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 for induced suppression of an immune response.

Description

[0001] This application claims priority to U.S. Provisional Application 63 / 513380, filed July 13, 2023, and U.S. Provisional Application 63 / 609429, filed December 13, 2023, the entire 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, this disclosure relates to the production of antigen-binding molecules, particularly in the context of therapeutic, preventative, diagnostic, imaging, and research applications. Background Technology

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

[0004] A key limitation of classic whole-protein targeted immunization approaches for antibody development is that they can only produce antibodies against the target site, with limited control over the antibody binding site (i.e., the target epitope on the target protein), and the antibody response primarily targets the immunodominant epitope. Alternative strategies, such as small antigen immunization, represent ideal binding regions of the target protein and offer better control over the antibody binding site. However, this comes at the cost of increased risk that the antibody may fail to bind to the target protein in vivo (i.e., it may not recognize proteins in their "native" conformation), for example, due to the unavailability of the antigen sequence or its different presentation in the native folded protein. Therefore, extensive screening is required to find antibodies with high binding affinity to the target in vivo, but success is not guaranteed.

[0005] Existing methods for producing antibodies through animal immunization cannot provide efficient and reliable production of monoclonal antibodies with the desired functional properties associated with their target antigens. Summary of the Invention

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

[0007] In some embodiments, the animal comprises an endogenous nucleotide sequence used to induce inhibition of the expression of one or more genes involved in triggering the primary humoral immune response or to inhibit the activity of their products.

[0008] In some embodiments, the animal contains endogenous nucleotide sequences used to induce inhibition of the expression of one or more genes involved in immunoglobulin isotype conversion, B cell maturation, and / or plasma cell and / or memory B cell production, or to inhibit the activity of their products.

[0009] In some embodiments, the animal comprises an endogenous nucleotide sequence for inducing inhibition of the expression of one or both genes selected from CD40 and / or CD40L, or for inhibiting the activity of their products. In some embodiments, the animal comprises an endogenous nucleotide sequence for inducing inhibition of the expression of CD40 and / or CD40L, or for inhibiting the activity of their products.

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

[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 triggering the primary humoral immune response.

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

[0013] In some embodiments, the endogenous nucleotide sequence that induces recombinase expression or activity encodes a conditional system for controlling recombinase expression or activity.

[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 comprises a target sequence of a recombinase located flanking one or more exons of CD40. In some embodiments, the endogenous nucleotide sequence comprises a target sequence of a recombinase located flanking one or more exons of CD40L.

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

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

[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 comprising or consisting of a nucleotide sequence having 60% or more identity with the nucleotide sequence of SEQ ID NO:6.

[0022] This disclosure also provides a method for producing antigen-binding molecules, the method comprising administering a peptide / polypeptide or a nucleic acid encoding a peptide / polypeptide to the animal described in this disclosure.

[0023] 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) Treat the animals to suppress their ability to elicit a primary immune response; and (iii) Administering the animal a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide contains the 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 the second peptide / polypeptide, wherein the second peptide / polypeptide comprises the 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 elicit a primary immune response.

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

[0026] This disclosure also provides a method for producing antigen-binding molecules, 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 animals to suppress their ability to elicit a primary immune response; and (iii) Administering the animal a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide contains the 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 the second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide to suppress its ability to elicit a primary immune response.

[0028] In some embodiments, treating the animal to suppress its ability to elicit a primary immune response includes administering an agent that inhibits the expression of one or more genes involved in eliciting a primary humoral immune response or the activity of their products. In some embodiments, treating the animal to suppress its ability to elicit a primary immune response includes administering an agent that reduces / blocks the stimulation, differentiation, activation, maturation, and / or proliferation of naïve B cells in the animal, reduces the number / proportion of naïve B cells in the animal, and / or inhibits immunoglobulin class switching and / or B cell maturation. In some embodiments, the agent inhibits the expression of one or both genes selected from CD40 and / or CD40L or the activity of their products.

[0029] In some embodiments, the reagent inhibits the activity of CD40 and / or CD40L. In some embodiments, the reagent is selected from or includes antibodies, antigen-binding molecules, peptides, decoy receptors, aptamers, blocking agents, or small molecules.

[0030] In some embodiments, the reagent inhibits the expression of CD40 and / or CD40L. In some embodiments, the reagent is or comprises 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 generating a hybridoma that produces an antigen-binding molecule capable of binding to a target protein / protein complex.

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

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

[0035] In some embodiments, the nucleic acid or multiple nucleic acids comprises a nucleotide sequence for inducing suppression of the primary humoral immune response.

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

[0037] In some embodiments, the nucleic acid or multiple nucleic acids comprise nucleotide sequences that are used to inducibly inhibit the expression of one or more genes involved in immunoglobulin isotype conversion and / or B cell maturation, or one or more genes involved in the stimulation, differentiation, activation and / or proliferation of naïve B cells, or to inhibit the activity of their products.

[0038] In some embodiments, the nucleic acid or multiple nucleic acids comprises a nucleotide sequence for inducibly inhibiting the expression of one or both genes selected from CD40 and / or CD40L or inhibiting the activity of their products.

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

[0040] This disclosure also provides one or more nucleic acids comprising a nucleotide sequence encoding all or part of a gene involved in triggering a primary humoral immune response, wherein the nucleotide sequence is flanked by target sequences of a recombinase.

[0041] In some embodiments, the nucleic acid / multiple nucleic acids further comprises a nucleotide sequence encoding a conditional system for controlling 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 comprising a target sequence of a recombinase located flanking one or more CD40 exons and / or one or more CD40L exons.

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

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

[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 comprises a nucleotide sequence, the nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or more identity with the nucleotide sequence of SEQ ID NO:6.

[0048] This disclosure also provides one or more vectors that contain nucleic acids or multiple nucleic acids as described in this disclosure.

[0049] This disclosure also provides a cell comprising one or more nucleic acids as described in this disclosure, or one or more vectors. In some embodiments, the cell comprises an endogenous nucleotide sequence of a gene or gene segment encoding one or more human immunoglobulins.

[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, a rat cell, or a rabbit cell.

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

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

[0053] This disclosure generally relates to the production of antibodies (particularly IgG antibodies, and ultimately preferably monoclonal IgG antibodies) having certain desired functional properties, said functional properties being products that bind to specific regions of a target protein / protein complex.

[0054] This can be achieved by the following steps: (i) immunizing an animal with a first peptide / polypeptide 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 the amino acid sequence of (a)) to elicit a primary immune response against the target amino acid sequence; (ii) inhibiting the animal's ability to elicit a primary immune response; and subsequently (iii) immunizing the animal with a second peptide / polypeptide containing the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0055] In some embodiments, this can be achieved by the following steps: (i) immunizing an animal (which contains an endogenous nucleotide sequence for inducing suppression of the primary humoral immune response) with a first peptide / polypeptide 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 that of (a)) to elicit a primary immune response against the target amino acid sequence; (ii) suppressing the animal’s ability to elicit a primary immune response; and subsequently (iii) immunizing the animal with a second peptide / polypeptide containing the target amino acid sequence or an amino acid sequence similar to that of the target amino acid sequence.

[0056] In some embodiments, this can be achieved by: (i) immunizing an animal with a first peptide / polypeptide 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)); (ii) immunizing the animal with a second peptide / polypeptide containing the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence; and subsequently (iii) inhibiting the animal's ability to elicit a primary immune response. In some cases, the animal may contain an endogenous nucleotide sequence for inducing inhibition of the primary humoral immune response.

[0057] Suppressing the ability of animals to elicit a primary immune response facilitates the generation of a secondary immune response against the target amino acid sequence upon administration of a second peptide / peptide. This secondary immune response is associated with the production of antibodies (particularly IgG antibodies) that can bind to their targets with high affinity.

[0058] In some embodiments, the second peptide / polypeptide is not the same as the first peptide / polypeptide.

[0059] Suppressing the primary immune response and / or promoting the secondary immune response Various aspects and embodiments of this disclosure relate to suppressing primary immune responses (e.g., primary humoral immune responses) in animals, for example, using reagents. In some embodiments, the animal comprises an endogenous nucleotide sequence for inducing suppression of the primary immune response.

[0060] The purpose of this method is to promote a secondary humoral immune response against the target amino acid sequence presented by the second peptide / peptide, rather than a primary humoral immune response against the region in the second peptide / peptide that is not presented by the first peptide / peptide.

[0061] That is, this method aims to suppress the primary humoral immune response against regions of the second peptide / peptide other than the target amino acid sequence, and / or enhance the secondary humoral immune response against the target amino acid sequence of the second peptide / peptide.

[0062] It should be clarified that this method is not intended to suppress the primary humoral immune response to the first peptide / peptide. Specifically, this method is not intended to prevent immunoglobulin isotype conversion caused by the administration of the first peptide / peptide, or to eliminate cells that are activated / stimulated by it and thus proliferate, or derivative cells of such cells (i.e., progeny of cells that are activated / stimulated by the administration of the first peptide / peptide).

[0063] In some embodiments, inhibiting the ability to elicit a primary humoral immune response does not inhibit the development of cells activated / stimulated by administration of the first peptide / peptide into cells expressing IgG, IgA, and / or IgE. In some embodiments, the development of cells activated / stimulated by administration of the first peptide / peptide into plasma B cells and / or memory B cells is uninhibited.

[0064] In some embodiments, suppressing the ability to elicit a primary humoral immune response does not eliminate cells expressing IgG, IgA, and / or IgE (e.g., cells proliferating due to activation / stimulation by administration of a first peptide / peptide, or cells expressing IgG, IgA, and / or IgE resulting from immunoglobulin isotype conversion). In some embodiments, plasma B cells and / or memory B cells (e.g., plasma B cells and / or memory B cells proliferating due to activation / stimulation by administration of a first peptide / peptide, or cells resulting from immunoglobulin isotype conversion) are not eliminated.

[0065] Upon introduction of the second peptide / polypeptide, the affinity maturation of memory B cells capable of generating antigen-binding molecules that recognize the target amino acid sequence is preferentially promoted compared to the primary humoral immune response generated against regions outside the target amino acid sequence.

[0066] In such embodiments, the reagent can suppress the initial immune response against a sequence in the second peptide / peptide that is not presented in the first peptide / peptide.

