be integrated into the genome of cells and can be administered to animals. The nucleic acids / vectors can provide recombinant expression of the peptides / peptides as described herein.

[0382] As used herein, "vector" refers to a nucleic acid molecule used as a means of transferring exogenous nucleic acids into cells. The vector may be a vector for expressing nucleic acids in cells. The aforementioned vector may include a promoter sequence operatively linked to a nucleotide sequence encoding a sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express peptides or polypeptides from the vectors disclosed herein. The term "operatively linked" may include placing the expression of the nucleic acid sequence under the influence or regulation of the regulatory sequence (thus forming an expression cassette) by covalently linking a selected nucleic acid sequence and a regulatory nucleic acid sequence (such as a promoter and / or enhancer). Thus, if the regulatory sequence is capable of influencing the transcription of the nucleic acid sequence, the regulatory sequence is operatively linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide. Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma-retroviral vectors such as vectors derived from mouse leukemia virus (MLV), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0383] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements required for protein expression from a vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector. ， For example, it may contain cytomegalovirus (CMV) or SV40 promoters to drive protein expression.

[0384] An immunological approach that induces an antibody response by administering nucleic acids encoding target peptides / peptides to subjects is described in an article by Aurisicchio et al. (2012) published in the Journal of Cell Physiology (J Cell Physiol 227: 3381-3388), the full text of which is incorporated herein by reference.

[0385] In some embodiments of the methods disclosed herein, nucleic acids encoding the peptides / polypeptides described herein are administered to animals. In the above embodiments, the peptides or polypeptides are expressed in animals following immunization.

[0386] application Various aspects of this disclosure relate to administering peptides / polypeptides or nucleic acids encoding peptides / polypeptides to animals. These aspects also include administering agents to animals for inducing inhibition of the animal's ability to initiate a primary immune response and / or promoting a secondary immune response in the animal.

[0387] The step of “administering” a peptide / polypeptide / nucleic acid to an animal includes introducing the peptide / polypeptide / nucleic acid into the animal once or multiple times. In some embodiments, “administering” a peptide / polypeptide / nucleic acid to an animal includes introducing one of peptide / polypeptide / nucleic acid 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 into the animal alone.

[0388] For example, in some embodiments, the method includes introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal in two separate doses, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal in two separate doses.

[0389] In some embodiments, the method includes introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal in one of three, four, five, or six steps, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal in one of one, two, three, or four steps.

[0390] In some embodiments, the method includes introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal at least twice, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal at least once.

[0391] Similarly, the step of “administering” an agent that causes suppression of the primary immune response and / or promotion of the secondary immune response includes introducing the agent into the animal once or multiple times. In some embodiments, “administering” an agent includes introducing the agent into the animal individually in one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0392] In some embodiments, the method includes introducing the reagent as defined herein into the animal in one, two, three, or four separate steps.

[0393] In some embodiments, in the application steps that include multiple introductions, the time interval between individual introductions is at least one of 24 hours, 36 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 7 days, 10 days, or 12 days. In some embodiments, in the application steps that include multiple introductions, the time interval between individual introductions is about 5-30 days, 7-20 days, for example, about 10-16 days. In some embodiments, in the application steps that include multiple introductions, the time interval between individual introductions is about 2-30 days, 5-20 days, for example, about 6-8 days.

[0394] The application steps described herein may be introduced using the same material or different materials, as long as the material meets the requirements for materials applied according to the application steps.

[0395] For illustrative purposes, the application of a first peptide / polypeptide (or nucleic acid encoding such peptide / polypeptide) as defined herein may include the simultaneous or sequential introduction of the same peptide / polypeptide, or two or more (e.g., one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-identical peptides or polypeptides, each peptide / polypeptide independently satisfying the requirements of the first peptide / polypeptide (or its encoding nucleic acid) as described herein. Similarly, the application of a second peptide / polypeptide (or its encoding nucleic acid) as defined herein may include the simultaneous or sequential introduction of the same peptide / polypeptide, or two or more (e.g., one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-identical peptides / polypeptides, each peptide / polypeptide independently satisfying the requirements of the second peptide / polypeptide (or its encoding nucleic acid) as described herein.

[0396] The individual introduction of the administration steps described herein may be sequential. That is, in some embodiments, the peptide / polypeptide / nucleic acid is introduced into the subject and then introduced individually after a given time interval.

[0397] In some embodiments, the peptide / peptide may be administered in the form of a reagent containing or expressing the peptide / peptide. In some embodiments, the peptide / peptide may be administered in the form of a reagent containing a nucleic acid encoding the peptide / peptide. In some embodiments, the nucleic acid may be administered in the form of a reagent containing the nucleic acid.

[0398] The reagent can be any reagent capable of delivering peptides / polypeptides / nucleic acids. In some embodiments, the reagent is a cell. In some embodiments, the reagent is a virus or virus-like particles.

[0399] In some embodiments, the peptide / polypeptide can be administered in a cellular form comprising or expressing the peptide / polypeptide. In some embodiments, the peptide / polypeptide can be administered in a cellular form comprising nucleic acid encoding the peptide / polypeptide. In some embodiments, the nucleic acid can be administered in a cellular form comprising the nucleic acid.

[0400] Cells expressing peptides / peptides can express the peptides / peptides endogenously. That is, the peptides / peptides can be encoded and / or expressed by the cell's nucleic acids before any nucleic acid encoding the peptide / peptide is introduced into the cell. Cells expressing peptides / peptides can also express the peptides / peptides exogenously. That is, the peptides / peptides can be encoded and / or expressed by nucleic acids introduced into the cell. In some embodiments, cells expressing peptides / peptides may be modified or have been modified to express or overexpress the peptides / peptides.

[0401] In some embodiments, the applied peptide / peptide / nucleic acid / cell / reagent is isolated or purified. As used herein, isolated or purified peptide / peptide / nucleic acid / cell / reagent refers to a composition comprising peptide / peptide, nucleic acid, cell / reagent, wherein at least 80%, 90%, 95%, 99%, or 100% (by weight) of the composition is a peptide / peptide / nucleic acid, cell, or reagent component of the composition.

[0402] In some embodiments, the peptide / polypeptide / nucleic acid is administered in an extract containing cells or proteins.

[0403] Peptides / polypeptides / nucleic acids / cells / reagents can be introduced into subjects in any suitable manner, as described in Antibodies: A Laboratory Manual (Second Edition, 2014) (edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press) (cited above in full), especially in Chapter 6.

[0404] Materials introduced into animals can be appropriately formulated based on the materials, route of introduction, animals, and desired response.

[0405] For example, peptides, polypeptides (optionally bound to carrier proteins such as KLH, BSA, or OVA), cells, and reagents can be diluted in sterile saline and can be used with adjuvants (e.g., complete or incomplete Freund's adjuvant). 、 Aluminum salts (such as aluminum sulfate (alum), aluminum phosphate, aluminum hydroxide) ）、 CpG or an adjuvant described by Lee and Nguyen in *Immune Netw.* (2015; 15(2):51-57) – the full text of which is incorporated herein by reference) is mixed to form a stable emulsion. Nucleic acids (optionally provided as liposomal nucleic acid complexes) can be injected in an aqueous solution of saline, introduced in an aqueous solution by pneumatic (jet) delivery, or coated onto gold microbeads and introduced via a gene gun.

[0406] When introducing material into an animal by injection, any suitable injection site can be used. For example, injection can be intraperitoneal, intravascular (e.g., intravenous or intraarterial), intradermal, subcutaneous, intramuscular, intraosseous, intrasheath, epidural, intracardiac, intra-articular, intracavernosal, and intravitreal injection. According to the method of this disclosure, preferred injection routes are intraperitoneal and intravascular (e.g., intravenous or intraarterial).

[0407] The appropriate amount or number of cells for a single introduction of peptide / polypeptide / nucleic acid / reagent can be readily determined by those skilled in the art, for example, by referring to *Antibodies: A Laboratory Manual (Second Edition, 2014)* (edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, formerly cited herein). Those skilled in the art can also readily determine the appropriate volume and concentration of the formulation used for introduction.

[0408] In some embodiments, the peptide / polypeptide / nucleic acid / cell can be formulated differently for different introduction and / or different administration steps. For example, in some embodiments, different carrier proteins or adjuvants can be used for different introduction and / or different administration steps. In some embodiments, according to the method of this disclosure, one or more adjuvants can be used for one or more introduction and / or administration steps, while different adjuvants or no adjuvants can be used for one or more other introduction and / or administration steps.

[0409] For example, in one embodiment of the method according to this disclosure, the step of administering a first peptide / polypeptide to a subject may include introducing the first peptide / polypeptide formulated with, for example, complete Freund's adjuvant and CpG into the subject, and the step of administering a second peptide / polypeptide to the subject may include introducing the second peptide / polypeptide formulated with, for example, incomplete Freund's adjuvant and CpG into the subject.

[0410] According to this disclosure, peptides / polypeptides / nucleic acids / cells / reagents can be introduced into animals in appropriate amounts to produce the desired response.

[0411] For example, introducing a peptide / peptide as defined herein into animals may include introducing one of 5 μg, 10 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, or 500 μg of peptide / peptide (e.g., all or each administration). In some embodiments, introducing a peptide / peptide as defined herein into animals may include introducing one of 5-500 μg, 10-200 μg, 20-80 μg, or ~50 μg of peptide / peptide into animals (e.g., all or each administration).

[0412] For example, introducing 5 μg, 10 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, or 500 μg of the agent (e.g., all or each time). In some embodiments, introducing the reagent as defined herein into an animal may include introducing one of 5-500 μg, 10-300 μg, 50-200 μg, or ~100 μg of the reagent into the animal (e.g., all or each time).

[0413] Introducing the reagents as defined herein into animals may include introducing one of the reagents at a concentration of 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, 20 mg / kg, or 25 mg / kg into animals (e.g., all or each time). In some embodiments, introducing the reagents as defined herein into animals may include introducing one of the reagents at a concentration of 1-25 mg / kg, 2-20 mg / kg, 5-15 mg / kg, or ~10 mg / kg into animals (e.g., all or each time). The administration steps of the methods disclosed herein may be sequential. That is, in some embodiments, the administration steps described herein are performed, and separate administration steps are performed after given time intervals.

[0414] The time interval between the sequentially performed administration steps is preferably a suitable time interval for the desired response in the animal. For example, in one embodiment of the method disclosed herein, sufficient time may be allowed for the animal to generate one or more antigen-binding molecules that bind to the first peptide / peptide (or nucleic acid encoding the peptide / peptide) after administration of the first peptide / peptide (or nucleic acid encoding the second peptide / peptide) as described herein and before administration of the second peptide / peptide (or nucleic acid encoding the second peptide / peptide).

[0415] In some embodiments, the time interval between the sequentially performed application steps is at least one of 24 hours, 36 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 7 days, 10 days, 14 days, 18 days, 21 days, or 28 days. In some embodiments, the sequentially performed application steps take approximately 3 to 21 days, for example, approximately 5 to 18 days, 7 days, or 28 days. to 16 days or 12-16 days. In some embodiments, the sequential application steps take approximately 14 days. In some embodiments, the sequential application steps take approximately 23 days. In some embodiments, the sequential application steps take approximately 30 days.

[0416] In embodiments where the application steps include introducing the material into the animal multiple times individually, the time intervals provided herein may be between the last introduction of an application step and the first introduction of a subsequent application step.

[0417] In some embodiments, the application steps of the methods disclosed herein may be performed simultaneously. That is, in some embodiments, the application steps described herein are performed simultaneously with another application step described herein, or immediately before or after it.

[0418] In certain embodiments, the methods of this disclosure involve simultaneously administering to an animal an agent capable of suppressing a primary immune response and / or capable of promoting a secondary immune response, as well as the second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) described herein.

[0419] The time interval between the application steps performed simultaneously is preferably less than one of 72 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours or 6 hours.

[0420] In some embodiments, when the administration steps are performed simultaneously, the reagents for each individual administration step are formulated together into a single formulation for administration to animals.

[0421] The administration steps described in this article, whether performed sequentially or simultaneously, can be introduced into animals via the same or different routes.

[0422] In some embodiments, an agent for suppressing primary immune responses and / or promoting secondary immune responses is administered in animals before, at the same time (e.g., simultaneously), and / or after administration of the second peptide / peptide. In some embodiments, the administration of the agent for suppressing primary immune responses and / or promoting secondary immune responses in animals is carried out before and / or after administration of the second peptide / peptide, for example, once or more within a maximum of 5 days before administration of the second peptide / peptide, and / or once or more within a maximum of 10 days after administration of the second peptide / peptide.

[0423] In some embodiments, in animals, an agent that inhibits the primary immune response and / or promotes the secondary immune response is administered 3 days and / or 1 day before administration of the second peptide / peptide, and 1, 3 and / or 6 days after administration of the second peptide / peptide.

[0424] In some embodiments, in animals, an agent that inhibits the primary immune response and / or promotes the secondary immune response is administered 1, 3 and / or 6 days after administration of the second peptide / polypeptide.

[0425] In aspects of this disclosure, in animals, including the administration of agents capable of suppressing primary immune responses and / or promoting secondary immune responses, it should be understood that the administration of said agents is for the purpose of suppressing primary immune responses to second peptides / peptides, and / or for the purpose of promoting secondary immune responses to them.

[0426] Similarly, it should be understood that the administration of the first peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) described herein, the administration of the reagent used to induce suppression of primary immune responses and / or promotion of secondary immune responses in animals, and the administration of the second peptide / polypeptide (or nucleic acid encoding the polypeptide) described herein, results in the primary immune response to the first peptide / polypeptide not being substantially suppressed, but the primary immune response to the second peptide or polypeptide being suppressed, and / or the secondary immune response being promoted.

[0427] In some implementations, the method includes one or more further application steps as described herein.

[0428] For the purposes of the following instructions, the application steps are as follows: (a) Administering the first peptide / polypeptide or nucleic acid encoding the first peptide / polypeptide to the animal described herein, wherein the first peptide / polypeptide comprises the target amino acid sequence; (b) Administering to animals agents used to suppress (e.g., induce suppression) primary immune responses and / or promote secondary immune responses in animals; and (c) Administering to an animal a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide, wherein the second peptide / polypeptide comprises a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence, and wherein the second peptide / polypeptide is different from the first peptide / polypeptide (e.g., larger than the first peptide / polypeptide).

[0429] In some embodiments, the method of this disclosure includes one or more combinations of the following administration steps described above: (1) (a) + (c) (2) (a)+(b)+(c) (3) (a) + (c) + (a) (4) (a) + (b) + (c) + (a) (5) (a)+(b)+(c)+(b)+(a) (6) (a) + (c) + (b) (7) (a)+(b)+(c)+(d) In some implementations, the application step is as follows: (a) Administering the first peptide / polypeptide or nucleic acid encoding the first peptide / polypeptide to the animal described herein, wherein the first peptide / polypeptide comprises the target amino acid sequence; (b) Administering to an animal a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide, wherein the second peptide or polypeptide comprises a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence, and wherein the second peptide / polypeptide differs from the first peptide / polypeptide (e.g., is larger than the first peptide / polypeptide); and (c) Administering to animals an agent that suppresses (e.g., induces suppression) the primary immune response and / or promotes the secondary immune response in the animals.

[0430] In some implementations, the application step is as follows: (a) Administering the first peptide / polypeptide or nucleic acid encoding the first peptide / polypeptide to the animal described herein, wherein the first peptide / polypeptide comprises the target amino acid sequence; (b) Administering to animals agents used to suppress (e.g., induce suppression) primary immune responses and / or promote secondary immune responses in animals; (c) Administering to an animal a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide, wherein the second peptide or polypeptide comprises a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence, and wherein the second peptide / polypeptide differs from the first peptide / polypeptide (e.g., is larger than the first peptide / polypeptide); and (d) Administering to animals agents that suppress (e.g., induce suppression) primary immune responses and / or promote secondary immune responses in animals.

[0431] In some embodiments, the method further includes an enhancement step. An enhancement step may include introducing the peptide / peptide / nucleic acid described herein into an animal without a carrier or adjuvant.

[0432] Enhancement steps are well known to those skilled in the art of immunology. Methods for producing antigen-binding molecules may include enhancement steps, such as increasing the titer before isolating the antigen-binding molecules or before obtaining cells for hybridoma generation.

[0433] Enhancement steps may be included in methods for generating immunity against proteins / pathogens, such as inducing a memory response.

[0434] In some embodiments, a boosting step is performed before isolating the antigen-binding molecule. In some embodiments, a boosting step is performed before obtaining B lymphocytes for hybridoma generation. In some embodiments, a boosting step is performed at least about 12 hours to 5 days, about 1-4 days, or about 2-3 days before obtaining B lymphocytes for hybridoma generation.

[0435] In some embodiments, the enhancer step includes administering to the animal a peptide / polypeptide / nucleic acid that has already been administered to the animal, for example, in the prior administration step of (a) or (c) above.

[0436] In some embodiments, the method includes performing more than one reinforcement step (e.g., one of 2, 3, 4, 5, or 6). The multiple reinforcement steps may be spaced at least 12 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours apart. In some embodiments, the reinforcement steps may be spaced approximately 12-48 hours apart, for example, approximately 24 hours.

[0437] The enhancement step may include introducing the peptide / polypeptide / nucleic acid by injection. In some embodiments, particularly those related to methods for generating antigen-binding molecules, the enhancement step may include injecting the peptide / polypeptide / nucleic acid into the abdomen of the animal.

[0438] Assessment of antigen-binding molecule production The methods disclosed herein may also include monitoring / evaluating the immune response in animals following one or more administration steps as described herein. In some embodiments, the methods include detecting the presence of an immune response capable of recognizing a target peptide / peptide / amino acid sequence.

[0439] In some embodiments, the method includes detecting the presence of an antigen-binding molecule capable of binding to a target peptide / peptide / amino acid sequence. In some embodiments, the method includes detecting the presence of immune cells / populations of immune cells capable of generating antigen-binding molecules capable of binding to the target peptide / peptide / amino acid sequence.

[0440] In some embodiments, the method includes detecting the presence of an antigen-binding molecule having one or more target functional properties, or detecting the presence of immune cells / populations of immune cells capable of producing the aforementioned antigen-binding molecule. Target functional properties may be, for example, the ability to antagonize or activate the function of a target protein / protein complex (e.g., catalytic activity, binding (e.g., protein-protein interactions, such as ligand-receptor binding or polymerization), signal transduction, transport, storage, structural support, etc.). The aforementioned functional properties of the antigen-binding molecule can be analyzed, for example, using appropriate assays for the aforementioned functions of the target protein / protein complex.

[0441] Immune responses can be analyzed using methods well known to those skilled in the art. For example, at an appropriate time after administration according to the methods described herein, samples (e.g., blood samples) can be obtained from the subject and analyzed for the production of antigen-binding molecules and / or the presence of cells exhibiting target characteristics (e.g., the production of antigen-binding molecules capable of recognizing the target antigen).

[0442] Methods for detecting and quantifying antigen-binding molecules are well known to those skilled in the art, for example in *Antibodies: A Laboratory Manual (Second Edition)* (Edward A. Greenfield, ed., Cold Spring Harbor Laboratory Press, 2014; formerly incorporated herein by reference), particularly in Chapter 15. For example, following one or more administration steps according to this disclosure, blood, plasma, serum, or ascites samples can be obtained from a subject and analyzed by, for example, ELISA or flow cytometry. As used herein, “plasma” refers to the liquid component of blood lacking cellular components and may be the liquid portion of blood obtained after the removal of blood cells. As used herein, “serum” refers to plasma lacking clotting factors (such as fibrinogen) and may be the liquid portion of blood obtained after the removal of fibrin clots and blood cells. As used herein, “ascites” refers to fluid obtained from the peritoneal cavity.

[0443] Immunoassays can be used to detect the production of antigen-binding molecules. They can be used to determine whether a sample obtained from a subject contains antigen-binding molecules capable of binding a given peptide or polypeptide.

[0444] In some embodiments, the method includes quantifying antigen-binding molecules in a sample. ， For example, by measuring antibody titers. Antibody titer is a measure of the amount of antibody produced by a subject that can recognize (i.e., bind to) a given antigen. Antibody titer is expressed as the reciprocal of the highest dilution of the test sample (such as a serum sample) that gives a positive result for the detection of the antigen, for example, in an immunologic assay.

[0445] As used herein, “binding” via an antigen-binding molecule refers to a specific interaction between the antigen-binding molecule and its homologous antigen. “Specific interaction” is not a non-specific interaction between an antibody and an antigen. Antigen-binding molecule: Antigen binding is mediated by non-covalent interactions (e.g., van der Waals forces, electrostatic interactions, hydrogen bonds, and hydrophobic interactions). Specifically, this interaction is the interaction between the antigen-binding site of the antigen-binding molecule and its homologous epitope in the antigen. An epitope is a portion of an antigen that contacts the antigen-binding molecule. Specifically, the epitope is the antigenic portion bound by the antigen-binding molecule. Epitopes are provided by the antigenic sequence of amino acids. Epitopes can be linear, consisting of a continuous sequence of amino acids (i.e., the primary amino acid sequence). Alternatively, epitopes can be conformational, consisting of a discontinuous sequence of amino acids from any antigenic amino acid sequence. The discontinuous amino acid sequence can be located in different regions of a peptide / polypeptide and can be located at very close sites when the antigen is folded, for example, when folded into its native structure.

[0446] The ability of a given antigen-binding molecule to bind a given protein can be analyzed using techniques well known to those skilled in the art, including ELISA, Western blotting (e.g., protein blotting), immunoprecipitation, surface plasmon resonance (SPR; see, for example, Hearty et al., *Methods in Molecular Biology*, 2012; Vol. 907: 411-442), or biomembrane interference (see, for example, Lad et al., *Journal of Biomolecular Screening*, 2015; Vol. 20(4): 498-507), and flow cytometry. These analyses can determine and quantify the interaction between the protein / domain and the antigen-binding molecule. These methods may involve expressing the protein / domain, contacting the expressed protein / domain with the antigen-binding molecule, and detecting the formation of a non-covalent complex between the protein / domain and the antigen-binding molecule.

[0447] The specific region of the given binding partner bound by the antigen-binding molecule can also be analyzed using methods well known in the art, including X-ray cocrystal analysis of antibody-antigen complexes, mass spectrometry hydrogen-deuterium exchange analysis, cryo-electron microscopy, peptide scanning, and mutagenesis localization. For example, the studies by Gershoni et al. published in BioDrugs, Vol. 21, No. 3, 2007, pp. 145-156, and the literature by Abbott et al. published in Immunology, Vol. 142, pp. 526-535, 2014, are included in this article by full citation.

[0448] Other items disclosed The present invention also provides one or more nucleic acids comprising the nucleotide sequence described herein.

[0449] In some embodiments, the nucleic acid / multiple nucleic acids (e.g., from other nucleic acids or naturally occurring biological materials) are purified or isolated. In some embodiments, the nucleic acid / multiple nucleic acids comprise or consist of DNA and / or RNA.

[0450] It should be understood that in some embodiments, the multiple nucleic acids according to this disclosure may comprise one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 9, or 10) nucleotide sequences of this disclosure. In the above embodiments, the multiple nucleotide sequences may independently conform to any embodiment of the nucleotide sequences described herein.

[0451] This disclosure also provides one or more vectors containing nucleic acids / multiple nucleic acids according to this disclosure.

[0452] The nucleic acid / multiple nucleic acids may be contained in a vector or multiple vectors. As used herein, "vector" refers to a nucleic acid molecule used to transfer exogenous nucleic acids into cells. A vector can be a carrier for expressing nucleic acids in cells. （ (i.e., expression vector). The vector may include a promoter sequence operatively linked to a nucleotide sequence encoding the expression sequence. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon well known in the art can be used.

[0453] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, and viral vectors (e.g., γ-). inverse Transcriptoviral vectors (such as vectors derived from mouse leukemia virus (MLV), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (such as yeast artificial chromosomes).

[0454] In some embodiments, the vector may be a eukaryotic vector, such as a vector contained in eukaryotic cells for expressing the desired element. In some embodiments, the vector may be a mammalian vector. ， For example, it may contain cytomegalovirus (CMV) or SV40 promoters.

[0455] This invention also provides a cell comprising or expressing nucleic acids / multiple nucleic acids or vectors / multiple vectors. The cell may be a eukaryotic cell, such as an animal cell. The cell may also be a non-human animal cell, such as a mouse, rat, hamster, camel, guinea pig, rabbit, goat, chicken, or primate. （ For example, cells from non-human primates (such as monkeys), sheep, donkeys, cattle, cats, dogs, pigs, or horses. In some embodiments, the cells are mammalian cells. （ For example, non-human mammalian cells. In some embodiments, the cells are derived from rodents (e.g., species of the genera *Mice*, *Rats*, or *Guinea Pigs*) or lagomorphs (e.g., species of the family *Leporidae*). In some embodiments, the cells are mouse cells.

[0456] In some embodiments, the cell is a pluripotent cell. ， For example, pluripotent cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are embryonic stem cells.

[0457] An embryo comprising cells according to the present disclosure is also provided. A blastocyst comprising cells according to the present disclosure is also provided.