[0067] Primary and secondary immune responses are components of the adaptive immune response. The adaptive immune response is described, for example, in Janeway's Immunobiology, 9th edition, Murphy et al., 2017 (Galland Science Press, Taylor-Francis Publishing Group), specifically in Part IV.

[0068] The primary immune response refers to the reaction of a subject's adaptive immune system after initial exposure to an antigen.

[0069] The triggering of the primary immune response typically involves antigen-presenting cells (APCs, such as dendritic cells) taking up, processing, and presenting antigens on MHC class II molecules. In the presence of appropriate co-stimulation, naïve T cells containing the MHC class II:peptide complex-specific T cell receptor (TCR) are activated and stimulated to proliferate.

[0070] The antigen also binds to the homologous B cell receptor (BCR) expressed on the surface of naïve B cells. The bound antigen is internalized, processed, and presented on the surface of B cells as an MHC class II:peptide complex. Effector follicular T helper cells (T cells) containing the MHC class II:peptide complex-specific TCR presented by the B cells... FH When stimulated, B cells produce cytokines (such as IL-4 and IL-21), which induce B cell proliferation and differentiation into plasma B cells and memory B cells.

[0071] The antibodies initially produced by the primary immune response are mainly IgM allotype antibodies, which bind to the target antigen with low affinity.

[0072] A secondary immune response refers to the reaction of a subject's adaptive immune system when the subject is exposed again to an antigen that has already elicited a primary immune response.

[0073] Following subsequent exposure, 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). The memory B cells internalize, process, and present the antigen, thereby activating memory T cells. FH cell.

[0074] Upon antigen binding, memory B cells in the germinal center undergo somatic hypermutation in region V, resulting in closely related B cell clones with different antigen affinities. B cells with high-affinity BCRs recognize antigens presented by follicular dendritic cells, process them, and present them to T cells. FH Cells, T FH Cells promote B cell survival by producing IL-21 and binding CD40. Activated B cells that mature in germinal centers also undergo immunoglobulin class switching, producing IgG, IgA, or IgE antibodies. B cells expressing high-affinity BCR differentiate into mature plasma B cells, producing large amounts of high-affinity antibodies against that antigen.

[0075] As used in this article, "humoral immune response" refers to an immune response involving the production of antibodies by B cells.

[0076] Primary humoral immune response can be characterized by: activation and / or maturation of naïve B cells (i.e., stimulation of naïve B cell proliferation, differentiation and / or immunoglobulin class switching), B cells producing IL-4 and / or IL-21, cells expressing IgM and / or IgD being converted to cells expressing IgG, IgE or IgA via immunoglobulin class switching, differentiation of naïve B cells into plasma B cells, differentiation of naïve B cells into memory B cells, and / or production of antibodies that bind to target antigens with low affinity (e.g., IgM antibodies).

[0077] B cell development is described in Pieper et al., J Allergy Clin Immunol. (2013) 131(4):959-71, the entire contents of which are incorporated herein by reference. B cell characteristics are described in Carsetti et al., Cytometry A.2022;101(2):131-139, the entire contents of which are incorporated herein by reference.

[0078] As used herein, "precursor cells of naïve B cells" refers to a cell type upstream of naïve B cells during B cell development. In some embodiments, the precursor cells of naïve B cells may be selected from: stem cells, progenitor B cells, early progenitor B cells, late progenitor B cells, pre-B cells, large pre-B cells, small pre-B cells, or immature B cells.

[0079] Stem cells may be hematopoietic stem cells, which can be characterized, for example, by expressing (e.g., surface expression) CD34 and / or not expressing (e.g., surface expression) CD10. Early progenitor B cells can be characterized by expressing (e.g., surface expression) CD10, CD43, CD45, and / or MHC class II molecules. Late progenitor B cells can be characterized by expressing (e.g., surface expression) CD19, CD40, CD43, CD45, and / or MHC class II molecules. Large pro-B cells can be characterized by expressing (e.g., surface expression) pro-B cell receptors, CD19, CD40, CD43, CD45, and / or MHC class II molecules. Small pro-B cells can be characterized by expressing (e.g., surface expression) pro-B cell receptors, CD19, CD40, CD45, and / or MHC class II molecules. Immature B cells can be characterized by expressing (e.g., surface expression) CD10, CD19, CD20, CD24, CD38, CD40, CD45, IgM and / or MHC class II molecules, and / or not expressing (e.g., surface expression) CD27.

[0080] As used herein, “naïve” B cells refer to mature B cells that have not yet been exposed to B cell BCR-specific antigens. Naïve B cells may also be referred to as mature naïve B cells or mature B cells. Naïve B cells can be characterized by expressing one or more of the following (e.g., on the cell surface): CD19, CD20, CD24, CD40, CD38, CD45, CD21, MHC class II molecules, IgM, and IgD. Naïve B cells can be characterized by not expressing (e.g., on the cell surface) CD27.

[0081] As used herein, "plasma" B cells refer to B cells that express a large number of soluble antibodies. Plasma B cells can be characterized by expressing one or more of the following (e.g., on the cell surface): CD27, CD38, CD138, CD78, CD126, CXCR4, and BCMA. Plasma B cells can be characterized by not expressing (e.g., on the cell surface) CD20 and / or CD24.

[0082] As used in this article, “memory” B cells refer to B cells that form in the germinal center after the primary immune response. Memory B cells can 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 molecules, and TACI.

[0083] The specific genes or proteins referenced in this specification include isotypes, fragments, variants, or homologs of that gene / protein from any species. It should be understood that, in aspects and embodiments involving a specific animal, the gene or protein refers to an appropriate homolog encoded by that animal's genome. For example, if the animal is a mouse, the gene / protein may be a mouse homolog of the relevant gene / protein. In another example of a transgenic mouse encoding a human homolog of a relevant gene / protein, the gene may be a human homolog of the relevant gene / protein.

[0084] Secondary humoral immune responses can be characterized as: activation of memory B cells (i.e., stimulation of memory B cell proliferation and / or differentiation), somatic hypermutation of memory B cells, differentiation of memory B cells into plasma B cells, and / or production of antibodies (e.g., IgG antibodies) that bind to target antigens with high affinity.

[0085] Certain aspects and embodiments of this disclosure relate to agents that can be used in animals to suppress primary humoral immune responses. Certain aspects and embodiments of this disclosure relate to animals comprising endogenous nucleotide sequences that can be used in these animals to induce suppression of primary humoral immune responses. It should be understood that the effect of such suppression is to support / promote secondary humoral immune responses in the animal.

[0086] This disclosure also provides reagents, such as those described herein, for use in the methods disclosed herein, i.e., for suppressing primary humoral immune responses in animals. Application of said reagents is also provided, i.e., for suppressing primary humoral immune responses in animals.

[0087] In this article, "inhibition" can also be referred to as "antagonism". Preparations that can inhibit a response / expression / activity can be called "inhibitors" or "antagonists" of the relevant response / expression / activity.

[0088] In some embodiments, the suppression of the primary humoral immune response as described in this disclosure includes one or more of the following: inhibiting the expression of genes involved in initiating the primary humoral immune response, inhibiting the activity of products of genes involved in initiating the primary humoral immune response, reducing the number / proportion of naïve B cells, reducing / blocking the stimulation, differentiation, activation, maturation and / or proliferation of naïve B cells, for example through T cells, inhibiting immunoglobulin class switching, inhibiting the expression of genes involved in immunoglobulin class switching, and / or inhibiting the activity of products of genes involved in immunoglobulin class switching.

[0089] As used herein, “expression” can refer to gene expression 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 quantitative real-time PCR (qRT-PCR) or by measuring mRNA levels using reporter gene-based methods. Similarly, protein expression can be measured using a variety of methods known to those skilled in the art, such as antibody-based methods like Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, or reporter gene-based methods.

[0090] As used herein, a factor / activity / cell type “involved in” a specific reaction / process (e.g., triggering an immune response, immunoglobulin class switching, etc.) refers to a factor / activity / cell type associated with the relevant reaction / process. This 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 this factor / activity / cell type.

[0091] Various aspects and embodiments of this disclosure relate to inhibiting the expression (i.e., gene expression or protein expression) of one or more genes involved in triggering the primary humoral immune response, and / or inhibiting the activity of the products of one or more genes involved in triggering the primary humoral immune response.

[0092] In a preferred embodiment, the one or more genes are not involved in or are not essential for initiating a secondary humoral immune response. In some embodiments, the one or more genes involved in initiating a primary humoral immune response are selected from CD40 and / or CD40L. In some embodiments, the one or more genes involved in initiating a primary humoral immune response are CD40.

[0093] Inhibiting the expression of a specific gene or protein may include: inhibiting the transcription of the gene, inhibiting the post-transcriptional processing (e.g., splicing) of the RNA transcribed from the gene, reducing the stability of the RNA transcribed from the gene, promoting the degradation of the RNA transcribed from the gene, inhibiting the translation of the RNA transcribed from the gene into a protein, inhibiting the post-translational processing of the polypeptide encoded by the gene, reducing the stability of the polypeptide encoded by the gene, or promoting the degradation of the polypeptide encoded by the gene.

[0094] Gene expression can be analyzed using methods well known to those skilled in the art. The level of RNA encoding a specific gene can be determined using techniques such as RT-qPCR. Protein expression can also be determined using methods well known to those skilled in the art. The level of a specific protein / its isoform can be determined using antibody-based methods, including Western blotting, immunohistochemistry / cytochemistry, flow cytometry, ELISA, etc.

[0095] Suppressing the expression of a specific gene or protein can reduce its expression level to less than 1 times the level observed in the unsuppressed state, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times. In some embodiments, the suppression of gene or protein expression inhibits greater than 5% of the expression level observed in the unsuppressed 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%.

[0096] Suppressing gene or protein expression can be achieved, for example, by altering / disrupting the nucleotide sequence of the gene, or by altering / disrupting the nucleotide sequence required for gene expression (e.g., a regulatory sequence that regulates gene expression). In some embodiments, suppressing gene or protein expression may include altering the nucleotide sequence, for example, by replacing, deleting, or inserting one or more nucleotides. For example, in certain aspects and embodiments of this disclosure, it is contemplated to suppress gene or protein expression by deleting all or part of the nucleotide sequence of the relevant gene.

[0097] In some embodiments, altering / destroying nucleotide sequences may include, for example, altering / deleting regulatory sequences of gene transcription (such as promoters, enhancers), introducing premature stop codons into gene transcription sequences, altering nucleotide sequences to encode truncated and / or nonfunctional gene products, or altering nucleotide sequences to encode misfolded and / or degraded gene products.