[0458] According to the present invention, the aforementioned nucleic acids, vectors, cells, embryos, and blastocysts can be used for the production of animals. Therefore, the present invention also provides an animal produced by intrauterine implantation of a blastocyst containing cells according to the present invention.

[0459] Preparation of peptides / polypeptides The peptides and polypeptides used in the methods described herein can be prepared according to methods well known to those skilled in the art.

[0460] Peptides can be prepared by chemical synthesis, such as liquid-phase or solid-phase synthesis. For example, peptides / peptides can be produced using, for instance, the research by Chandrudu et al. published in *Molecules*, Vol. 18, 2013, pp. 4373-4388, which is incorporated herein by reference in its entirety. Alternatively, peptides / peptides can be generated through recombinant expression. Molecular biology techniques suitable for the recombinant production of peptides / peptides are well known in the art, such as those described by Green and Sambrook in *Molecular Cloning: A Laboratory Manual (4th Edition)* (Cold Spring Harbor Laboratory Press, 2012) and in *Nature Methods*, Vol. 5, No. 2, 2008, pp. 135-146, both of which are incorporated herein by reference in their entirety.

[0461] For recombinant production according to this disclosure, any cell suitable for expressing peptides / polypeptides can be used. The cells can be prokaryotes or eukaryotes. In some embodiments, the cells are prokaryotic cells, such as archaea or bacterial cells. In some embodiments, the bacteria can be Gram-negative bacteria, such as Enterobacteriaceae, e.g., Escherichia coli. In some embodiments, the cells are eukaryotic cells, such as yeast cells, plant cells, insect cells, or mammalian cells, such as CHO, HEK (e.g., HEK293), HeLa, or COS cells. In some embodiments, the cells are CHO cells that transiently or stably express peptides.

[0462] In some cases, the cells are not prokaryotic cells because some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. Furthermore, the expression levels may be very high in eukaryotes, and the protein can be more easily purified from eukaryotes using appropriate tags. Specific plasmids that enhance the secretion of the peptide / polypeptide into the culture medium can also be utilized.

[0463] In some embodiments, the polypeptide can be prepared by cell-free protein synthesis (CFPS) techniques, for example, using the article by Zemella et al. in ChemBioChem, Vol. 16, No. 17, 2015, pp. 2420-2431, the full text of which is incorporated herein by reference.

[0464] Production may involve culturing or fermenting eukaryotic cells modified to express the target peptide / peptide. The culture or fermentation can be carried out in a bioreactor providing appropriate nutrient supply, air / oxygen, and / or growth factors. Secreted proteins can be collected by isolating the culture medium / fermentation broth from the cells, extracting the protein components, and isolating individual proteins to separate the secreted peptide / peptide. Culture, fermentation, and isolation techniques are well known to those skilled in the art, for example, as described in Green and Sambrook's *Molecular Cloning: A Laboratory Manual* (4th edition, cited above in full).

[0465] A bioreactor comprises one or more containers capable of culturing cells. Culturing in the bioreactor can occur continuously, with reactants continuously flowing into the reactor and cultured cells continuously flowing out. Alternatively, the culturing can be batch-processed. The bioreactor monitors and regulates environmental conditions (e.g., pH, oxygen, inflow and outflow rates) and agitation within the containers to provide optimal conditions for the cultured cells.

[0466] After culturing cells expressing the peptide / peptide, the target peptide / peptide can be isolated. The protein can be isolated from the cells using any suitable method well known in the art. To isolate the peptide, it may be necessary to separate the cells from the nutrient medium. If the peptide / peptide is secreted from the cells, the cells can be separated from the medium containing the secreted target peptide / peptide by centrifugation. If the target peptide / peptide is collected intracellularly, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with lysis buffer, and cell disruption (e.g., ...). like (Through acoustic waves, rapid freeze-thaw cycles, or permeation pyrolysis).

[0467] Then, it may be necessary to separate the target peptide / peptide from the supernatant or culture medium, which may contain other protein and non-protein components. A common method for separating protein components from the supernatant or culture medium is precipitation. Proteins with different solubilities precipitate under different concentrations of precipitating agents, such as ammonium sulfate. For example, water-soluble proteins are extracted under low concentrations of precipitating agents. Therefore, by adding different concentrations of precipitating agents, proteins with different solubilities can be distinguished. Dialysis can then be used to remove ammonium sulfate from the separated proteins.

[0468] Other methods well-known in the art for distinguishing different proteins include ion-exchange chromatography and size chromatography. These can be used as alternatives to precipitation or performed after precipitation.

[0469] Once the target peptide / peptide is isolated from the culture, it may be necessary or desirable to concentrate the peptide / peptide. Many methods for protein concentration, such as ultrafiltration or lyophilization, are well-known in the art.

[0470] Sequence identity To determine the percentage of identity between two or more aligned amino acid or nucleic acid sequences, various methods well known to those skilled in the art can be employed. This can be achieved using publicly available computer software, such as pairwise and multiple sequence alignment, in various ways known to those skilled in the art. For example, publicly available computer software such as ClustalOmega (published by Söding in *Bioinformatics*, Vol. 21, pp. 951-960, 2005), T-coffee (published by Notredame et al. in *Journal of Molecular Biology*, Vol. 302, pp. 205-217, 2000), Kalign (published by Lassmann and Sonnhammer in *BMC Bioinformatics*, Vol. 6, pp. 298, 2005), and MAFFT (published by Katoh and Standley in *Molecular Biology & Evolution*, Vol. 30, No. 4, pp. 772-780, 2013) is preferred. When using the aforementioned software, default parameters, such as space penalties and expansion penalties, are preferably used.

[0471] sequence *** The present invention includes combinations of the described aspects and preferred features, unless such combinations are clearly not permitted or explicitly avoided.

[0472] The features disclosed in the foregoing description or the following claims or drawings, expressed in their particular form or in terms of implementing the disclosed function or in terms of the manner or process of obtaining the disclosed result, may be used alone or in any combination of the foregoing features to implement various forms of the invention.

[0473] To avoid any doubt, any theoretical explanations provided herein are offered to improve the reader's understanding. The inventors do not intend to be bound by any of these theoretical explanations.

[0474] Any chapter headings used in this document are for organizational purposes only and should not be construed as limiting the topics described.

[0475] In this specification, including the following claims, unless the context otherwise requires, the words “comprising” and “including” and variations such as “containing,” “comprising,” and “including” should be understood to imply the inclusion of an element or step or a combination of elements / steps, but do not exclude the presence of any other element or step or combination of elements / steps.

[0476] When this paper discloses nucleic acid sequences, its reverse complementary sequences are also explicitly considered. Furthermore, when this paper discloses nucleic acid sequences encoding polypeptides, equivalent polypeptide-coding sequences resulting from genetic code degeneracy are also explicitly considered.

[0477] It must be noted that the singular forms “a,” “an,” and “the” used in the specification and appended claims include plural references unless the context clearly specifies otherwise. A range may be expressed herein as from “about” one particular value and / or to “about” another particular value. When the above range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, it should be understood that the particular value forms another implementation by using the antecedent “about.” The term “about” in relation to numerical values ​​is optional, for example, indicating + / - 10%.

[0478] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the disclosed products can exist in vitro, ex vivo, or in vivo. The term "in vitro" ” The term is intended to include experiments on materials, biological substances, cells, and / or tissues under laboratory conditions or in culture. " "In vivo" is intended to include experiments and procedures performed on complete multicellular organisms. In some implementations, methods performed in vivo can be performed on non-human animals. "Ex vivo" refers to things that exist outside of an organism or occur outside of an organism. ，For example, outside the human or animal body, it refers to cells in tissues (such as whole organs) or extracted from an organism.

[0479] For information on standard molecular biology techniques, see Sambrook, J., Russell, DW, in *Molecular Cloning, A Laboratory Manual*, 3rd edition, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0480] Various aspects and embodiments of this disclosure will now be discussed. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference in their entirety. While the invention has been described in conjunction with exemplary embodiments described below, many equivalent modifications and variations will be understood by those skilled in the art when this disclosure is given. Therefore, the exemplary embodiments of the invention described above are to be considered illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention. Brief description of the attached diagram The principles of the present invention will now be discussed with reference to the accompanying drawings, which illustrate the embodiments and experiments.

[0482] Figure 1A and 1B As described in Example 8.1, a schematic diagram of (1A) the targeted genomic mouse site, the targeted vector, the targeted allele (after insertion and integration of the targeted vector), and the constitutive knock-in allele for generating Cd79a-CreERT2 knock-in mice; and (1B) the targeted vector, as shown in the nucleotide sequence of SEQ ID NO:1.

[0483] Figure 2 As described in Example 8.2, this diagram illustrates the target genomic mouse locus, target vector, target allele (after insertion and integration into the target vector), conditional knockout allele, and Cre recombination-derived knockout allele used to generate Ighm-Ighd CKO mice.

[0484] Figure 3 The immunization, administration, and sampling timeline is used to assess the suppression of the primary immune response by using anti-IgD antibodies to deplete IgM+IgD+ naive B cells.

[0485] Figure 4A and 4B The graphs show the proportion of B cells in the spleen, lymph nodes, and PBMCs before (4A) and after (4B) anti-IgD treatment, compared to the isotype control antibody. ****p<0.0001, ***p<0.001 are based on multiple comparison tests.

[0486] Figure 5 The graphs show the antigen-immune response. All mice received two initial doses of EGFR-hFc, followed by a third dose of PBS (left), EGFR-hFc (middle), or CD33-mFc (right). Data are presented as mean + / - standard error. N=9 on days 7, 14, and 26. n=6 on days 31, 35, and 40. n=3 on day 50.

[0487] Figure 6 A graph showing the percentage of B cells in which CreERT2-mediated ZsGreen allele recombination was detected in B cells isolated from bone marrow, spleen, and lymph nodes, and a graph showing the percentage of lox-Stop-lox-ZsGreen allele homozygous mice (Gt(ROSA)26Sor) within a specified number of days after treatment with tamoxifen at 100 mg / kg body weight or 200 mg / kg body weight. tm6（CAG -ZsGreen1）Hze ) and Cd79a CreERT2 A graph showing the percentage of CreERT2-mediated ZsGreen allele recombinant non-B cells in heterozygous mouse tissues.

[0488] Figure 7 The results showed that, at a specified number of days after treatment with 100 mg / kg body weight of tamoxifen or the vector control (corn oil), homozygous lox-Stop-lox-ZsGreen alleles ( Gt(ROSA)26Sor tm6（CAG-ZsGreen1）Hze )and Cd79a CreERT2 A graph showing the percentage of B cells with CreERT2-mediated ZsGreen allele recombination detected in B cells isolated from the bone marrow, spleen, and lymph nodes of heterozygous mice.

[0489] Figure 8A and 8B The results showed treatment with tamoxifen at 200 mg / kg body weight. Ighm-Ighd flox / flox ； Cd79a + / CreERT2 mice 60 A schematic diagram and image showing the in vivo knockout of Ighm exons 1-6 and Ighd exons 1-3 in CreERT2-mediated B cells after hours. (8A) shows the tamoxifen-induced CreERT2-mediated knockout. Ighm Exons 1-6 and Ighd Before (top) and after (bottom) exon 1-3 gene region knockout. IghmIghd flox A schematic diagram of the locus structure. It shows the structure used for IghmIghd.flox 、IghmIghd KO The sites of the forward and reverse primers for amplicon PCR amplification in the control region. (8B) After separation by agarose gel electrophoresis, the amplicon was treated with 200 mg / kg body weight of tamoxifen (TAM) or vector control (corn oil). treatment IghmIghd flox / flox ;Cd79a+ / CreERT2 60 hours after mice ， Genomic DNA obtained from cells isolated from bone marrow was used to amplify IghmIghd. flox 、IghmIghd KO Images of the PCR reaction products from primers used for the amplicon in the control region. Showing IghmIghd. flox 、IghmIghd KO The expected sites of amplicon in the control region.

[0490] Figure 9 The results show that, as determined by flow cytometry, IghmIghd... flox / flox Cd79a+ / CreERT2 mice were administered tamoxifen or corn at a dose of 200 mg / kg body weight. Oil (Vector control) After 60 hours of treatment, Cd19 was obtained from the spleen and lymph nodes. + Histograms and bars representing the expression of IgM and IgD in cells.

[0491] Figure 10A and 10B The results showed that repeated administration of tamoxifen resulted in a reduction in homozygous individuals isolated from the lox-Stop-lox-ZsGreen allele ( Gt(ROSA)26Sor tm6（CAG-ZsGreen1）Hze )and Cd79a CreERT2 Schematic and graph showing the maintenance of CreERT2-mediated ZsGreen allele recombination in B cells of bone marrow, spleen, and lymph nodes in heterozygous mice. (10A) shows a schematic of the timeline of administration of tamoxifen and the vector (corn oil) to mice in arms 1 and 2. (10B) shows a graph showing the percentage of B cells with CreERT2-mediated ZsGreen allele recombination detected in B cells isolated from bone marrow, spleen, and lymph nodes in mice in arms 1 and 2 at specified days after administration of the initial dose of tamoxifen.

[0492] Figure 11 Displaying the... IghmIghd flox / flox ; Cd79a + / CreERT2 In mice, it is used to maintain B cell specificity and CreERT2-mediated... Ighm Exons 1-6 and Ighd A schematic diagram of the timeline for tamoxifen administration following the knockout of exons 1-3.

[0493] Figure 12 The results show that, as determined by NGS sequencing, samples were obtained from IghdIghm on days 24 and 55. flox / flox Cd79a + / CreERT2 Bar charts showing the diversity of VH genes in B cells from samples collected from mice and BALB / c mice.

[0494] Figures 13A to 13D Displays data collected at the specified time points (13A and 13C). IghdIghm flox / flox Cd79a + / CreERT2 Histograms showing the binding of antibodies in mouse and (13B and 13D) BALB / c mouse serum to untransfected CHO cells (control), cells transiently expressing the membrane-bound form of the target protein (ECD w / protease cleavage site), or cells transiently expressing the membrane-bound form of the target protein soluble extracellular domain (ECD w / o protease cleavage site).

[0495] Figure 14A and 14B The results, obtained using the ELISA method, indicate that the virus originates from... IghdIghm flox / flox ; Cd79a + / CreERT2 A diagram showing the binding of two representative antibodies from two antibody clones produced by mice to the full-length extracellular domain of the target protein (containing the protease cleavage site, hollow circles). Figure 14A The closed circles in the diagram indicate the binding of the isotype control antibody.

[0496] Figures 15A to 15F (15A) shows that after tamoxifen-induced gene knockout, BALB / c mice and IghdIghm flox / flox Cd79a + / CreERT2 Flow cytometry data on the proportion of IgG+ and CD45R+ B cells in (IgMIgDcKO) mice. (15B) Showing the difference between BALB / c mice and IgMIgD before tamoxifen-induced gene knockout. f / f IgG in mice + B cell quantification and IgG levels in BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene knockout. + A graph showing the quantitative distribution of B cells. (15C) shows BALB / c mice with IgMIgD prior to tamoxifen-induced gene knockout. f / f IgG in mice +B cell quantification and IgG levels in BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene knockout. + B220 + A graph showing the quantitative distribution of B cells. (15D) This graph shows the B220 levels in lymph nodes (LN) and spleen (SP) of BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene knockout. + A graph showing the quantitative distribution of B cells. (15E) shows BALB / c mice with IgMIgD prior to tamoxifen-induced gene knockout. f / f IgG in mice + B220 + B cell quantification and IgG levels in BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene knockout. + B220 + A graph showing the quantitative distribution of B cells. (15F) shows the B220 levels in lymph nodes (LN) and spleen (SP) of BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene knockout. + A chart for B cell quantification.

[0497] Figure 16 The graph shows the binding of antibodies produced in serum from immunization of BALB / c or NZBWF1 mice, as measured by ELISA, to the recombinant human DLL3-Fc-tagged protein. Serum was collected on day 38 post-immunization.

[0498] Figure 17 The graph shows the binding of antibodies produced by immunization in the serum of BALB / c or NZBWF1 mice, as measured by ELISA, to two doses of KLH-conjugated 13-monomer peptide. Serum was collected on day 25 post-immunization.

[0499] Figures 18A-18C (18A) An image of the PCR reaction product using primers used to amplify CreERT2-mediated... IghdIghm flox / flox The 2328bp wild-type (wt) or 2458bp flox (fl) amplicons detectable in the absence of locus recombination, and in CreERT2-mediated of IghdIghm flox / flox The 232bp IgMIgDcKO amplicon, detectable after locus recombination, was separated by agarose gel electrophoresis and administered at 200 mg / kg body weight (tamoxifen) over a 40-day cycle. Ten days after the last treatment, the amplicon was taken from BALB / c or... IghdIghm flox / flox Cd79a + / CreERT2Genomic DNA was obtained from cells isolated from the spleen and ear, and the treatment consisted of 5 doses of tamoxifen. Flow cytometry and quantification were performed on B cells expressing IgM and IgD collected from lymphoid tissue of tamoxifen-induced transgenic mice (IgM and IgDcKO) and compared with wild-type mice.

[0500] Figure 19 An immunization strategy for generating target epitope-specific antibodies for each target protein. "Antigen 1" refers to a peptide containing the amino acid sequence of the target epitope within the target protein. "Antigen 2" refers to the full-length target protein containing the target epitope.

[0501] Figure 20A and 20B (20A) A graph showing the binding of antibodies produced in the serum of IgMIgDcKO mice and wild-type BALB / c mice to target protein 1 as defined in Example 19, as determined by ELISA. (20B) FACS analysis of the binding profiles of antibodies produced in the serum of IgMIgDcKO and wild-type mice against target protein 1 as defined in Example 19.

[0502] Figure 21A and 21B (21A) A graph showing the binding of antibodies generated in the serum of IgMIgDcKO mice and wild-type BALB / c mice as determined by ELISA. (21B) FACS analysis of the binding profile of antibodies generated in the serum of IgMIgDcKO and wild-type mice as defined in Example 19.

[0503] Figure 22 FACS analysis showed that the recombinant antibody was specific for the full-length extracellular domain (ECD) or epitope knockout (ΔEp) extracellular domain of target protein 1 as defined in Example 19.

[0504] Figure 23A and 23B The results, as determined by ELISA, show the efficacy of [the treatment] for [a specific disease / condition] derived from [a specific IghdIghm flox / flox ; Cd79a + / CreERT2 A graph showing the dose-response curves of two representative mouse antibody clones binding to the full-length extracellular domain of the target protein 3 as defined in Example 19.

[0505] Example Example 1: Transgenic mice with IGHM and IGHD inducible knockout Transgenic mice with inducible knockout of IGHM and IGHD gene homologs were produced.

[0506] In short, mouse embryonic stem cells modified via CRISPR / Cas9-mediated gene editing (as described, for example, by Lee et al. in Drug Discovery Today: Disease Models, 2016, Vol. 20, pp. 13-20) contain cells located in the IGHM and IGHD The loxP target sequence flanking the gene exon, and under the regulation of a promoter that provides expression in B-cell lineage cells, encodes CreERT. Modified embryonic stem cells were used to generate transgenic mice by introducing them into blastocysts and subsequently implanting them into the uterus for pregnancy.

[0507] By administering tamoxifen, the expression of IgM and IgD in the resulting transgenic mice can be induced to be knocked out.

[0508] Example 2: Transgenic mice encoding human immunoglobulin genes with induced knockout of IGHM and IGHD Transgenic mice encoding human immunoglobulin genes were produced, and mouse homologs of these mice were induced to knock out the IGHM and IGHD genes.

[0509] In short, embryonic stem cells derived from transgenic mice encoding human immunoglobulin genes were modified via CRISPR / Cas9-mediated gene editing (as described by Lee et al. in Drug Discovery Today: Models of Disease, 2016, Vol. 20, pp. 13–20). The modified embryonic stem cells were used to generate transgenic mice by introducing them into blastocysts and subsequently implanting them into the uterus to induce pregnancy.

[0510] The resulting transgenic mice produced mice with fully human variable regions (i.e., VH and VL). sequence The antibody series, in which the expression of mouse IgM and IgD can be induced to knock out by administration of tamoxifen.

[0511] Example 3: Antibody production using transgenic mice with induced knockout of IGHM and IGHD The mice prepared as described in Examples 1 and 2 were used to prepare antibodies capable of binding to naturally occurring target amino acid sequences.

[0512] Peptides containing the target sequence of the target protein's extracellular domain were synthesized using standard methods. The extracellular domain of the target protein was recombinantly expressed and purified in Chinese hamster ovary (CHO) cells or HEK293 cells.

[0513] The peptide was conjugated to KLH and hepatitis B core antigen (HBc) carriers for immunization. The conjugation used succinimide-6-((β-maleimide propionylamino)hexanoate) (SMPH) as a linker between the peptide and the protein carrier. Successful conjugation with the protein carrier was confirmed by SDS-PAGE analysis.

[0514] Mice were first immunized with 50 μg of peptide (representing a small fragment of the extracellular domain of the target protein [first injection]).

[0515] Following immunization with the peptide [first injection]: (a) A group of mice were induced to knock out IgM and IgD by administration of tamoxifen (intraperitoneal injection); and (b) Another group of mice did not receive treatment to induce IgM and IgD knockout.

[0516] Subsequently, both groups of mice were immunized with 50 μg of a polypeptide, which represents the full-length amino acid sequence of the extracellular domain of the target protein [second injection].

[0517] After the second injection, mice were immunized twice more with the same peptide or polypeptide as the second injection [third and fourth injections].

[0518] After the third and fourth injections, mice were given one to three booster injections, the same peptides or polypeptides that were immunized during the second injection [booster injection].

[0519] Injection sites include the armpits, groin, feet, back, and abdomen.

[0520] Different injectable drugs use different formulations, as shown below: First injection: Peptide + Freund's complete adjuvant Second injection: ECD + Freund's incomplete adjuvant Third and fourth injections: ECD + Freund's incomplete adjuvant Enhancer injection: ECD (with or without adjuvant) Example 4: ELISA analysis of antibody production Following the final booster injection, serum was collected from the mice, and the antibodies bound to: (1) Peptides, and (2) Extracellular domain (ECD) of the target protein.

[0521] Analysis was performed using enzyme-linked immunosorbent assay (ELISA).

[0522] In short, the ELISA plate was coated overnight at 4°C with peptide or ECD (1 μg / ml in PBS). After coating, the ELISA plate was washed with sashing buffer (0.05% Tween 20 in 1x PBS), then blocked for 1 hour at room temperature with 1% BSA in 1x PBS, followed by three washes with washing buffer.

[0523] Serum was collected from mice, and serial dilutions were added to the wells of an ELISA plate. The plate was then incubated at room temperature for 1 hour. After washing with washing buffer, horseradish peroxidase (HRP)-conjugated antibody was added to the wells, and the plate was incubated at room temperature for 1 hour.

[0524] The ELISA plate was then developed at room temperature with TMB substrate solution for 10 minutes. Development was terminated by adding 2M sulfuric acid (H2SO4), and absorbance (OD) was measured at 450 nm within 30 minutes after termination.

[0525] Example 5: Hybridoma generation Hybridoma formation and antibody analysis of hybridoma formation are as follows.

[0526] Fusion (Day 1): Mice were dissected under sterile conditions to obtain spleens and lymph nodes, and single-cell suspensions of these tissues were prepared.

[0527] Cells fuse with myeloma cells via polyethylene glycol (PEG) fusion or electrofusion. For PEG fusion, the ClonaCell-HY hybridoma cloning kit was used, and cells were fused according to the manufacturer's instructions (Stem Cell Technologies, Canada). Fusion cells were cultured overnight in ClonaCell-HY medium C (Stem Cell Technologies, Canada) at 37°C in a 5% CO2 incubator. The next day, the fusion cells were centrifuged and resuspended in 10 ml of ClonaCell-HY medium C, then gently mixed with 90 ml of semi-solid methylcellulose-based ClonaCell-HY medium D (Stem Cell Technologies, Canada) containing HAT components, and seeded into 96-well plates. Cells were allowed to grow in a 37°C, 5% CO2 incubator. After 7–10 days, individual hybridoma clones were identified, and antibody-producing hybridomas were selected by screening the supernatant using an enzyme-linked immunosorbent assay (ELISA).

[0528] Alternatively, for electrofusion, use a NEPA21 super electroporator and fuse cells according to the manufacturer's experimental protocol (Nepagene). Restore fusion cells overnight in ClonaCell-HY medium C (Stem Cell Technologies, Canada) at 37°C in a 5% CO2 incubator. The next day, centrifuge the fusion cells and resuspend them in 1 ml of ClonaCell-HY medium C, then gently mix with 90 ml of semi-solid methylcellulose-based ClonaCell-HY medium D (Stem Cell Technologies, Canada) containing HAT components and 500 μg of FITC-labeled anti-mouse antibody (Jackson Immunoresearch). Then seed the plates into 8 to 16 x 6-well plates. Allow colonies to grow for 7 days in a 37°C 5% CO2 incubator. Scan colonies for FITC fluorescence, pick using a Clonepix (Fortebio) device, and transfer to 96-well plates containing AOF medium. Allow the picked colonies to grow for 5 days, then screen the supernatant by enzyme-linked immunosorbent assay (ELISA).

[0529] Second round of ELISA: On day 13, a second round of ELISA was performed on the cell culture supernatant from the 96-well plate.