[0098] The act of altering / disrupting nucleotide sequences to suppress / prevent gene or protein expression is known as gene "knockout".

[0099] Nucleotide sequence disruption can be achieved through homologous recombination or targeted nucleic acid modification using site-specific nucleases (SSNs, also referred to as “gene editing systems” in this paper).

[0100] Homologous recombination modification involves the exchange of nucleic acid sequences via cross-events guided by homologous sequences, and has been reviewed in such references as *Mortensen Curr Protoc Neurosci* (2007), Chapter 4, Unit 4.29, and Vasquez et al., PNAS. (2001) 98(15): 8403-8410, both of which are incorporated herein by reference in their entirety. The homologous sequence is flanked by all or part of the nucleotide sequence to be disrupted. The recombination process can be catalyzed by a recombinase. For example, in certain aspects and embodiments of this disclosure, the disruption of nucleotide sequences via homologous recombination between loxP sequences catalyzed by Cre recombinase is considered.

[0101] Disruption of nucleotide sequences via homologous recombination can be achieved in animals, for example involving the Cre-LoxP, Flp-FRT, and Dre-rox systems as described above.

[0102] A review of gene editing using SSNs was published in, e.g., Eid and Mahfouz, Exp Mol Med. (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 sites of desired nucleic acid sequences. DSBs can be repaired by error-prone non-homologous end joining (NHEJ), in which the ends of the break are rejoined, often accompanied by nucleotide insertions or deletions. Alternatively, DSBs can be repaired by highly homologous directed repair (HDR), in which a DNA template with homologous ends to the break site is provided and introduced into the DSB site. SSNs can be engineered to generate target nucleic acid sequence-specific DSBs, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeats / CRISPR-associated protein 9 (CRISPR / Cas9) systems.

[0103] A systematic review of ZFNs was conducted, for example, by Umov et al., Nat Rev Genet. (2010) 11(9):636-46, the entire contents of which are incorporated herein by reference. ZFNs contain a programmable zinc finger DNA-binding domain and a DNA-cutting domain (e.g., the FokI endonuclease domain). The DNA-binding domain can be identified by screening zinc finger arrays that can bind to target nucleic acid sequences. A systematic review of TALENs was conducted, for example, by Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, the entire contents of which are incorporated herein by reference. TALENs contain a programmable DNA-binding TALE domain and a DNA-cutting domain (e.g., the FokI endonuclease domain). The TALE contains a repeating domain consisting of 33-39 amino acid repeats, which are identical except for two residues at positions 12 and 13 of each repeat (i.e., repeat variable double residues (RVDs)). Each RVD determines the binding of the repeat sequence to nucleotides in the target DNA sequence according to the following relationship: "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). A review of CRISPR / Cas9 and related systems (such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3) is available, for example, in Nakade et al., Bioengineered (2017) 8(3):265-273, the entire contents of which are incorporated herein by reference. These systems contain an endonuclease (such as Cas9, Cpf1, etc.) and a single-stranded guide RNA (sgRNA) molecule. The sgRNA can be modified to target the endonuclease activity to the target nucleic acid sequence.

[0104] As used herein, the “product” of a gene may, for example, refer to nucleic acids transcribed from the gene, or peptides / polypeptides (including peptides / polypeptides modified with chemical groups, such as carbohydrate and / or lipid groups) produced by translating (and, where appropriate, further post-translational processing) nucleic acids transcribed from the gene.

[0105] Inhibiting the activity of a specific 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.

[0106] Inhibiting the activity of a specific gene product can reduce it to less than 1 times the activity level observed in the uninhibited state, for example, ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times. In some embodiments, inhibiting the activity of a specific gene product suppresses greater than 5% of the activity level 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%.

[0107] In some embodiments, the reagent can reduce the expression of a target gene or protein (i.e., a gene or protein involved in initiating a primary immune response) and / or reduce the activity of the target gene or protein. In certain circumstances, the reagent can inhibit, degrade, silence, knock down, reduce, or otherwise decrease the expression and / or activity of a gene or protein involved in initiating a primary immune response.

[0108] The reagent may have one or more of the following properties for the target gene or target protein (i.e., the gene or protein involved in triggering 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 the protein; isolating the protein; competing for the protein's binding site; and / or blocking the protein's activity.

[0109] In some embodiments, the reagent can inhibit the expression of one or both genes selected from CD40 and / or CD40L, or inhibit the activity of their products.

[0110] Such reagents can be used to treat the animals described herein. In some embodiments, as described herein, the animals contain endogenous nucleotide sequences for inducing suppression of the primary immune response. In some embodiments, the animals do not contain endogenous nucleotide sequences for inducing suppression of the primary immune response.

[0111] In some embodiments, the reagent is an antibody or an antigen-binding molecule (both referred to herein as "antigen-binding molecules"), such as an anti-CD40 or anti-CD40L antibody. In some cases, the antigen-binding molecule specifically targets a protein involved in initiating a primary immune response, such as CD40 or CD40L. In some cases, the antigen-binding molecule can specifically bind to a protein involved in initiating a primary immune response, such as CD40 or CD40L. In some cases, the antigen-binding molecule is an anti-CD40 or anti-CD40L antigen-binding molecule.

[0112] The antigen-binding molecule may be an antagonistic antigen-binding molecule that can inhibit or reduce the biological activity of target proteins (such as CD40 or CD40L) involved in triggering the primary immune response.

[0113] The antigen-binding molecule can bind to specific target regions of target proteins (such as CD40 or CD40L) involved in initiating a primary immune response. The antigen-binding domain of the antigen-binding molecule can bind to linear epitopes of target proteins (such as CD40 or CD40L) involved in initiating a primary immune response, the epitopes consisting of a continuous amino acid sequence (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding domain molecule can bind to conformational epitopes of target proteins (such as CD40 or CD40L) involved in initiating a primary immune response, the epitopes consisting of discontinuous amino acid sequences within the amino acid sequence.

[0114] The antigen-binding molecule may be a multispecific antigen-binding molecule. “Multispecific” means that the antigen-binding molecule can specifically bind to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains, for example, comprising different VH and VL). Multispecific antigen-binding molecules may be provided in any suitable form, such as the form described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, the entire contents of which are incorporated herein by reference.

[0115] In some embodiments, the antigen-binding molecule binds to a target protein (e.g., CD40 or CD40L) involved in triggering the primary immune response and another target (e.g., an antigen other than the target protein), thus being at least bispecific. The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants.

[0116] The ability of a specific peptide to specifically bind to a particular molecule or another specific peptide / peptide can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR, see Hearty et al., MethodsMol Biol 2012, 907:411-442), bio-layer interference (see Lad et al., J Biomol Screen. 2015, 20(4): 498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. These analyses allow for the measurement and quantification of binding to a specific molecule. In some embodiments, binding may be a reaction detected in a specific experiment. Binding affinity can be expressed as a dissociation constant (KD).

[0117] The peptide / peptide region bound by the antibody can be determined by those skilled in the art using a variety of methods known in the art, including X-ray cocrystallization analysis of antibody-antigen complexes, peptide scanning, mutation localization, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based “protective” methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, the entire contents of which are incorporated herein by reference.

[0118] In some embodiments, the antigen-binding molecule inhibits the interaction between two binding partners (such as CD40 and CD40L). The ability of the antigen-binding molecule to inhibit the interaction between two binding partners can be determined by analyzing the downstream functional results of such interaction in a suitable assay, for example by detecting protein generation during the reaction using ELISA, Western blotting, or electrophoresis.

[0119] Those skilled in the art can produce suitable antigen-binding molecules using techniques such as those described herein or those known in the art (see Chiu and Gilliland, Curr Opin Struct Biol. 2016, 38:163-173; Jakobovits A, Curr Opin Biotechnol. 1995 Oct;6(5):561-6; and Brüggemann M et al., Arch Immunol Ther Exp(Warsz). 2015; 63(2): 101-108). A suitable technique is phage display, see Hammers and Stanley, J Invest Dermatol. 2014, 134(2): e17 and Bazan J et al., Hum Vaccin Immunother. 2012, 8(12): 1817-1828. Antigen-binding polypeptide chains can also be prepared by chemical synthesis (see Chandrudu et al., Molecules (2013), 18: 4373-4388), recombinant expression (see Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, 2012, and Nat Methods. (2008); 5(2): 135-146), or cell-free protein synthesis (CFPS; see Zemella et al., Chembiochem (2015) 16(17): 2420-2431), all of which are incorporated herein by reference. The antigen-binding molecules can be monoclonal, i.e., a homogeneous group of 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 *Monoclonal Antibodies: A manual of techniques*, H. Zola (CRC Press, 1988) and *Monoclonal Hybridoma Antibodies: Techniques and Applications*, J.G. Hurrell (CRC Press, 1982). For a discussion of chimeric antibodies, see Neuberger et al. (Part II, Eighth International Symposium on Biotechnology, 1988, pp. 792-799). Suitable polyclonal antibodies can also be prepared using methods well-known in the art.

[0120] The antigen-binding portion can be a part of the antibody (e.g., the Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment [ScFv]). Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments, can also be used as / provided for genetically engineered antibodies and antibody fragments. The fact that the variable heavy chain (VH) domain and variable light chain (VL) domain of antibodies are involved in antigen recognition was first confirmed by early protease digestion experiments and further confirmed by studies on the "humanization" of rodent antibodies. Fusing a rodent-derived variable domain with a human-derived constant domain allows the resulting antibody to retain the antigen specificity of the rodent parent antibody (Morrison et al., (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0121] The antibody and antigen binding fragments described in this disclosure include a complementarity-determining region (CDR) of an antibody capable of binding to the relevant target molecule, i.e., one or more proteins as described herein that are involved in triggering the primary humoral immune response.

[0122] The reagent may be nucleic acid-based or contain nucleic acid elements. The reagent may promote gene expression silencing through RNA-mediated interference or antisense degradation mechanisms (such as via RNase H).

[0123] In some embodiments, the reagent is or comprises an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid (such as a DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid (such as mRNA that can be translated into a protein, such as the FHR protein) and is capable of reducing target nucleic acid transcription (e.g., transcription from DNA to mRNA), reducing target nucleic acid (e.g., mRNA) translation, or altering transcript splicing (e.g., via single-stranded morpholine oligonucleotides). Antisense nucleic acids can be single-stranded (e.g., space bodies) or double-stranded (e.g., siRNA). Antisense nucleic acids are capable of hybridizing with target nucleic acids (e.g., target mRNA) via Watson-Crick base pairing (e.g., selective hybridization). In some cases, antisense nucleic acids specifically bind to target nucleic acids. In some cases, antisense nucleic acids hybridize with target nucleic acid sequences (e.g., mRNA) under stringent hybridization conditions. In some cases, antisense nucleic acids hybridize with target nucleic acids (e.g., mRNA) under moderately stringent hybridization conditions.

[0124] The nucleotide sequence of an antisense nucleic acid is highly complementary to that of the target nucleic acid, enabling it to bind or hybridize with the target nucleic acid. Therefore, if technicians know the sequence of the target nucleic acid, designing a suitable antisense nucleic acid that can hybridize with the target to achieve the desired effect is simple and routine.

[0125] The target RNA can be an mRNA that encodes a protein involved in triggering the primary immune response (such as CD40 or CD40L).

[0126] In some cases, the reagents can promote RNA interference (RNAi). RNAi utilizes small double-stranded RNA molecules to degrade target mRNA. Non-limiting examples of antisense nucleic acids used as reagents as described in this invention include: siRNA (including its derivatives or precursors, such as nucleotide analogs), short hairpin RNA (shRNA), microRNA (miRNA; including its long primary transcripts (pri-miRNAs) and partially processed 60-70 base pair hairpin transcripts (pre-miRNAs)), saRNA (small activating RNA), and small nucleolar RNA (snoRNA), or certain derivatives or precursors thereof. Antisense nucleic acid molecules can stimulate RNA interference (RNAi).

[0127] siRNA nucleic acids are approximately 21-25 nucleotides in length and contain a guide strand that hybridizes with the target mRNA and a complementary guest strand (e.g., each complementary sequence of a double-stranded siRNA is 21-25 nucleotides in length, and the length of a 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 described in, for example, Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616. Suitable siRNA molecules suitable for the methods of this invention can be designed using methods known in the art, see, for example, Elbashire et al., Nature, 2001 411:494-8; Amarzguioui et al., Biochem. Biophys. Res. Commun. 2004 316(4):1050-8; and Reynolds et al., Nat. Biotech. 2004, 22(3):326-30. Details regarding the preparation of siRNA molecules can be found on the websites of several commercial vendors, such as Ambion, Dharmacon, GenScript, Invitrogen, and OligoEngine. A BLAST alignment procedure can typically be used to check the matching of any potential siRNA candidate sequence with other nucleic acid sequences or nucleic acid sequence polymorphisms (see the National Library of Medicine website). Generally, multiple siRNAs need to be generated and screened to obtain effective drug candidates (see U.S. Patent No. 7,078,196). siRNAs can be produced from vector expression and / or by chemical or synthetic methods. Synthetic RNAi are available from commercial sources, such as Invitrogen (Carlsbad, California); RNAi vectors are also available from commercial sources, such as Invitrogen.

[0128] 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 (pre-miRNAs) of 60-70 nucleotides, and further processed in the cytoplasm into small double-stranded nucleic acids that can interact with RISC and target mRNAs. miRNAs contain a "seed sequence" necessary for binding to the target mRNA. The "seed sequence" typically consists of six nucleotides located at positions 2-7 of the 5' end of the miRNA.

[0129] In some embodiments, the formulation 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 (such as CD40 or CD40L) as described herein that participates in initiating a primary immune response. In some embodiments, the formulation comprises an siRNA molecule containing a guide strand that is complementary to or hybridizes with a portion of an mRNA sequence encoding all or part of an mRNA sequence encoding a protein (such as CD40 or CD40L) as described herein that participates in initiating a primary immune response. In some embodiments, the formulation comprises a miRNA molecule (primary, precursor, or mature miRNA) containing a seed sequence capable of hybridizing with a portion of an mRNA sequence encoding all or part of an mRNA sequence encoding a protein (CD40 or CD40L) as described herein that participates in initiating a primary immune response.

[0130] In some cases, the reagent is a single-stranded antisense oligonucleotide (ASO). ASO modifies the expression of the target RNA by altering splicing or recruiting RNase H to degrade the target RNA. When an ASO binds to the target RNA, RNase H recognizes the resulting DNA:RNA hybrid. ASOs are typically 18–30 base pairs in length. Many ASOs are designed as chimeras (containing a mixture of bases with different chemical properties) or spacers (containing a central DNA portion surrounded by “wings” of modified bases). ASOs are described in publications such as Scoles et al., Neurol Genet. 2019 Apr; 5(2): e323.

[0131] Antisense nucleic acids may contain naturally occurring nucleotides or modifications, such as thiophosphate bonds, phosphoryl diamide bonds, methoxyethyl nucleotide modifications (e.g., 2-MOE), "locked" nucleic acids (e.g., LNA), peptide nucleic acids (PAN), and / or 5'-methylcytosine modifications.

[0132] In some embodiments, the reagent comprises an antisense oligonucleotide capable of hybridizing with a portion of the mRNA sequence of a protein (such as CD40 or CD40L) that is involved in initiating a primary immune response as described herein.

[0133] As described herein, antisense nucleic acids may comprise or consist of nucleotide sequences that have complementarity with their target nucleic acids 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 based on the full length of the antisense nucleic acid and / or all or part of the target nucleic acid to which the antisense nucleic acid is bound.

[0134] 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) capable of binding (e.g., with high affinity and specificity) to proteins, peptides, and small molecules. Aptamers typically have well-defined secondary or tertiary structures due to their tendency to form complementary base pairs, and thus can often fold into diverse and complex molecular structures. These three-dimensional structures are crucial for the binding affinity and specificity of the aptamer, and specific three-dimensional interactions drive the formation of the aptamer-target complex. In vitro, aptamers can be screened from large random sequence libraries through exponential enrichment of ligand systems (SELEX, described in Ellington AD, Szostak JW, Nature 1990, 346:818-822; Tuerk C, Gold L., Science 1990, 249:505-510), or by developing SOMAmers (slow dissociation rate modified aptamers) (Gold L et al., (2010), biomarker discovery based on aptamer proteomics, PLoS ONE 5(12):e15004). SOMAmers are short single-stranded deoxy oligonucleotides that possess protein-like properties due to their functional groups mimicking amino acid side chains. Applications of SELEX and SOMAmers techniques include, for example, adding functional groups mimicking amino acid side chains to expand the chemical diversity of aptamers. This allows for the enrichment and identification of high-affinity aptamers targeting specific targets.

[0135] Aptamers can be DNA or RNA molecules, and can be single-stranded or double-stranded. Aptamers may contain chemically modified nucleic acids, such as chemically modified sugar groups, phosphate groups, and / or bases. Such modifications can enhance the stability of the aptamer or improve its resistance to degradation, and may include modifications to the 2' site of the ribose.

[0136] Aptamers can be synthesized by methods well known to those skilled in the art. For example, aptamers can be prepared chemically (e.g., on a solid-phase support). Solid-phase synthesis can be performed using phosphoramide chemistry. In short, a solid-supported nucleotide is detrimethylated and then coupled with a suitably activated nucleoside phosphoramide to form a phosphite triester bond. This may be followed by a capping reaction, followed by oxidation of the phosphite triester with an oxidizing agent (usually iodine). This cycle is repeated to assemble the aptamer (see Sinha, ND; Biernat, J.; McManus, J.; Köster, H., Nucleic Acids Res. 1984, 12, 4539; and Beaucage SL; Lyer, RP, (1992), Tetrahedron 48 (12): 2223).

[0137] Aptamers can be peptides that have been screened or modified to bind to specific target molecules. A review of peptide aptamers and methods for their generation and identification can be found in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101, the entire contents of which are incorporated herein by reference. Peptide aptamers can optionally have a minimum length of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and a maximum length 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 polypeptide aptamers may optionally be 2-30, 2-25, 2-20, 5-30, 5-25, or 5-20 amino acids in length.

[0138] Aptamers may have K on the order of nanomolars or picomolars. D For example, below 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM or 100 pM.

[0139] Aptamers or SOMAmers suitable for the purposes described herein can bind to proteins (CD40 or CD40L) involved in initiating primary immune responses as described herein. Aptamers or SOMAmers suitable for the purposes described herein can specifically bind to proteins (CD40 or CD40L) involved in initiating primary immune responses as described herein. The aptamers can inhibit the function of proteins involved in initiating primary immune responses (such as CD40 or CD40L), for example, by blocking their binding to homologous binding chaperones or ligands.

[0140] The reagent may be a chelating agent, for example, targeting proteins involved in initiating the primary immune response as described herein. The reagent may be a protein molecule.

[0141] The reagent may be a small molecule. For example, the small molecule may bind to proteins involved in initiating the primary immune response as described herein and prevent / reduce their normal function and / or prevent / reduce their interaction with homologous binding chaperones. The small molecule may prevent / reduce the proper folding of the target protein.

[0142] 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 a primary immune response as described herein. The receptor may be a receptor (including fragments and derivatives thereof) for proteins involved in initiating a primary immune response as described herein. A decoy receptor can recognize and bind to a specific ligand but may not be able to transduce signals or activate a subsequent response. A decoy receptor may bind to proteins involved in initiating a primary immune response as described herein to form a complex. A decoy receptor may act as an inhibitor of a protein by blocking / reducing the ability or effectiveness of binding a protein involved in initiating a primary immune response as described herein to its receptor. A decoy receptor may act as an inhibitor of a protein by binding to a binding chaperone of the protein involved in initiating a primary immune response (e.g., in a region normally bound by a binding chaperone), thereby blocking the interaction between the protein and one or more binding chaperones. For example, the formulation may be a molecule that binds to CD40L and may block / reduc its interaction with CD40.

[0143] Decoy receptors can be soluble (non-membrane-bound) or membrane-bound (e.g., expressed on the cell surface). Decoy receptors can be presented or applied to the surface of nanocarriers, such as nanoparticles, liposomes, microbeads, polymers, metal particles, dendritic polymers, nanotubes, or micron-sized silica rods (see Wilczewska AZ et al., Pharmacol Rep. 2012, 64(5):1020-1037).