[0530] Amplification / Cryopreservation: On day 14, cells from wells showing positive results in the second round of ELISA were transferred to wells in a 24-well plate for culture and expansion. When the cultures were nearly confluent, the cells were harvested and cryopreserved.

[0531] Example 6: Peptide and ECD-binding antibody with hybridoma Compared to mice that did not have IgM and IgD knocked out before the second injection (i.e., group (b) mice), mice immunized according to the experimental protocol (where tamoxifen was administered before the second injection to induce IgM and IgD knockout) (i.e., group (a) mice - see Example 3) produced higher titers of antibodies that were able to bind to the extracellular domains of the target protein.

[0532] No difference was observed between mice in groups (a) and (b) in terms of antibody titers that could bind the peptide.

[0533] Compared to group (b), group (a) had a higher proportion of antibodies in mouse serum that could bind to the target protein, including peptides and extracellular domains.

[0534] Compared to group (b), a greater proportion of mice in group (a) developed hybridomas, and the antibodies produced by these hybridomas showed binding to the peptides and extracellular domains of the target protein.

[0535] Example 7: Hybridoma producing antibodies that bind to the peptide and ECD Cells from wells that showed a positive signal in the ELISA were diluted to approximately one cell per well of a multi-well plate and cultured in vitro for 1–2 weeks.

[0536] Then, as described in Example 4, the cell culture supernatant in the ELISA wells was combined with: (1) Target recombinant Fc marker protein; (2) Optionally, Fc may be used alone; and (3) Peptides that produce antibodies.

[0537] Cells from wells containing antibodies that can bind only to (1) and (3) were diluted to approximately one cell per well in a multi-well plate and cultured in vitro for 1–2 weeks.

[0538] The cell culture supernatant from the wells was then analyzed by ELISA as described above. The cells in these wells were considered monoclonal.

[0539] Mice immunized according to the experimental protocol, including mice that were given tamoxifen prior to the second injection to induce IgM and IgD knockout (i.e., (a)). Group Mice (see Example 3) generate hybridomas that produce peptides and extracellular domains containing peptides. of The success rate of the monoclonal antibody was greater than that of mice in which IgM and IgE were not knocked out before the second injection (i.e., group (b) mice).

[0540] The cells from the wells were transferred to T25 culture flasks for culture and expansion. After 3-4 weeks, the cells were frozen in liquid nitrogen for storage or used to produce antibodies in ascites fluid.

[0541] Example 8: Exemplary transgenic mice Example 8.1 Composition Cd79a-CreERT2 Knock-in mice Mice containing an endogenous nucleotide sequence were generated, which provided a constitutive knock-in of CreERT2 at Cd79a.

[0542] Targeting based on mouse Cd79a transcript (NCBI reference sequence: NM_007655.4) carrier The insertion of the target carrier is schematically shown in... Figure 1A In the middle, including (from 5' to 3'): A short homologous arm of ~3kb containing exon 1 of Cd79a, which Explicit The child has a mutated translation start codon; A puromycin resistance gene, PuroR, is located on the flank of the FRT site (which provides for translocase-mediated excision); The first 7 nucleotides of exon 2 of Cd79a acid,Next is the Kozak sequence and ORF of CreERT2; Cd79a 3'UTR and human growth hormone polyadenylation signal (hGHpA; to prevent transcriptional readthrough); A long homology arm of ~6kb, comprising exons 3 through 5 of Cd79a and Arhgef1; and Nucleic acid encoding thymidine kinase.

[0543] The target carrier is schematically represented in Figure 1B The nucleotide sequence of the target vector is shown in SEQ ID NO:1.

[0544] The targeting vector was transfected into cells of the Balb / c embryonic stem cell line. Embryonic stem cell clones containing successfully integrated sequences were identified by positive selection for puromycin resistance and negative selection for thymidine kinase activity. The selected cells were then treated to achieve translocase-mediated PuroR excision, yielding… born Mature knock-in allele.

[0545] Embryonic stem cells containing knock-in alleles were used to produce transgenic mice by microinjection into the blastocysts of Balb / c mice (e.g., Sumiyama et al., PLoS One, 2018, Vol. 13, No. 9, p. e0203056), which were then implanted into female Balb / c mice for pregnancy.

[0546] The Cd79a-CreERT2 mice constitutively express CreERT2 in B-cell lineage cells under the regulation of the Cd79a promoter.

[0547] Example 8.2 Conditional Ighm and Ighd Knockout mice A mouse containing an endogenous nucleotide sequence was generated that provided inducible knockout of Ighm and Ighd.

[0548] Based on mouse Ighm transcripts (Ensembl reference sequence: ENSMUST00000177715) and mouse Ighd transcripts (Ensembl reference sequence: ENSMUST00000194162). Targeting vector insertion is as follows: Figure 2 As shown, including (from 5' to 3'): A short homologous arm of ~4kb; lie in loxPThe regions flanking the site include: the neomycin resistance gene NeoR, which is located flanking the FRT site (providing its translocase-mediated excision); exons 1 to 6 of Ighm; exons 1 to 3 of Ighd; and the puromycin resistance gene PuroR, which is located flanking the F3 site (providing its translocase-mediated excision). A homologous arm ~6kb long; and Nucleic acid encoding thymidine kinase.

[0549] The nucleotide sequence of the target region of the vector is shown in SEQ ID NO:2.

[0550] The targeting vector was transfected into cells of the Balb / c embryonic stem cell line. Embryonic stem cell clones containing successfully integrated sequences were identified by positive selection for neomycin and puromycin resistance and negative selection for thymidine kinase activity. Selected cells were then treated to achieve translocase-mediated NeoR and PuroR excision, generating mature conditional knockout alleles.

[0551] Cre recombinase-mediated exon 1 to 6 (including the proximal promoter) of Ighm is expected to result in loss of function, and exon 1 to 3 (and the proximal promoter) of Ighd is expected to block Ighd transcription.

[0552] Embryonic stem cells containing conditionally knocked-out alleles were used to produce transgenic mice by microinjection into the blastocysts of Balb / c mice (e.g., Sumiyama et al., PLoS One, 2018, Vol. 13, No. 9, p. e0203056), which were then implanted into female Balb / c mice for pregnancy.

[0553] The Ighm-Ighd CKO Mice were provided with conditional knockout of Cre expression for Ighm and Ighd.

[0554] Will Ighm-Ighd CKO Mice were crossed with Cd79a-CreERT2 knock-in mice described in Example 8.1 to obtain... IghmIghd flox Allele homozygous mice and Cd79a CreERT2 Allelic heterozygous mice, and knockout of Ighm and Ighd expression in B cells after tamoxifen treatment. 。 The resulting transgenic mice have the following genotypes: IghmIghd flox / flox Cd79a + / CreERT2 。

[0555] Example 9: Suppressing the primary immune response by using anti-IgD antibodies to deplete IgM+IgD+ immature B cells Experiments were conducted to test the ability of anti-IgD antibody therapy to limit the response of primary B cells to a second antigen.

[0556] The anti-IgD antibody binds to a population of B cells expressing IgD on their surface. Treatment with the anti-IgD antibody leads to the depletion of immature B cells expressing both IgM and IgD. Studies have found that removing IgM before immunization... + IgD + Double-positive B cell populations can significantly reduce an animal's ability to initiate a primary immune response against a neoantigen.

[0557] The timeline of immunization, administration, and sampling is as follows: Figure 3 As shown.

[0558] On day 1, 8-9 week old mice were immunized with the primary antigen EGFR-hFc (50 μg in complete Freund's adjuvant) to induce a primary response to EGFR. On day 7, mice were immunized again with EGFR-hFc (50 μg in incomplete Freund's adjuvant) to enhance the primary response to EGFR. On day 30, mice were immunized a third time with the same antigen (EGFR-hFc, 50 μg in incomplete Freund's adjuvant) or a neoantigen (CD33-mFc, 50 μg in incomplete Freund's adjuvant), or with an equal volume of PBS injected in incomplete Freund's adjuvant.

[0559] Immunized mice were intraperitoneally injected with five doses of 10 mg / kg anti-IgD antibody (anti-mouse IgDa; clone AMS-9.1) or isotype antibody (IgGaK). The five doses were administered 1 day and 3 days before the third immunization, and 1 day, 3 days and 6 days after the third immunization (days 27, 29, 31, 33 and 36 of the entire experimental timeline).

[0560] The proportion of B cells in the spleen, lymph nodes, and peripheral blood mononuclear cells (PBMCs) of mice was measured one day before anti-IgD antibody treatment on day 26, and four and fourteen days after the last dose of anti-IgD treatment on days 40 and 50. Cells were isolated and labeled with CD45R VioBlue, IgM-APC, and IgD-FITC conjugate antibodies, and the proportions of IgM+, IgD+, IgM+IgD+, and IgM-IgD-B cell populations were analyzed by FACS.

[0561] Mouse serum was collected on days 7, 14, 26, and 31 (assessing the primary response to EGFR) and days 36, 40, and 50 (assessing the primary response to CD33 and the secondary response to EGFR). Antibodies binding to EGFR-His and CD33-His proteins in serum were assessed in an ELISA assay, followed by detection with a secondary anti-mouse IgG-HRP antibody and the colorimetric substrate 3,3′,5,5′-tetramethylbenzidine. Samples were initially diluted 1:100 and then serially diluted 3-fold to obtain 11 dilutions for the ELISA assay. Reactions to the antigens were plotted as dilution factors at 50% maximum binding.

[0562] result Figure 4 shows the proportion of B cells in the spleen, lymph nodes, and PBMCs before (4A; day 26) and after (4B; days 40 and 50) anti-IgD treatment.

[0563] Anti-IgD treatment led to the depletion of IgM+IgD+ B cells in the spleen, lymph nodes, and PBMCs. A 40-60% reduction in IgM+IgD+ cells was observed on day 4 and day 40 following the last anti-IgD treatment. A simultaneous increase in the IgM+ population was also observed. By day 50, the proportion of IgM+IgD+ cells had recovered to over 80% of the control group, although the decrease in IgM+IgD+ cell numbers and the increase in IgM+ cell numbers on days 40 and 50 remained significant (p<0.0001, Tukey multiple comparison test).

[0564] Figure 5 Showing according to Figure 3 The timeline shows the immune responses observed after two immunizations with EGFR-hFc to generate a primary immune response, and a third immunization with PBS (left), EGFR-hFc (middle), or CD33-mFc (right).

[0565] Secondary immune responses were observed only after the third administration of EGFR on day 36 (top-middle figure). Mice treated with anti-IgD antibodies on days 36 and 40 showed a 78% reduction in EGFR response compared to the isotype control mice, and an 88% reduction compared to the control mice on day 50.

[0566] Treatment with anti-IgD antibody on days 40 and 50 reduced the primary response to CD33 by 26% and 56% respectively, compared to the control group (bottom right figure).

[0567] Therefore, treatment with anti-IgD antibodies can lead to the depletion of IgM+IgD+B cells and reduce the primary response to neoantigens, with little effect on secondary responses.

[0568] Example 10: Carrying Cd79a CreERT2 CreERT2 activity in constitutively knock-in allele transgenic mice Characterization Lox-Stop-lox-ZsGreen allele homozygous mice (Gt(ROSA)26Sor) tm6（CAG-ZsGreen1）Hze ） and Cd79a CreERT2 Heterozygous mice to assess B cell specificity in vivo cre / lox Induction of recombinant expression. In the mice described above, tamoxifen-induced CreERT2 expression is expected to lead to B-cell-specific expression of ZsGreen.

[0569] In short, 6-8 week old mice were administered a single intraperitoneal injection of tamoxifen at a dose of 100 mg / kg or 200 mg / kg body weight. Mice were euthanized on days 1, 2, 4, and 5 post-treatment for analysis. Cells isolated from bone marrow, spleen, lymph nodes, and blood were stained with Zombie NIR reagent and anti-mouse Cd45r antibody and evaluated by flow cytometry to determine Cd45r ZsGreen fluorescence. + B cells and Cd45r - The proportion of non-B cells.

[0570] The results are as follows Figure 6 As shown, Cre-mediated recombination specifically produced ZsGreen fluorescence in Cd45r+ B cells across all lymphoid tissues. ZsGreen fluorescence was detected in 60–90% of total Cd45r+ B cells in bone marrow, spleen, lymph nodes, and blood, while less than 5% of Cd45r-non-B cells in these tissues showed ZsGreen fluorescence. Effective B cell-specific Cre-mediated recombination was achieved in a dose-dependent manner within 2 days following tamoxifen treatment. These data suggest that cells carrying single... Cd79a CreERT2 Allelic transgenic mice can achieve effective, tamoxifen-induced, B cell-specific, cre / lox-mediated gene knockout in a dose-dependent manner.