[0144] Methods for detecting whether a decoy receptor competitively binds to a target protein may include SPR (see Hearty et al., Methods Mol Biol 2012, 907:411-442), competitive ELISA assays, or solid-binding assays. Other suitable methods are also known in the art.

[0145] Agents that can 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 more than one of the above categories. For example, antigen-binding molecules or decoy receptors may also act as chelating agents.

[0146] Any reagents described herein may optionally be isolated and / or substantially purified.

[0147] Reduce the production of plasma cells and memory B cells Various aspects and embodiments of this disclosure relate to reducing and / or blocking stimulation of early-stage B cells (e.g., B cells that have not yet been exposed to the corresponding BCR-specific antigen, or B cells that have been exposed to the antigen but have not yet differentiated or proliferated into plasma cells or memory B cells).

[0148] The aim is to inhibit / block the co-stimulation of B cells by activated T cells, thereby preventing B cell proliferation, differentiation, and activation into antibody-secreting cells (such as plasma B cells or memory B cells). This inhibits the initiation of adaptive humoral immunity by preventing the formation of functional germinal centers and reduces / prevents the animal's ability to elicit a primary humoral immune response upon subsequent attacks.

[0149] This protocol is not intended to inhibit the stimulation, activation, differentiation, and / or proliferation of B cells or their derivatives (i.e., progeny of cells activated / stimulated to proliferate by administration of the first peptide / peptide) that are stimulated to proliferate by administration of the first peptide / peptide.

[0150] In some embodiments, the treatment of animals to inhibit / block the stimulation, activation, differentiation, and / or proliferation of B cells is performed after a period of time sufficient for the B cells to complete the stimulation / activation / differentiation / proliferation due to the administration of the first peptide / polypeptide. In some embodiments, such animal treatment is performed after a period of time sufficient for the cells stimulated / activated / differentiated / proliferated (or derived cells of such cells) by the administration of the first peptide / polypeptide to differentiate into plasma B cells and / or memory B cells.

[0151] In some embodiments, the treatment of animals to induce inhibition / blockage of B cell stimulation, activation, differentiation, and / or proliferation is performed after a period of time sufficient for the B cells (or their derivatives) to complete stimulation / activation / differentiation / proliferation upon administration of the first peptide / polypeptide. In some embodiments, such animal induction treatment is performed after a period of time sufficient for the cells stimulated / activated / differentiated / proliferated (or their derivatives) upon administration of the first peptide / polypeptide to differentiate into plasma B cells and / or memory B cells.

[0152] In some embodiments, inhibiting / blocking / reducing the stimulation, activation, differentiation, and / or proliferation of B cells does not involve inhibiting / blocking / reducing cells expressing IgG, IgE, or IgA. In some embodiments, inhibiting / blocking / reducing the stimulation, activation, differentiation, and / or proliferation of B cells does not involve inhibiting / blocking / reducing plasma B cells and / or memory B cells.

[0153] In some embodiments, inhibition / blocking / reduction of B cell stimulation, activation, differentiation, and / or proliferation is achieved by inhibiting the expression of one or more factors expressed by such cells. For example, inhibition of B cell stimulation, activation, differentiation, and / or proliferation can be achieved by inhibiting the expression of CD40 and / or CD40L (both of which are involved in regulating all stages of the B cell response to antigens) (see Janeway CA Jr et al., *Immunobiology: The Immune System in Health and Disease*, 5th ed., New York: Garland Science Press; 2001. Activation of B cells by arming helper T cells, the entire contents of which are incorporated herein by reference).

[0154] Various aspects and embodiments of this disclosure relate to inhibiting the generation (i.e., differentiation, proliferation) of plasma B cells or memory B cells in response to antigen attack.

[0155] Inhibit the generation of B cells to reduce / prevent the animal's ability to elicit a primary humoral immune response to an antigen after a subsequent attack.

[0156] This is not intended to consume / remove cells or their derivative cells that have been activated / stimulated by the application of the first peptide / polypeptide and thus proliferate (i.e., the progeny of cells that have been activated / stimulated by the application of the first peptide / polypeptide).

[0157] In some embodiments, the treatment of animals to inhibit the generation of plasma cells or memory B cells is performed after a period of time sufficient for the cells (or their derivatives) that have proliferated due to the administration of the first peptide / peptide to complete immunoglobulin class switching (i.e., conversion to cells expressing IgG, IgE, or IgA). In some embodiments, the treatment of animals to inhibit the generation of plasma cells or memory B cells is performed after a period of time sufficient for the cells that have been activated / stimulated (or their derivatives) by the administration of the first peptide / peptide to differentiate into plasma B cells and / or memory B cells.

[0158] In some embodiments, the treatment of animals to induce inhibition of plasma cell or memory B cell generation is performed after a period of time sufficient for the cells (or their derivatives) that have proliferated due to the administration of the first peptide / peptide to complete immunoglobulin isotype conversion (i.e., conversion to cells expressing IgG, IgE, or IgA). In some embodiments, the treatment of animals to induce inhibition of plasma cell or memory B cell generation is performed after a period of time sufficient for the cells that have been activated / stimulated (or their derivatives) by the administration of the first peptide / peptide to differentiate into plasma B cells and / or memory B cells.

[0159] In some embodiments, inhibiting the generation of plasma cells or memory B cells does not involve reducing the number / proportion of cells expressing IgG, IgE, or IgA. In some embodiments, inhibiting the generation of plasma cells or memory B cells does not involve reducing the number / proportion of plasma B cells and / or memory B cells generated by the administration of the first peptide / peptide.

[0160] Inhibit immunoglobulin isotype switching Various aspects and embodiments of this disclosure relate to the inhibition of immunoglobulin isotype switching. Its primary objective is to eliminate, during subsequent immunization, the animal's ability to elicit an IgG response to a region of the second peptide / peptide that differs from the first peptide / peptide.

[0161] Some aspects and embodiments of this disclosure include inhibiting the expression (gene expression or protein expression) of genes involved in immunoglobulin isotype conversion, and / or inhibiting the activity of products of genes involved in immunoglobulin isotype conversion.

[0162] Immunoglobulin class switching is also known as "isotype switching" and "class switching recombination". For related reviews, please see Stavnezer and Schrader, J Immunol. (2014) 193(11): 5370-5378, the full contents of which are incorporated herein by reference.

[0163] Mature naïve B cells express both IgM and IgD. Activation through antigen binding leads to cell proliferation, and if the cells encounter suitable factors (such as IL-4 or CD40L), class switching reorganization is triggered via cytokine receptor and CD40 signaling, switching from expressing IgM and IgD to expressing IgG, IgE, or IgA. During class switching, the constant regions of the immunoglobulin heavy chain change, but the variable regions remain unchanged; therefore, antigen specificity remains constant.

[0164] Immunoglobulin class switching involves replacing the μ and δ heavy chain constant (CH) regions of expressed Ig with γ, ε, or α CH regions via deletion recombination between two different switching (S) regions. Class switching recombination (CSR) is initiated by activation-induced cytidine deaminase (AICDA), which converts cytosine in the S region to uracil. Uracil is then removed via two DNA repair pathways, resulting in mutations, single-strand DNA breaks, and double-strand DNA breaks required for CSR.

[0165] Various aspects and embodiments of this disclosure relate to inhibiting the immunoglobulin class conversion of B cells expressing IgM and / or IgD to cells expressing IgG, IgE, and / or IgA. In certain embodiments, this disclosure relates to inhibiting the immunoglobulin class conversion of B cells expressing IgM and / or IgD to B cells expressing IgG.

[0166] This is not intended to inhibit immunoglobulin isotype conversion in cells or their derivatives (i.e., progeny of cells that have been activated / stimulated by the administration of the first peptide / peptide) that have proliferated.

[0167] In some embodiments, the treatment of the animal to inhibit (e.g., induce inhibition) immunoglobulin isotype conversion is performed after a period of time sufficient for the cells (or their derivatives) activated / stimulated by the administration of the first peptide / peptide to complete immunoglobulin isotype conversion (i.e., conversion to cells expressing IgG, IgE, or IgA). In some embodiments, the treatment of the animal to inhibit (e.g., induce inhibition) immunoglobulin isotype conversion is performed after a period of time sufficient for the cells activated / stimulated by the administration of the first peptide / peptide (or their derivatives) to differentiate into plasma B cells and / or memory B cells.

[0168] In some embodiments, inhibiting immunoglobulin isotype conversion does not involve inhibiting immunoglobulin isotype conversion of cells (or their derivatives) that have been activated / stimulated to proliferate by administration of a first peptide / peptide.

[0169] CD40 / CD40L signaling plays a crucial role in immunoglobulin class switching. CD40 is expressed on B cells, while CD40L is expressed on activated T cells. It has been demonstrated that CD40 knockout mice do not produce IgG responses to T cell-dependent antigens (Kawabe et al., Immunity (1994) 1: 167-178), and that B cells from CD40 knockout mice do not undergo isotype switching in vitro upon stimulation with sCD40L and IL-4 (Castigli et al., PNAS (1994) 91(25): 12135-12139). Furthermore, CD40L knockout mice fail to produce antigen-specific IgG1 responses after thymus-dependent antigen immunization (Xu et al., Immunity (1994) 1:423-431). Therefore, inhibiting the expression of CD40 and / or CD40L, or the activity of their products, will impair immunoglobulin class switching.

[0170] In some embodiments, inhibiting immunoglobulin class switching includes inhibiting the expression of genes or proteins of factors (e.g., cytokines, cell surface proteins, etc.) involved in T cell expression during immunoglobulin class switching. In some embodiments, inhibiting immunoglobulin class switching includes inhibiting the activity of factors (e.g., cytokines, cell surface proteins, etc.) involved in T cell expression during immunoglobulin class switching.

[0171] In some embodiments, inhibiting immunoglobulin class switching includes inhibiting the expression or activity of one or more factors involved in immunoglobulin class switching. In some embodiments, inhibiting immunoglobulin class switching includes inhibiting the expression of one or both of CD40 and / or CD40L, or the activity of their products.

[0172] It should be understood that inhibition of immunoglobulin class switching can also be achieved through methods such as reducing the number / proportion of naïve B cells, inhibiting / blocking the stimulation, differentiation, activation, maturation, and / or proliferation of naïve B cells, or inhibiting T cell-mediated helper effects, as described above. In other words, immunoglobulin class switching can be inhibited by consuming / removing cell populations that would otherwise undergo immunoglobulin class switching.

[0173] 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).

[0174] 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), the animals containing endogenous nucleotide sequences for inducing suppression of primary immune responses.

[0175] In some embodiments, the methods of this disclosure include administering a reagent as disclosed above, which can induce suppression of the primary immune response and / or promote a secondary immune response in animals. In some embodiments, the reagent can induce suppression of the primary immune response in animals.

[0176] In some embodiments, the reagent can effectively inhibit the primary immune response and / or promote the secondary immune response in animals. In some embodiments, the reagent can inhibit the expression of genes involved in initiating the primary humoral immune response, inhibit the activity of products of genes involved in initiating the primary humoral immune response, reduce the number / proportion of naïve B cells, reduce / inhibit the stimulation, differentiation, activation, maturation and / or proliferation of naïve B cells through, for example, T cell-mediated co-stimulation, inhibit immunoglobulin isotype conversion, inhibit the expression of genes involved in immunoglobulin isotype conversion, and / or inhibit the activity of products of genes involved in immunoglobulin isotype conversion.

[0177] In some embodiments, the reagent may induce the expression or activity of one or more factors, thereby inhibiting the primary immune response and / or promoting the secondary immune response.

[0178] Various aspects and embodiments of this disclosure relate to the use of such reagents in methods for producing antigen-binding molecules, such as those described herein.

[0179] In some embodiments, the reagent can inhibit the expression (gene expression or protein expression) of genes involved in initiating the primary humoral immune response. In some embodiments, the reagent can inhibit the expression (gene expression or protein expression) of genes involved in immunoglobulin isotype switching. In some embodiments, the reagent can inhibit the expression (gene expression or protein expression) of genes involved in stimulating the proliferation, differentiation, and / or activation of naïve B cells into antibody-secreting cells.

[0180] In some embodiments, the reagent can alter / destroy the nucleotide sequence of a target gene (i.e., a gene involved in triggering a primary immune response) or alter / destroy the nucleotide sequence required for target gene expression (e.g., regulatory sequences that regulate target gene expression, transcription factors).

[0181] 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 recombinases capable of altering / destructing the nucleotide sequence of a target gene (i.e., a gene involved in initiating a primary immune response).

[0182] In some embodiments, the animal contains an endogenous nucleotide sequence encoding a tamoxifen / 4-hydroxytamoxifen control system for controlling the activity of the recombinase.

[0183] In aspects and embodiments of using such systems, the agent capable of suppressing the primary immune response and / or promoting the secondary immune response may be tamoxifen / 4-hydroxytamoxifen. Similarly, methods involving such systems, as disclosed herein, may include administering tamoxifen / 4-hydroxytamoxifen to an animal to suppress the animal's ability to elicit a primary immune response.

[0184] In some embodiments, agents capable of suppressing the primary immune response and / or promoting the secondary immune response may be administered to animals multiple times. In some embodiments, the agents are administered at selected doses and / or frequencies that achieve the desired level of suppression of the primary immune response or the level of promotion of the secondary immune response.

[0185] For example, in an animal containing an endogenous nucleotide sequence that can be used to block the expression of one or more genes involved in initiating a primary humoral immune response via recombinase-mediated inhibition, and an endogenous nucleotide sequence for inducing recombinase expression or activity, the reagent can be administered multiple times to induce recombinase expression or activity to maintain a sufficient level of recombinase expression / activity, thereby persistently blocking the expression of one or more genes involved in initiating a primary humoral immune response. For example, Example 12 of this document describes the introduction of Cd40... flox / flox ;Cd79a + / CreERT2Mice were repeatedly administered tamoxifen to maintain the knockout of Cd40 gene expression in B cells.

[0186] In some embodiments, within the context of the methods described herein, an agent capable of suppressing the primary immune response and / or promoting the secondary immune response is administered to the animal more than once, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more times. Each administration of the agent is separated by a predetermined time interval, which may be 2 to 14 days, for example, 3 to 12 days, 5 to 10 days, or 5 to 9 days. In some embodiments, after the initial administration of the reagent to the animal, subsequent administrations may be given every 2 days, 3 days (±1 day), 4 days (±1 or 2 days), 5 days (±1 or 2 days), 6 days (±1 or 2 days), 7 days (±1, 2 or 3 days), 8 days (±1, 2 or 3 days), 9 days (±1, 2 or 3 days), 10 days (±1, 2 or 3 days), 11 days (±1, 2 or 3 days), 12 days (±1, 2, 3 or 4 days), 13 days (±1, 2, 3 or 4 days), or 14 days (±1, 2, 3 or 4 days).

[0187] nucleotide sequence Various aspects and embodiments of this disclosure relate to animals containing endogenous nucleotide sequences for inducing suppression of the primary humoral immune response.

[0188] As used herein, nucleotide sequences “for” inducing suppression of the primary humoral immune response may encode one or more factors that are involved in (e.g., desired or permitted) inducing suppression of the primary humoral immune response.

[0189] In some embodiments, the nucleotide sequence is used to site-specifically recombinase-mediatedly inhibit the expression of one or more genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) involved in triggering the primary humoral immune response.

[0190] In some embodiments, the nucleotide sequence is used for site-specific recombinase-mediated repression of one or both of the genes CD40 and / or CD40L.

[0191] Site-specific recombinase (SSR) systems for repressing gene expression are well known in the art. Such systems include, for example, the Cre-LoxP, Flp-FRT, and Dre-rox systems, described in Branda and Dymecki, Developmental Cell (2004) 6(1): 7-28, and Kim et al., Lab Anim Res. (2018) 34(4): 147-159, the entire contents of which are incorporated herein by reference. Variants of such SSR systems and other SSR systems are also well known in the art and can be similarly used to repress the expression of target genes.

[0192] Targeted disruption of nucleotide sequences can be achieved by providing the target sequences of recombinases on both sides (i.e., upstream / 5' and downstream / 3') of the entire or partial nucleotide sequence of a gene or the nucleotide sequence required for gene expression. In the presence of the corresponding recombinase, homologous recombination occurs between the target sequences, thereby disrupting the nucleotide sequence of the gene or the nucleotide sequence required for gene expression.

[0193] In the Cre-loxP system, Cre recombinases bind to inverted repeats of loxP target sequences and promote nucleotide recombination and excision between loxP target sequences. As used herein, “Cre recombinase” refers to any peptide / peptide having Cre recombinase catalytic activity. Cre recombinases may comprise the amino acid sequence of UniProtKB Q71TG5-1, v1, or an amino acid sequence with 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 UniProtKBQ71TG5-1, v1. Cre recombinases include, for example, fusion proteins of Cre recombinases (including CreERT and CreERT2 as described below).

[0194] 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 inversions, insertions, and translocations of nucleotide sequences.

[0195] SSR-mediated gene knockout, particularly using the Cre-loxP system, is described in Kim et al., Lab Anim Res. (2018) 34(4): 147-159, which is incorporated herein by reference.

[0196] It should be understood that alterations / disruptions made using an SSR system as described in this disclosure may suppress / prevent gene or protein expression. In some embodiments, alterations / disruptions made using an SSR system may suppress / prevent the production of products encoded by the unchanged nucleotide sequence of the gene.

[0197] In some embodiments, alterations / disruptions made using an SSR system reduce / prevent transcription of the gene by introducing an early stop codon into the transcribed sequence of 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.

[0198] In some embodiments, the nucleotide sequence encoding factors disclosed herein are used to disrupt the expression of one or more genes involved in triggering the primary humoral immune response in a site-specific recombinase-mediated manner.

[0199] In some embodiments, the nucleotide sequence comprises a target sequence of a recombinase located flanking all or part of the nucleotide sequence of a gene involved in inducing a primary humoral immune response. In some embodiments, the nucleotide sequence comprises a target sequence of a recombinase located flanking all or part of the nucleotide sequence required for gene expression.

[0200] In some embodiments, the target sequence of the recombinase is a loxP sequence, and the recombinase is a Cre recombinase. In some embodiments, 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.

[0201] In some embodiments, the nucleotide sequences flanking the recombinase target sequences are nucleotide sequences essential for the expression of the gene product. In some embodiments, the nucleotide sequences flanking the recombinase target sequences encode all or part of one or more exons of the gene. In some embodiments, the nucleotide sequences flanking the recombinase target sequences encode all or part of a regulatory sequence that regulates the expression of the gene, such as a promoter or enhancer.

[0202] It should be understood that "the target sequences of the recombinase are located on both sides of a specific nucleotide sequence" means that these target sequences are provided on both sides of that specific nucleotide sequence. That is, in the endogenous nucleotide sequences of this disclosure, they are located at the 5' end and 3' end of that specific nucleotide sequence. For example, the endogenous nucleotide sequences of this disclosure contain the following nucleotide sequence arrangements: 5'-[Target sequence of recombinase]-[Target gene nucleotide sequence / all or part of the nucleotide sequence required for expression of the target gene]-[Target sequence of recombinase]-3' In some embodiments, the target sequence of the recombinase may be located within approximately 5, 10, 50, 100, 250, 500, or 1000 bases of the first and / or last base of the target gene nucleotide sequence / nucleotide sequence required for target gene expression. That is, in some embodiments, the last base of the target sequence of the recombinase may be located within approximately 5, 10, 50, 100, 250, 500, or 1000 bases of the first base of the target gene nucleotide sequence / nucleotide sequence required for target gene expression, and / or the first base of the target sequence of the recombinase may be located within approximately 5, 10, 50, 100, 250, 500, or 1000 bases of the last base of the target gene nucleotide sequence / nucleotide sequence required for target gene expression.

[0203] In some aspects and embodiments, the endogenous nucleotide sequence of this disclosure further comprises a nucleotide sequence encoding a recombinase, i.e., a recombinase corresponding to the target sequence of the relevant recombinase. For example, in an embodiment where the target sequence of the recombinase is a loxP sequence, the endogenous nucleotide sequence further comprises a nucleotide sequence encoding a Cre recombinase.

[0204] The endogenous nucleotide sequence may further include a nucleotide sequence encoding a regulatory sequence (such as a promoter and / or enhancer) for recombinase expression. This regulatory nucleotide sequence is operatively linked to the nucleotide sequence encoding the recombinase. In some embodiments, the endogenous nucleotide sequence encodes an expression cassette for the recombinase.

[0205] In various aspects and embodiments of this disclosure, endogenous nucleotide sequences are used to induce suppression of the primary humoral immune response. Suppression of the primary humoral immune response is inducible, for example, in response to a specific chemical or physical treatment.