[0571] In further experiments, the response kinetics of cre / lox-mediated gene knockout in mice were evaluated.

[0572] In short, 6-8 week old mice were intraperitoneally injected with a single dose of tamoxifen at 100 mg / kg body weight or corn oil (carrier control). Mice were euthanized at 1, 2, 6, 11, and 17 days post-treatment for analysis. Cells isolated from bone marrow, spleen, lymph nodes, and blood were stained with Zombie NIR reagent and anti-mouse Cd45r antibody and evaluated by flow cytometry to determine Cd45r with ZsGreen fluorescence. +The proportion of B cells.

[0573] The results are as follows Figure 7 As shown. Cd45r in the spleen, bone marrow, lymph nodes, and blood. + Tamoxifen effectively induced Cre-mediated recombination in B cells, resulting in the production of ZsGreen fluorescence. Recombinant ZsGreen fluorescence (Cd45r) was detected in all tissue types at day 2. + The highest proportion of B cells was observed. Recombinant Cd45r was observed in the spleen, lymph nodes, and blood on day 17 post-treatment. + The total number of B cells decreased slightly (~10%). Two days after tamoxifen injection, recombinant Cd45r was observed in the bone marrow. + The proportion of B cells decreased rapidly. By day 17, B cells in the bone marrow were mainly composed of ZsGreen-negative, non-recombinant cells. These data suggest that cells carrying a single Cd79a... CreERT2 Allele-derived transgenic mice can achieve effective, tamoxifen-induced, B-cell-specific, cre / lox-mediated gene knockout. Although all lymphoid tissues showed a high response to tamoxifen-induced CreERT2 activity, a high proportion of recombinant B cells survived for more than 10 days in peripheral lymphoid tissues after tamoxifen treatment, but the proportion of recombinant cells in bone marrow cells decreased more rapidly.

[0574] Example 11: CreERT2-induced Ighm-Ighd flox / flox ; Cd79a + / CreERT2 Ighm and Characterization of Ighd gene knockout Tamoxifen induces Ighm-Ighd flox / flox ; Cd79a + / CreERT2 Knockout of Ighm and Ighd in mice (as described in Example 8) was evaluated. Briefly, 3-week-old mice were intraperitoneally injected with a single dose of 200 mg / kg body weight of tamoxifen or corn oil (vector control). Mice were euthanized 60 hours after treatment for analysis. Genomic DNA was isolated from bone marrow cells and used for genotyping PCR. Three sets of primers were used to detect the presence of... Ighm Exons 1-6 and Ighd Knockout of exons 1-3 (representative examples shown in the figure) 8A The first primer set provides CreERT2-mediated... IghmIghd flox Amplification of a detectable 554bp amplicon in the absence of locus recombination ( IghmIghd flox (Amplicon). The second set of primers provides the CreERT2-mediated... IghmIghd flox Following locus recombination, the amplification of the 232bp amplicon (IghmIghd) can be detected. KO(Amplicon). The third set of primers provided amplification of the 187bp control amplicon, mediated by CreERT2. IghmIghd flox Recombination at the locus (control region) can be detected both in the presence and absence of the locus.

[0575] The results are as follows Figure 8B As shown. The control amplicon was intact and was detected in both tamoxifen and vector-treated mice. IghmIghd KO The amplicon was detected only in cells isolated from mice treated with tamoxifen. IghmIghd flox Amplicon assays were detected only in cells isolated from mice treated with corn oil. These data indicate that... IghmIghd flox / flox ; Cd79a + / CreERT2 Tamoxifen treatment in mice achieved the goal of containing Ighm Exons 1-6 and Ighd Knockout of the (floxed) region surrounded by loxP sites in exons 1-3.

[0576] In further experiments, the expression of IgM and IgD in peripheral lymphoid tissue B cells was analyzed to assess tamoxifen-induced [therapeutic effects] at the cellular level. IghmIghd flox / flox ; Cd79a + / CreERT2 Knockout of Ighm and Ighd in transgenic mice.

[0577] In short, 3-week-old IghmIghd flox / flox ; Cd79a + / CreERT2 Mice were intraperitoneally injected with a single dose of tamoxifen or corn oil (carrier control) at 200 mg / kg body weight. After 60 hours of tamoxifen treatment, mice were euthanized for analysis. Cells isolated from the spleen and lymph nodes were stained with Zombie NIR reagent, anti-mouse Cd19 antibody, anti-mouse IgM antibody, and anti-mouse IgD antibody, and the expression of Cd19, IgM, and IgD on the cells was assessed by flow cytometry.

[0578] The results are as follows Figure 9 As shown. IgM isolated from the spleen and lymph nodes of mice treated with the vector. - IgD - Cd19 + The proportion of these substances in B cells isolated from the spleen and lymph nodes of mice treated with tamoxifen was more than five times higher than that in mice treated with tamoxifen. These data suggest that tamoxifen treatment leads to... IghmIghd flox / flox ; Cd79a+ / CreERT2 In mice, the expression of Ighm and Ighm was severely disrupted, thereby inhibiting the uptake of B cells. IgM and IgD The expression.

[0579] Example 12: Maintaining high levels of CreERT2-induced knockout The study investigated the ability of repeated administration of tamoxifen to maintain high levels of CreERT2-mediated knockout of loxP-surrounded (floxed) sites.

[0580] In short, 6-8 week old lox-Stop lox-ZsGreen allele homozygous mice ( (Gt(ROSA)) 26Sor tm6（CAG-ZsGreen1）Hze ） Tamoxifen was administered intraperitoneally at a dose of 200 mg / kg body weight. The animals were then divided into two groups (representatively shown in...). Figure 10A In arm 1, mice were injected with four additional doses of tamoxifen at a dose of 100 mg / kg body weight every 5 to 9 days. In arm 2, mice were injected with corn oil (carrier control) at the same schedule. Mice were euthanized on days 3, 10, 17, and 32 after the first administration of tamoxifen for analysis. Cells isolated from bone marrow, spleen, and lymph nodes were stained with Zombie NIR reagent and anti-mouse Cd45r antibody and evaluated by flow cytometry to determine Cd45r with ZsGreen fluorescence. + The proportion of B cells.

[0581] The results are as follows Figure 10B As shown. During the experiment, recombinant B cells were reduced by ~90% in the bone marrow and by ~25% in the spleen and lymph nodes of mice receiving only a single initial dose of tamoxifen. Throughout the experiment, mice in arm 1 (which received repeated injections of tamoxifen every 5–9 days) maintained a high proportion of recombinant B cells in all lymphoid tissues. These data demonstrate that a high proportion of B cells can be maintained over time by regularly repeated administration of tamoxifen, accompanied by CreERT2-mediated knockout of loxP-surrounded (floxed) sites.

[0582] In further experiments, tamoxifen was repeatedly administered... IghmIghd flox / flox ; Cd79a + / CreERT2 The ability of mice (as described in Example 8) to maintain high levels of Ighm and Ighd knockout in B cells was investigated (see Example 8). Figure 11 ).

[0583] In short, 3-week-old IghmIghd flox / flox ; Cd79a + / CreERT2 Mice were administered tamoxifen intraperitoneally at a dose of 200 mg / kg body weight. Additional doses of tamoxifen (100 mg / kg body weight) were given every 5 to 10 days. Euthanasia was performed on days 3, 10, 17, and 32 following the initial tamoxifen administration. Cells isolated from the spleen and lymph nodes were stained with Zombie NIR reagent, anti-mouse Cd19 antibody, anti-mouse IgM, and anti-mouse IgD antibody, and the expression of Cd19, IgM, and IgD on the cells was assessed by flow cytometry.

[0584] Cd19 isolated from the spleen, lymph nodes, and bone marrow of tamoxifen-treated mice at all time points + A high proportion of B cells are Ighm - and Ighd - This suggests that repeated administration of tamoxifen at regular intervals can maintain high levels of CreERT2-mediated Ighm knockout and Ighd expression in B cells over time.

[0585] Example 13: Using transgenic mice with induced knockout of Ighm and Ighd to obtain specific targets for the target protein regional antibodies The inventors used transgenic mice provided with Ighm and Ighd inducible knockouts to obtain antibodies that bind to the membrane proximal protease cleavage site within the extracellular domain of the target protein.

[0586] 8-9 weeks old IghmIghd flox / flox ; Cd79a + / CreERT2 Mice (as described in Example 8) or BALB / c mice were immunized on days 0 and 7 with a peptide containing a protease cleavage site ('antigen 1'; 50 μg in complete Freund's adjuvant) to induce a primary response. On days 24 and 25, mice were intraperitoneally injected with 300 mg / kg body weight of tamoxifen to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the intact extracellular domain of the protein (including the protease cleavage site, 'antigen 2'; 50 μg in complete Freund's adjuvant). On days 34, 42, and 52, mice were injected with three additional doses of 200 mg / kg body weight of tamoxifen to maintain B cell knockout. Ighm and Ighd Knockout.

[0587] On day 28 (“first time point”, during induction) Ighd and Ighm (before knockout and immunization with the protein's intact extracellular domains) and day 63 ("second time point", during induction) Ighd and IghmPeripheral lymphocyte samples were collected from mice after knocking out and immunizing with the intact extracellular domain of the protein. The V gene of IgG+ B cells that bind to the extracellular domain of the target protein was used to analyze the binding of antibodies generated in mice to (i) the soluble cleaved form of the extracellular domain (i.e. lacking protease cleavage sites) and (ii) the non-cleaved form of the extracellular domain (i.e. lacking protease cleavage sites).

[0588] In short, antibody gene sequences of B cells from samples collected on days 28 and 63 were obtained via NGS sequencing and clustered into clonoids using Cell Ranger (B cells belonging to a common lineage calculated based on their antibody gene sequences). The antibody sequences were then aligned against an internal antibody database to determine the use of the V(D)J gene. Sequences were processed to remove all clones with non-functional or multifunctional heavy and / or light chain sequences. The resulting antibody amino acid sequences were then aligned with their closest germline sequences to quantify the number of somatic hypermutations (SHMs) based on the number of distinct residues from the identified germline sequences. For simplicity, the analysis assumed no reverse mutations in the germline residues.

[0589] Figure 12 The results showed that, before and after the induction of Ighd and Ighm knockout, [the following was observed]. IghmIghd flox / flox ; Cd79a + / CreERT2 The diversity of VH gene use was preserved in mice (i.e., relative to wild-type corresponding mice).

[0590] The table below illustrates that Ighd and Ighm knockout does not prevent somatic hypermutation. In fact, in IghmIghd flox / flox ; Cd79a + / CreERT2 The number of somatic hypermutations in the VH and VL genes of representative antibody clones produced by mice that specifically bind to the cleavage sites of target proteins increased at the second time point (i.e., after Ighd / Ighm gene knockout). *Representative clones The data showed that somatic hypermutation was maintained during B cell affinity maturation in IgMIgDcKO mice.

[0591] The ability of antibodies in serum collected at time points 1 and 2 to bind to either the soluble extracellular domain of the target protein (lacking protease cleavage sites) or the full-length extracellular domain of the target protein (including protease cleavage sites) was assessed.

[0592] In short, CHO cells were transiently transfected with either (i) a membrane-bound plasmid encoding the target protein (containing a protease cleavage site) or (ii) a soluble plasmid containing the target protein's extracellular domain (lacking a protease cleavage site) and a native transmembrane domain (for membrane localization and surface expression). Untransfected CHO cells served as a negative control. Cells were harvested 3 x 10⁻⁶ cells per cell type after 18 hours. 4 Cells / well were co-incubated with 50 µL of 3-fold serial dilution (starting with a 1:200 dilution in PBS) of mouse serum obtained at the specified time for 1 hour at 4°C. Cells were then washed twice with 150 µL of FACS buffer (1xPBS + 1% FBS) and resuspended for 20 minutes at 4°C in 50 µL of Alexa Fluor 647 AffiniPure goat anti-mouse IgG (Jackson Immuno, 0.1% dilution) antibody. Cells were washed twice with FACS buffer and resuspended in 40 µL of a final volume of DAPI solution, but not in 40 µL of DAPI-free FACS buffer, except for unstained wells. Samples were then run on an iQue3 flow cytometer, and data were analyzed using FlowJo v10.8.1 software.

[0593] The results are as follows Figures 13A to 13D As shown. Source: IghdIghm flox / flox ; Cd79a + / CreERT2 mouse serum in Ighd After knocking out Ighm and immunizing with antigen 2, the mice did not contain antibodies that could bind the soluble extracellular domain of the target protein (lacking protease cleavage sites), while the serum of BALB / c mice immunized with antigen 2 contained antibodies that bound the protein.