[0206] In embodiments using an SSR system, SSR-mediated gene expression repression can be induced by increasing the level or activity of the relevant recombinase, for example by administering the recombinase and / or increasing the expression of the recombinase.

[0207] In embodiments using endogenous nucleotide sequences, the endogenous nucleotide sequence contains a target sequence of a recombinase located flanking all or part of the nucleotide sequence of a gene involved in inducing a primary humoral immune response or flanking all or part of the nucleotide sequence required for gene expression. The expression of the relevant gene can be induced to be suppressed by administering the recombinase to the animal, administering the nucleic acid (such as a vector) encoding the recombinase to the animal, and / or treating the animal to increase the expression or activity of the recombinase.

[0208] It should be understood that increasing recombinase levels and / or recombinase activity in cells containing endogenous nucleotide sequences (which contain the target sequence of the recombinase located flanking the target nucleotide sequence) can promote recombination, thereby disrupting the target gene and subsequently inhibiting its expression.

[0209] In some embodiments, the upregulation of recombinase expression / activity is chemically induced. Chemically induced SSR-mediated gene knockout is described in Kim et al., Lab Anim Res. (2018) 34(4): 147-159 (incorporated above by reference).

[0210] In some embodiments, the endogenous nucleotide sequence encodes a conditional system for controlling the expression or activity of recombinases.

[0211] In aspects and embodiments of using such conditional systems, the reagents capable of inhibiting the primary immune response and / or promoting the secondary immune response may be reagents that induce recombinase expression or activity.

[0212] In some embodiments, the endogenous nucleotide sequence comprises a nucleotide sequence encoding a conditional expression system for controlling recombinase expression.

[0213] "Conditional expression," also referred to as "inducible expression" in this paper, refers to gene / protein expression that depends on specific conditions, such as the presence of specific reagents. Conditional expression systems are well-known in this field and were reviewed in Ryding et al., Journal of Endocrinology (2001) 171, 1-14, the entire contents of which are incorporated herein by reference.

[0214] Conditional expression systems include those that use tetracycline to control transcriptional activation, such as the Tet-On and Tet-Off systems.

[0215] The Tet-On system uses a nucleic acid encoding a reverse tetracycline transactivation (rtTA) protein, a fusion protein formed by a tetracycline repressor (TetR) protein and a VP16 activation domain, wherein mutations at four amino acid sites in the tetracycline repressor protein reverse the response to tetracycline / doxycycline. In the absence of tetracycline (or its derivatives such as doxycycline), rtTA does not bind to the TetO operator sequence, and the polypeptide is not expressed. In the presence of tetracycline / doxycycline, rtTA binds to the TetO sequence in TRE, activating transcription of downstream nucleic acids of the promoter. The Tet-On system is described in Das et al., Curr Gene Ther. (2016) 16(3):156-67 (the entire contents of which are incorporated herein by reference), including systems using optimized rtTA variants, such as the Tet-On Advanced system (using the rtTA variant protein rtTA2). s -M2) and Tet-On 3G system.

[0216] The Tet-On Advanced system is also described in Urlinger et al., Proc. Natl. Acad. Sci. USA (2000) 97(14):7963-8 (the entire contents of which are incorporated herein by reference), and the Tet-On 3G system is described in Zhou et al., Gene Ther. 13(19):1382-1390 (the entire contents of which are incorporated herein by reference).

[0217] The Tet-Off system uses a nucleic acid encoding a tetracycline transactivator (tTA) protein, a fusion protein formed by a tetracycline repressor and the VP16 activation domain of an HSV protein. In the absence of tetracycline (or its derivatives such as doxycycline), tTA binds to the TetO operator sequence, which constitutes the tetracycline response element (TRE) upstream of a minimal promoter (such as a CMV promoter). The tTA binds to the TetO sequence in the TRE, activating 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 Bujard et al., Proc. Natl. Acad. Sci. USA (1992) 89(12):5547-51 (the entire contents of which are incorporated herein by reference).

[0218] Other tetracycline control systems include the T-REx conditional expression system, described in Yao et al., Human Gene Therapy (1998) 9(13): 1939-1950 (the entire contents of which are incorporated herein by reference). In the T-REx system, TetR is expressed under the control of the CMV promoter; 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, its binding to TetR releases TetR from the TetO2 sequence, thereby relieving transcriptional repression in the target region.

[0219] In some embodiments, the endogenous nucleotide sequences of this disclosure comprise nucleotide sequences encoding elements of a system for conditional expression of recombinases. In some embodiments, the endogenous nucleotide sequences encode a tetracycline / doxycycline-regulated transcriptional activation system for controlling recombinase expression.

[0220] In aspects and embodiments of using such systems, the agent capable of suppressing the primary immune response and / or promoting the secondary immune response may be tetracycline / doxycycline. Similarly, methods of this disclosure involving such systems may include administering tetracycline / doxycycline to an animal to suppress the animal's ability to elicit a primary immune response.

[0221] In some embodiments, the endogenous nucleotide sequence encodes a conditional system for controlling recombinase activity.

[0222] In some embodiments, the recombinase encoded by the endogenous nucleotide sequence includes a portion for inducing regulation of 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 controlling the recombinase's contact with the target sequence.

[0223] The systemic description of inducible recombinase activity is found in Kim et al., Lab Anim Res. (2018) 34(4): 147-159 (incorporated above by reference). The tamoxifen-inducible Cre system employs a fusion protein containing a Cre recombinase fused to an estrogen receptor (ER-LBD) with a mutant ligand-binding domain, termed CreER recombinase. CreER is normally localized in the cytoplasm of cells expressing this fusion protein in the form of HSP90 binding. However, binding to synthetic steroids (such as tamoxifen or 4-hydroxytamoxifen) disrupts the interaction between HSP90 and CreER, allowing CreERT (i.e., CreER-tamoxifen) to translocate to the nucleus, where it binds to the 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 than CreER.

[0224] In some embodiments, nuclear translocation of the recombinase is inducible. In some embodiments, the recombinase comprises a portion that includes or is composed of an estrogen receptor (ER-LBD) having a mutant ligand-binding domain. 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.

[0225] In some embodiments, the endogenous nucleotide sequence encodes a tamoxifen / 4-hydroxytamoxifen control system for controlling recombinase activity.

[0226] In aspects and embodiments of using such systems, 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 elicit a primary immune response.

[0227] The expression of recombinases can be controlled by regulatory sequences that drive expression in hematopoietic cells, such as those driving expression in B-cell lineage cells. Therefore, in some embodiments, an endogenous nucleotide sequence encodes a recombinase that is controlled by a regulatory sequence (e.g., a promoter) driving expression in hematopoietic cells. In some embodiments, an endogenous nucleotide sequence encodes a recombinase that is controlled by a regulatory sequence (e.g., a promoter) driving expression in B-cell lineage cells.

[0228] In some embodiments, the expression of the recombinase may be controlled by cell type or tissue-specific regulatory sequences. For example, the expression of the recombinase may be controlled by cell type or tissue-specific promoters or enhancers. In this way, the expression of the recombinase and the resulting SSR-mediated gene knockout can be restricted to target cells or tissues. Therefore, in some embodiments, endogenous nucleotide sequences encoding recombinases are controlled by cell type or tissue-specific regulatory sequences, such as cell type or tissue-specific promoters.

[0229] In some embodiments, the endogenous nucleotide sequence encoding the recombinase is controlled by a hematopoietic cell or tissue-specific regulatory sequence, such as a hematopoietic cell or tissue-specific promoter. In some embodiments, the endogenous nucleotide sequence encoding the recombinase is controlled by a B-cell lineage-specific regulatory sequence, such as a B-cell lineage-specific promoter.

[0230] In some embodiments, the endogenous nucleotide sequence is controlled by the CD79A promoter encoding a recombinase (e.g., a Cre recombinase, such as CreERT2).

[0231] In some embodiments, the endogenous nucleotide sequence encodes a target sequence of a recombinase, said target sequence (e.g., a loxP sequence) located 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 selected from CD40 and / or CD40L.

[0232] In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences removes exons flanking the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences removes the complete coding sequence of the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences removes the transcription promoter of the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences removes / destroys one or more splice donor and / or acceptor sites encoded by the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences removes the translation start codon for RNA translation of the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences introduces frameshift mutations into the nucleotide sequence of the relevant gene. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences results in a truncated and / or nonfunctional form of the protein encoded by the relevant gene at that locus. In some embodiments, recombinase-mediated excision of regions flanked by recombinase target sequences results in nonsense-mediated degradation of RNA transcribed from that locus.

[0233] In some embodiments, the endogenous nucleotide sequence encodes the target sequence of the recombinase (e.g., loxP The target sequence is located flanking one or more exons of CD40. In some embodiments, the endogenous nucleotide sequence encodes the target sequence of the recombinase, which is located flanking one or more exons of CD40, specifically exons 2, 3, 4, and 5. In some embodiments, the endogenous nucleotide sequence encodes the target sequence of the recombinase, which is located flanking exons 2 through 5 of CD40. In some embodiments, recombinase-mediated excision of the region flanking the target sequence of the recombinase introduces a frameshift mutation in the nucleotide sequence encoding CD40. In some embodiments, recombinase-mediated excision of the region flanking the target sequence of the recombinase results in the CD40 locus encoding a truncated and / or nonfunctional form of CD40. In some embodiments, recombinase-mediated excision of the region flanking the target sequence of the recombinase results in nonsense-mediated degradation of RNA transcribed from the CD40 locus.

[0234] In some embodiments, the endogenous nucleotide sequence encodes a target sequence located flanking the region of CD40 as shown in SEQ ID NO:3. In some embodiments, the endogenous nucleotide sequence is used to cleave the region of CD40 as shown in SEQ ID NO:3.

[0235] In some embodiments, the endogenous nucleotide sequence comprises or consists of a nucleotide sequence having 60% or higher identity with the nucleotide sequence of 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% identity with the nucleotide sequence of SEQ ID NO:4. In some embodiments, the endogenous nucleotide sequence comprises or consists of the nucleotide sequence of SEQ ID NO:4.

[0236] In some embodiments, after recombinase-mediated excision, the CD40 locus comprises a nucleotide sequence with 60% or higher 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% nucleotide sequence identity to SEQ ID NO:5. In some embodiments, after recombinase-mediated excision, the CD40 locus comprises the nucleotide sequence of SEQ ID NO:5.