[0594] In further experiments, from IghdIghm flox / flox ; Cd79a + / CreERT2 Twenty of the most representative cloned antibody gene sequences obtained from mice were cloned into plasmids for expression as chimeric mouse / human antibodies (including mouse VH and VL regions, and human IgG1 CH1, hinge, CH2 and CH3 regions (heavy chain) and human κCL (light chain)). These chimeric antibodies were expressed in ExpiCHO™ (Thermo Fisher) cells and purified by Protein A affinity chromatography for subsequent characterization.

[0595] The ability of antibodies to bind to protease cleavage sites was assessed by ELISA. In short, wells of polypropylene plates were coated with 1 μg / ml of neutral avidin (Invitrogen), incubated overnight at 4°C, washed with 1x phosphate-buffered saline (PBS) containing 1% BSA, and blocked for 2 hours at room temperature. The plates were then washed twice with 1x PBS containing 0.05% Tween 20, and 1 μg / ml of (i) a biotinylated, His-labeled soluble extracellular domain of the target protein (lacking protease cleavage sites) or (ii) a biotinylated, His-labeled full-length extracellular domain of the target protein (including protease cleavage sites) was added. The plates were washed three times with 1x PBS containing 0.05% Tween 20 and dried between each step. Nine dilutions of the antibody (serially diluted with 1x PBS + 1% BSA, starting at 10 μg / ml) were then added, and the plates were incubated for 1 hour at room temperature. After washing three times with 1x PBS containing 0.05% Tween 20 and drying between each step, the HRP-conjugated goat anti-human secondary antibody (Invitrogen) was prepared by diluting it 1:7000 with 1x PBS containing 1% BSA and added to a plate. The plate was incubated in the dark at room temperature for 1 hour. The plate was then colorimetrically detected using the substrate 3,3′,5,5′-tetramethylbenzidine (Turbo TMB; Pierce, USA). The reaction was terminated with Invitrogen ELISA stop solution (catalog number #SS04), and the optical density (OD value) was measured at 450 nm using a BioTek PowerWave HT microplate spectrophotometer.

[0596] Figure 14A and 14B This shows the results obtained by ELISA from... IghdIghm flox / flox ; Cd79a + / CreERT2 Dose-response curves of two representative antibody clones obtained from mice binding to the full-length extracellular domain of the target protein (containing the protease cleavage site). Both showed high affinity binding to the target antigen (shown on...). Figure 14A The antibody's EC50 is 2.01 x 10⁻⁶. -11 M, shown in Figure 14B The antibody has an EC50 of 3.81 x 10⁻⁶. -11 M).

[0597] In summary, these data indicate that providing Ighm and Ighd Inducible knockout transgenic mice can be used to obtain a diverse, high-affinity antibody library for a given target protein region.

[0598] Example 14: The IgG type conversion reaction initiated before gene knockout following tamoxifen-induced conditional gene knockout. It should be maintained The inventor used the provided Ighm and Ighd Transgenic mice with induced knockout were used to investigate whether IgG type switching responses were maintained after tamoxifen-induced conditional gene knockout.

[0599] 8-9 weeks old IghmIghd flox / flox ; Cd79a + / CreERT2 Mice (as described in Example 8) or BALB / c mice were immunized on days 0 and 7 with a peptide containing a protease cleavage site ('antigen 1'; 50 μg in complete Freund's adjuvant) to induce a primary response. On days 24 and 25, mice were intraperitoneally injected with 300 mg / kg body weight of tamoxifen to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the intact extracellular domain of the protein (including the protease cleavage site 'antigen 2'; 50 μg in incomplete Freund's adjuvant). On days 34, 42, and 52, mice were injected with three additional doses of 200 mg / kg body weight of tamoxifen to maintain B cell knockout. Ighm and Ighd Knockout.

[0600] Peripheral lymph node samples were collected from mice on day 28 (“time point 1”, before induction of Ighd and Ighm knockout and immunization with intact extracellular domains of the proteins) and day 63 (“time point 2”, after induction of Ighd and Ighm knockout and immunization with intact extracellular domains of the proteins). Cells isolated from the lymph nodes were stained with Zombie NIR reagent for anti-mouse CD45R, IgG1, IgG2a, and IgG2b, and IgG+ and CD45R were assessed by flow cytometry. + The proportion of B cells.

[0601] The results are as follows Figure 15A and 15B As shown. Before tamoxifen-induced gene knockout, normal wild-type BALB / c and IgMIgD... f / f There was no significant difference in the proportion of IgG+ B cells in mouse lymph nodes. After tamoxifen-induced gene knockout, the proportion of IgG+ B cells was similar in normal BALB / c mice and IgMIgD conditional knockout (IgMIgDcKO) mice. Figure 15B and 15C ).

[0602] Further research showed that, compared with wild type, tamoxifen-induced gene knockout resulted in lower levels of CD45R in the peripheral lymphoid tissue of IgMIgDcKO mice. + The total number of B cells decreased ( Figure 15DLN = lymph nodes; SP = spleen.

[0603] Repeat the above experiment using the second target protein. 8-9 weeks old. IghdIghm flox / flox ; Cd79a + / CreERT2 Mice (as described in Example 8) or BALB / c mice were immunized on days 0 and 7 with a peptide containing a specific target domain of the protein ('antigen 3'; 50 μg in complete Freund's adjuvant) to induce a primary response. On days 24 and 25, mice were intraperitoneally injected with 300 mg / kg body weight of tamoxifen to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the intact extracellular domain of the protein (including the target domain, 'antigen 4'; 50 μg in incomplete Freund's adjuvant). On days 34, 42, and 52, mice were injected with three additional doses of 200 mg / kg body weight of tamoxifen to maintain B cell knockout. Ighm and Ighd Knockout.

[0604] Peripheral lymph node samples were collected from mice on day 28 (“time point 1”, before induction of Ighd and Ighm knockout and immunization with intact extracellular domains of the proteins) and day 63 (“time point 2”, after induction of Ighd and Ighm knockout and immunization with intact extracellular domains of the proteins). Cells isolated from the lymph nodes were stained with Zombie NIR reagent for anti-mouse CD45R, IgG1, IgG2a, and IgG2b, and IgG+ and CD45R were assessed by flow cytometry. + The proportion of B cells.

[0605] The results are as follows Figure 15E and 15F As shown. Before tamoxifen-induced gene knockout, normal wild-type BALB / c and IgMIgD... f / f IgG in mouse lymph nodes + There was no significant difference in the proportion of B cells. Following tamoxifen-induced gene knockout, IgG levels in normal BALB / c mice and IgMIgD conditional knockout (IgMIgDcKO) mice were significantly different. + The proportion of B cells was similar ( Figure 15E Compared to wild-type mice, tamoxifen-induced gene knockout resulted in increased CD45R levels in the spleen of IgMIgDcKO mice. + The total number of B cells decreased, but there was no decrease in the number of B cells in the lymph nodes. Figure 15F LN = lymph nodes; SP = spleen.

[0606] The table below shows that the epitope-specific clonal type generated by the antigen immune response at the first time point was not lost after Ighd and Ighm knockout. The same epitope-specific clonal type was present in mice both before and after Ighd and Ighm knockout induction. This indicates that the epitope-specific clonal type was maintained in IgMIgDcKO mice. In summary, these results indicate that in IgMIgDcKO mice, tamoxifen-induced conditional gene knockout maintains the immune IgG type conversion response initiated before gene knockout.

[0607] Example 15: NZBWF1 mice exhibit a stronger immune response to antigens that are highly homologous to themselves. The inventors used a highly immune-responsive mouse strain (NZBWF1) to analyze responses to antigens that are highly homologous to themselves.

[0608] BALB / c or NZBWF1 mice aged 6-12 weeks were immunized with recombinant human DLL3-His labeled protein. On day 38 post-immunization, the antibody titer against human DLL3-Fc labeled protein in the animal serum was analyzed by indirect enzyme-linked immunosorbent assay (ELISA).

[0609] The results are as follows Figure 16 As shown, the homology between human DLL3 and mouse DLL3 proteins is 85.8%. Compared with BALB / c mice, NZBWF1 mice showed a stronger titer against the antigen.

[0610] These results indicate that NZBWF1 mice respond to protein antigens and produce strong antibody titers against antigens that are highly homologous to themselves (>80%).

[0611] Example 16: NZBWF1 mice produce a stronger antibody response to antigens with poor immunogenicity. The inventors used a highly immunoreactive mouse strain (NZBWF1) to analyze responses to antigens with poor immunogenicity.

[0612] BALB / c or NZBWF1 mice aged 6–10 weeks were immunized with two doses of a 13-monomer peptide conjugated with KLH, 7 days apart. On day 21 post-immunization, the antibody titers against the biotin-bound peptide in the animal serum were analyzed by indirect enzyme-linked immunosorbent assay (ELISA).

[0613] The results are as follows Figure 17 As shown, NZBWF1 mice produced stronger titers against peptide antigens compared to BALB / c mice. These results indicate that NZBWF1 mice respond to small peptide immunity and produce antibody titers against antigens with poor immunogenicity.

[0614] Example 17: Additional characterization of transgenic mice with induced knockout of IGHM and IGHD Tamoxifen induces IghmIghd flox / flox ; Cd79a + / CreERT2 The knockout of Ighm and Ighd in mice (as described in Example 8.2) was further evaluated in mice immunized and treated with tamoxifen as described in Example 3. Briefly, on days 0 and 7, at 8–9 weeks of age... IghmIghd flox / flox ; Cd79a + / CreERT2 Mice (as described in Example 8) or BALB / c mice were immunized with a peptide containing a protease cleavage site ('antigen 1'; 50 μg in complete Freund's adjuvant) to induce a primary response. On days 24 and 25, mice were intraperitoneally injected with 300 mg / kg body weight of tamoxifen to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the intact extracellular domain of the protein (including the protease cleavage site 'antigen 2'; 50 μg in incomplete Freund's adjuvant). On days 34, 42, and 52, mice were injected with three additional doses of 200 mg / kg body weight of tamoxifen to maintain B cell knockout. Ighm and Ighd Knockout.

[0615] Ten days after the final tamoxifen treatment, mice were euthanized for analysis. Genomic DNA was isolated from cells derived from the spleen and ears and used for genotyping PCR. (The last sentence appears to be incomplete and possibly refers to a separate topic: "In CreERT2-mediated...") IghmIghd flox In the absence of locus recombination, the first primer set can be detected as being used to amplify either the 2328 bp wild-type or the 2458 bp IghmIghd. flox / flox Amplicon. In CreERT2-mediated... IghmIghd flox After locus recombination, the second primer set was detected to amplify a 232bp amplicon, indicating the knockout of the (floxed) Ighm-Ighd locus surrounded by the loxP site.

[0616] Genotyping revealed B cell specificity and tamoxifen-induced Ighm and Ighd Conditional knockout Figure 18AThis demonstrates the presence of a 232 bp band in B cells isolated from the spleen of tamoxifen-induced transgenic mice (IgMIgD cKO(fl / fl+tamoxifen)). The 232 bp band was absent in B cells isolated from the spleen of mice never treated with tamoxifen or from the spleen of wild-type mice treated with tamoxifen. Furthermore, the 232 bp band was not detected in ear cells from either wild-type or IgMIgD cKO mice, demonstrating the specificity of induced knockout on B cells.

[0617] Flow cytometry analysis of B cells collected from lymphoid tissue showed that, compared with wild-type mice, tamoxifen-induced transgenic mice (IgMIgD-cKO) had significantly higher levels of IgM in their lymphoid tissue. + IgD + 50-60% of B cells were knocked out. Figure 18B and 18C ).

[0618] In summary, these results indicate that tamoxifen-induced Ighm and Ighd Conditional knockout is specific to B cells.

[0619] Example 18: Transgenic mice with inducible knockout of IGHM and IGHD improved the detection rate of targeted epitope antibodies. Three target proteins were used to analyze the production of epitope-specific antibodies. The results are summarized below.

[0620] Figure 19 An immunization strategy for generating target epitope-specific antibodies against each target protein (“POI”) is shown.

[0621] The table below shows the percentage (%) of target epitope-specific serum produced in IgMIgDcKO mice compared to wild-type mice.

[0622] Target protein 1 Target protein 2 The ability of antibodies to bind to protease cleavage sites was assessed by ELISA. In short, wells of polypropylene plates were coated with 1 μg / ml of neutral avidin (Invitrogen), incubated overnight at 4°C, washed with 1x phosphate-buffered saline (PBS) containing 1% BSA, and blocked for 2 hours at room temperature. The plates were then washed twice with 1x PBS containing 0.05% Tween 20, and 1 μg / ml of (i) a biotinylated, His-labeled soluble extracellular domain of the target protein (lacking protease cleavage sites) or (ii) a biotinylated, His-labeled full-length extracellular domain of the target protein (including protease cleavage sites) was added. The plates were washed three times with 1x PBS containing 0.05% Tween 20 and dried between each step. Nine dilutions of the antibody (serially diluted with 1x PBS + 1% BSA, starting at 10 μg / ml) were then added, and the plates were incubated for 1 hour at room temperature. After washing three times with 1x PBS containing 0.05% Tween 20 and drying between each step, an HRP-conjugated goat anti-human secondary antibody (Invitrogen) was prepared by diluting the plate 1:7000 with 1x PBS containing 1% BSA and added to the plate. The plate was incubated in the dark at room temperature for 1 hour. The plate was then colorimetrically detected using the substrate 3,3′,5,5′-tetramethylbenzidine (Turbo TMB; Pierce, USA). The reaction was terminated with Invitrogen ELISA stop solution (catalog number #SS04), and the optical density (OD value) was measured at 450 nm using a BioTek PowerWave HT microplate spectrophotometer.

[0623] FACS analysis was also performed to assess serum binding profiles. Briefly, CHO cells were transiently transfected with either (i) a membrane-bound plasmid encoding the target protein (containing a protease cleavage site) or (ii) a soluble plasmid containing the target protein's extracellular domain (lacking a protease cleavage site) and a native transmembrane domain (for membrane localization and surface expression). Untransfected CHO cells served as a negative control. Cells were harvested 3 x 10⁻⁶ cells per cell type after 18 hours. 4Cells / well were co-incubated with 50 µL of 3-fold serial dilution (starting with a 1:200 dilution in PBS) of mouse serum obtained at the specified time and incubated at 4°C for 1 h. Cells were then washed twice with 150 µL of FACS buffer (1xPBS + 1% FBS) and resuspended for 20 min at 4°C in 50 µL of Alexa Fluor 647 AffiniPure goat anti-mouse IgG (Jackson Immuno, 0.1% dilution) antibody. Cells were washed twice with FACS buffer and resuspended in 40 µL of a final volume of DAPI solution, but not in 40 µL of DAPI-free FACS buffer, except for unstained wells. Samples were then run on an iQue3 flow cytometer, and data were analyzed using FlowJo v10.8.1 software.

[0624] The results for target protein 1 (POI1) are as follows: Figure 20A and 20B As shown, the results for target protein 2 (POI2) are as follows: Figure 21A and 21B .

[0625] The table below shows the rate of obtaining epitope-specific antibody clones from clonal B cell screening of POI1 and the third target protein (POI3).

[0626] Target protein 1 Target protein 3 Figure 22 The specificity of the recombinant antibodies, as determined by FACS analysis of POI1, was demonstrated. The ability of representative recombinant antibodies derived from IgMIgDcKO mice to bind to cells expressing full-length target proteins (including protease cleavage sites) or target protein types lacking protease cleavage sites was evaluated using FACS analysis. The results showed that these antibodies are specific for targeting protease cleavage sites and do not bind to other domains in the full-length protein.

[0627] Figure 23A and 23B This shows the results obtained by ELISA assay, from IghdIghm flox / flox ; Cd79a + / CreERT2 Dose-response curves of two representative antibody clones obtained in mice binding to the full-length extracellular domain of POI3 (including the protease cleavage site). Both showed high affinity binding to the target antigen (shown on...). Figure 23A The antibody's EC50 is 7.40 x 10⁻⁶.-11 M, shown in Figure 23B The antibody has an EC50 of 3.96 x 10⁻⁶. -11 M).

[0628] Example 20: Generating High Immunity IghdIghm flox / flox ; Cd79a + / CreERT2 mice By carrying IghdIghm flox / flox and Cd79a + / CreERT2 Alleles (in a BALB / c background) (e.g., mice produced in Example 8.2) were mated with autoimmune strains NZB or NZW mice, and then NZB and NZW were crossed to produce NZBWF1 progeny mice carrying biallelic alleles.

[0629] By administering tamoxifen, the expression of IgM and IgD in the resulting transgenic mice can be induced to be knocked out.

Claims

1. An animal containing an endogenous nucleotide sequence that induces inhibition of the primary humoral immune response.

2. The animal of claim 1, wherein the animal comprises an endogenous nucleotide sequence that induces inhibition of the expression of one or more genes involved in initiating a primary humoral immune response or inhibits the activity of their products.

3. The animal according to claim 1 or 2, wherein the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression of one or more genes involved in immunoglobulin isotype conversion and / or B cell maturation, or inhibits the activity of their products.

4. The animal according to any one of claims 1 to 3, wherein the animal comprises an endogenous nucleotide sequence that inducibly inhibits the expression of one or both of the genes selected from IGHM and IGHD or inhibits the activity of their products.

5. The animal according to any one of claims 1 to 4, wherein the animal comprises an endogenous nucleotide sequence that, through recombinase-mediated interference with the expression of one or more genes involved in initiating a primary humoral immune response.

6. The animal according to any one of claims 1 to 5, wherein the endogenous nucleotide sequence comprises a target sequence of a recombinase located flanking all or part of the nucleotide sequence of a gene involved in initiating a primary humoral immune response.

7. The animal of claim 6, wherein the animal comprises an endogenous nucleotide sequence that provides inducible expression or activity of the recombinase.

8. The animal of claim 7, wherein the endogenous nucleotide sequence providing inducible expression or activity of the recombinase encodes a conditional system for regulating the expression or activity of the recombinase.

9. The animal according to any one of claims 6 to 8, wherein the target sequence of the recombinase is a loxP sequence, and wherein the recombinase is a Cre recombinase.

10. The animal according to any one of claims 1 to 9, wherein the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

11. The animal according to any one of claims 1 to 10, wherein the animal is a mouse, rat, or rabbit.

12. The animal according to any one of claims 1 to 11, wherein the endogenous nucleotide sequence comprises an endogenous nucleotide sequence encoding a recombinase target sequence located flanking one or more exons of IGHM and IGHD.

13. The animal according to any one of claims 1 to 12, wherein the animal comprises an endogenous nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:4, or is composed of therefrom.

14. The animal according to any one of claims 1 to 13, wherein the animal comprises an endogenous nucleotide sequence encoding a conditional system for regulating the expression and / or activity of Cre recombinase.

15. The animal of claim 14, wherein the expression of the Cre recombinase is controlled by a promoter that drives expression in B-cell lineage cells.

16. The animal according to any one of claims 1 to 15, wherein the animal comprises an endogenous nucleotide sequence having 60% or more nucleotide sequence identity with, or is composed of, SEQ ID NO:

6.

17. A method for producing an antigen-binding molecule, comprising applying a peptide / polypeptide or a nucleic acid encoding a peptide / polypeptide to an animal according to any one of claims 1 to 16.

18. The method of claim 17, wherein the method comprises: (i) administering to an animal a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) Treating animals to suppress their ability to initiate a primary immune response; and (iii) administering a second peptide / polypeptide or nucleic acid encoding a second peptide / polypeptide to an animal, wherein the second peptide / polypeptide contains a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

19. The method of claim 17, wherein the method comprises: (i) administering to an animal a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) administering to an animal a second peptide / polypeptide or a nucleic acid encoding a second peptide or polypeptide, wherein the second peptide / polypeptide comprises a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence; and (iii) Treating animals to suppress their ability to initiate a primary immune response.

20. The method of claim 18 or 19, wherein treating the animal to suppress its ability to initiate a primary immune response comprises administering a reagent for inducing recombinase expression or activity.

21. A method for producing an antigen-binding molecule, wherein the method comprises: (i) administering to an animal a first peptide / polypeptide or a nucleic acid encoding the first peptide / polypeptide, wherein the first peptide / polypeptide contains a target amino acid sequence; (ii) Treating animals to suppress their ability to initiate a primary immune response; and (iii) administering a second peptide / polypeptide or nucleic acid encoding a second peptide / polypeptide to an animal, wherein the second peptide / polypeptide contains a target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

22. The method of claim 21, wherein the method comprises treating the animal before, during, and / or after administration of the second peptide / polypeptide or nucleic acid encoding the second peptide / polypeptide to suppress its ability to initiate a primary immune response.

23. The method according to any one of claims 18 to 22, wherein treating the animal to suppress its ability to initiate a primary immune response comprises administering an agent that inhibits the expression of one or more genes involved in initiating a primary humoral immune response or inhibits the activity of their products.

24. The method according to any one of claims 18 to 23, wherein treating the animal to suppress its ability to initiate a primary immune response comprises administering an agent that reduces the number / proportion of naive B cells in the animal, reduces the number / proportion of cells expressing IgM and / or IgD in the animal, and / or inhibits immunoglobulin isotype switching and / or B cell maturation.

25. The method according to claim 23 or 24, wherein the reagent inhibits the expression of one or both genes selected from IGHM and IGHD or inhibits the activity of their products.

26. The method according to any one of claims 23 to 25, wherein the reagent inhibits the activity of IgM and / or IgD.

27. The method according to any one of claims 23 to 26, wherein the reagent is selected from or includes antibodies, antigen-binding molecules, peptides, decoy receptors, aptamers, chelating agents, or small molecules.

28. The method according to any one of claims 23 to 25, wherein the reagent inhibits the expression of IGHM and / or IGHD.

29. The method according to any one of claims 23 to 25 or claim 28, wherein the reagent is selected from or includes RNAi, siRNA, antisense nucleic acid, antisense oligonucleotide or gene editing system.

30. The method according to any one of claims 21 to 29, wherein the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

31. The method according to any one of claims 21 to 30, wherein the animal is a mouse, rat, or rabbit.

32. The method according to any one of claims 17 to 31, wherein the method further comprises generating a hybridoma that generates an antigen-binding molecule capable of binding to a target protein / protein complex.

33. The method according to any one of claims 17 to 32, wherein the method further comprises isolating one or more antigen-binding molecules capable of binding to a protein comprising a target amino acid sequence.

34. The method according to any one of claims 17 to 33, wherein the method further comprises formulating an antigen-binding molecule capable of binding to a protein comprising a target amino acid sequence into a pharmaceutical composition.

35. A nucleic acid or multiple nucleic acids comprising a nucleotide sequence that induces inhibition of the primary humoral immune response.

36. The nucleic acid or multiple nucleic acids according to claim 35, comprising a nucleotide sequence that induces the expression of one or more genes involved in initiating a primary humoral immune response or inhibits the activity of their products.

37. The nucleic acid or multiple nucleic acids according to claim 35 or 36, comprising a nucleotide sequence that inducibly inhibits the expression of one or more genes involved in immunoglobulin isotype conversion and / or B cell maturation or inhibits the activity of their products.

38. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 37, comprising a nucleotide sequence, said nucleotide sequence inducibly inhibiting the expression of one or both genes selected from IGHM and IGHD or inhibiting the activity of their products.

39. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 38, comprising a nucleotide sequence, said nucleotide sequence interfering with the expression of one or more genes involved in initiating a primary humoral immune response via a recombinase-mediated mechanism.

40. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 39, comprising a nucleotide sequence encoding all or part of a nucleotide sequence of a gene involved in initiating a primary humoral immune response, flanked by a target sequence of a recombinase.

41. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 40, further comprising a nucleotide sequence encoding a conditional system for regulating recombinase expression or activity.

42. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 41, wherein the target sequence of the recombinase is a loxP sequence, and wherein the recombinase is a Cre recombinase.

43. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 42, comprising a nucleotide sequence, said nucleotide sequence comprising a target sequence of a recombinase located flanking one or more exons of IGHM and IGHD.

44. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 43, comprising a nucleotide sequence, said nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:

4.

45. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 44, comprising a nucleotide sequence encoding a conditional system for regulating the expression and / or activity of Cre recombinase.

46. ​​The nucleic acid or multiple nucleic acids according to claim 45, wherein the expression of the Cre recombinase is controlled by promoter regulation that drives expression in B-cell lineage cells.

47. The nucleic acid or multiple nucleic acids according to any one of claims 35 to 46, comprising a nucleotide sequence, said nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or more nucleotide sequence identity with SEQ ID NO:

6.

48. A vector or multiple vectors comprising the nucleic acid or multiple nucleic acids as described in any one of claims 35 to 47.

49. A cell comprising one or more nucleic acids according to any one of claims 35 to 47, or one or more vectors according to claim 48.

50. The cell of claim 49, wherein the cell comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

51. The cell according to claim 49 or 50, wherein the cell is a mammalian cell.

52. The cell according to any one of claims 49 to 51, wherein the cell is an embryonic stem cell.

53. The cell according to any one of claims 49 to 52, wherein the cell is a mouse cell, a rat cell, or a rabbit cell.

54. The animal according to any one of claims 1 to 16, or the method according to claims 17 to 34, wherein the animal is a hyperimmune mouse or a mouse with a hyperimmune phenotype.

55. The animal according to any one of claims 1 to 16 or 54, or the method according to claims 17 to 34, wherein the animal is a humanized mouse.

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