[0237] In some embodiments, the endogenous nucleotide sequence encodes a recombinase, such as a Cre recombinase. In a preferred embodiment, the Cre recombinase is CreERT2.

[0238] In some embodiments, the endogenous nucleotide sequence is controlled by a conditional system encoding a recombinase (e.g., a Cre recombinase, such as CreERT2) for regulating recombinase expression and / or activity. In some embodiments, the endogenous nucleotide sequence encodes a tamoxifen / 4-hydroxytamoxifen control system for regulating the activity of the recombinase.

[0239] In some embodiments, the expression of the recombinase is controlled by a regulatory sequence (e.g., a promoter) that drives expression in hematopoietic cells. In some embodiments, the expression of the recombinase is controlled by a regulatory sequence (e.g., a promoter) that drives expression in B-cell lineage cells. In some embodiments, the expression of the recombinase is controlled by the CD79A promoter.

[0240] In some embodiments, the endogenous nucleotide sequence comprises a nucleotide sequence with 60% or higher identity to the nucleotide sequence of SEQ ID NO:6, for example, with ≥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% identity to the nucleotide sequence of SEQ ID NO:4. In some embodiments, the endogenous nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:6.

[0241] animal This disclosure relates to animals used to produce antigen-binding molecules, wherein the primary immune response of the animal can be suppressed, as described herein.

[0242] Various aspects and embodiments of this disclosure relate to animals comprising endogenous nucleotide sequences for inducing suppression of the primary immune response. It should be understood that these endogenous nucleotide sequences are used to induce suppression of the primary humoral immune response in the animal.

[0243] In various aspects and embodiments of this disclosure, the animal may contain endogenous nucleotide sequences for inducing suppression of the primary humoral immune response as described in any of the embodiments herein.

[0244] The animals described in this disclosure can be individuals / subjects of any animal species. In a preferred embodiment, the animals are non-human animals.

[0245] The animal is preferably an individual / subject of a species commonly used to produce antibodies via 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).

[0246] In some embodiments, the animal is an individual / subject of a species belonging to the order Rodentia (e.g., Rhinoceros, Rat, or Guinea Pig) or Lagomorpha (e.g., Leporidae). In some embodiments, the animal is a mouse, rat, or rabbit.

[0247] In some embodiments, the animal is a mouse (i.e., in some embodiments, the animal is an individual / subject of a species of the genus *Mice*; for example, an individual / subject of the species *House Mouse*).

[0248] In some embodiments, the animal is a rat (e.g., an individual / subject of a species of the genus Rat; such as an individual / subject of a species of brown rat or black rat).

[0249] In some embodiments, the animal is a rabbit (e.g., an individual / subject of a species of the genus *Raphis*; e.g., an individual / subject of the species *Raphis*).

[0250] The animals described in this disclosure may have a genome containing a nucleotide sequence for inducing suppression of the primary humoral immune response. This nucleotide sequence for inducing suppression of the primary humoral immune response is preferably contained in the animal's genomic DNA. That is, the nucleotide sequence is integrated into or constitutes part of the genomic DNA of the animal's cells.

[0251] Animals as described in this disclosure may have a genome or contain genomic DNA containing nucleotide sequences for inducing suppression of the primary humoral immune response, or may contain endogenous nucleotide sequences for inducing suppression of the primary humoral immune response.

[0252] Animals as described in 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 for inducing suppression of the primary humoral immune response.

[0253] Animals as described in this disclosure may contain multiple (e.g., one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) endogenous nucleotide sequences, each conforming to the embodiments of endogenous nucleotide sequences described herein. In such embodiments, multiple endogenous nucleotide sequences can be used to inducibly inhibit the expression or activity of different genes / their products. For example, animals as described in this disclosure may contain endogenous nucleotide sequences for inducing inhibition of CD40 expression or the activity of its products.

[0254] In some embodiments, the animal comprises an endogenous nucleotide sequence for inducing inhibition of the expression of one or more genes involved in triggering a primary humoral immune response or the activity of their products. In some embodiments, the animal comprises an endogenous nucleotide sequence for inducing inhibition of the expression of one or both of CD40 and / or CD40L or the activity of their products.

[0255] In some embodiments, the animal contains endogenous nucleotide sequences for inducing knockout of one or more genes involved in triggering the primary humoral immune response. In some embodiments, the animal contains endogenous nucleotide sequences for inducing knockout of one or both of CD40 and / or CD40L.

[0256] As used herein, “inducible knockout” refers to induced gene knockout, such as in response to specific chemical or physical treatments. Inducible knockout can also be called “conditional knockout.” Inducible gene knockout technology is described in Kim et al., Lab Anim Res. (2018) 34(4): 147-159. Knockout can be induced by treatments that result in increased expression or activity levels of factors mediating gene knockout. For example, knockout can be mediated by site-specific recombinase (SSR) systems and can be induced by treatments that result in increased expression or activity levels of the relevant recombinase, which achieves gene knockout 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) that regulate expression in the target cell type or tissue.

[0257] In some embodiments, the animal described in this disclosure comprises an endogenous nucleotide sequence encoding a target sequence (e.g., a loxP sequence) of a recombinase located flanking one or more exons of one or more genes involved in initiating a primary humoral immune response. In some embodiments, the one or more genes involved in initiating a primary humoral immune response are CD40 and / or CD40L.

[0258] In some embodiments, the animal contains an endogenous nucleotide sequence encoding a target sequence (e.g., a loxP sequence) of a recombinase flanking one or more exons of CD40. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a target sequence of a recombinase flanking one or more exons of CD40, specifically exons 2, 3, 4, and 5. In some embodiments, the animal contains an endogenous nucleotide sequence encoding a target sequence of a recombinase flanking exons 2 through 5 of CD40. In some embodiments, recombinase-mediated excision of regions flanking the target sequence of the recombinase introduces a frameshift mutation in the nucleotide sequence encoding CD40. In some embodiments, recombinase-mediated excision of regions flanking the target sequence of the recombinase results in the CD40 locus encoding a truncated and / or nonfunctional form of CD40. In some embodiments, recombinase-mediated excision of regions flanking the target sequence of the recombinase results in nonsense-mediated degradation of RNA transcribed from the CD40 locus.

[0259] In some embodiments, the animal comprises an endogenous nucleotide sequence encoding a target sequence located flanking a region of CD40 as shown in SEQ ID NO:3. In some embodiments, the animal comprises an endogenous nucleotide sequence for excising a region of CD40 as shown in SEQ ID NO:3.

[0260] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or higher identity with the nucleotide sequence of SEQ ID NO:4, for example, having ≥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% identity with the nucleotide sequence of SEQ ID NO:4. In some embodiments, the animal contains an endogenous nucleotide sequence that comprises or is composed of the nucleotide sequence of SEQ ID NO:4.

[0261] In some embodiments, after recombinase-mediated excision, the animal comprises a nucleotide sequence with 60% or higher identity to the nucleotide sequence of 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% identity to the nucleotide sequence of SEQ ID NO:5. In some embodiments, after recombinase-mediated excision, the animal comprises the nucleotide sequence of SEQ ID NO:5.

[0262] In some embodiments, the animal described herein may contain one or more endogenous nucleotide sequences as described herein for inducing suppression of the primary immune response. In some embodiments, the animal may contain 1, 2, 3, 4, 5, 6, 7, 9, or 10 endogenous nucleotide sequences as described herein. In such embodiments, the plurality of endogenous nucleotide sequences may each independently conform to any of the embodiments of endogenous nucleotide sequences described herein. In some embodiments where the animal contains a plurality of endogenous nucleotide sequences as described herein, each endogenous nucleotide sequence may be used to induce suppression of different genes involved in triggering the primary humoral immune response (e.g., CD40 and / or CD40L).

[0263] In some embodiments, the animal described in this disclosure contains an endogenous nucleotide sequence encoding a recombinase (e.g., Cre recombinase). In a preferred embodiment, the Cre recombinase is CreERT2.

[0264] In some embodiments, the animals described in this disclosure contain an endogenous nucleotide sequence encoding a recombinase (e.g., a Cre recombinase, such as CreERT2), and the recombinase is controlled by a conditional system for regulating the expression and / or activity of the recombinase. In some embodiments, the animals of this disclosure contain an endogenous nucleotide sequence encoding an endogenous nucleotide sequence encoding a tamoxifen / 4-hydroxytamoxifen control system for regulating the activity of the recombinase.

[0265] In some embodiments, the expression of the recombinase in the animal is controlled by a regulatory sequence (e.g., a promoter) that drives expression in hematopoietic cells. In some embodiments, the expression of the recombinase in the animal is controlled 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 the animal is controlled by the CD79A promoter.

[0266] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or higher identity with the nucleotide sequence of SEQ ID NO:6, for example, having ≥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% identity with the nucleotide sequence of SEQ ID NO:6. In some embodiments, the animal contains an endogenous nucleotide sequence that comprises or is composed of the nucleotide sequence of SEQ ID NO:6.

[0267] Animals containing endogenous nucleotide sequences for inducing suppression of the primary humoral immune response may contain such endogenous nucleotide sequences, which are genetically engineered to contain such endogenous nucleotide sequences. Therefore, in some embodiments, the animal is a genetically engineered animal containing endogenous nucleotide sequences for inducing suppression of the primary humoral immune response. Genetically engineered animals may also be referred to as transgenic animals.

[0268] Methods of genetically engineering animals to contain desired nucleotide sequences are well known to those skilled in the art, as described, for example, in Huijbers, Methods Mol Biol (2017) 1642:1-19, Sumiyama et al., PLoS One (2018) 13(9):e0203056, and Asfaw et al., Cogent Food & Agriculture (2019) 5(1):1686802, all of which are incorporated herein by reference. Such methods include, for example, pronuclear microinjection (described in Pu et al., Methods Mol Biol (2019) 1874:17-41, all of which are incorporated herein by reference), and genetic modification of germ cells, zygotes, or embryos mediated by the SSN system (described in Lee et al., Drug Discovery Today: Disease Models (2016) 20: 13-20, all of which are incorporated herein by reference).

[0269] Methods for producing genetically engineered animals include, for example, transfecting a nucleic acid sequence into embryonic stem cells via homologous recombination to achieve genome integration, screening cells in which the nucleic acid sequence has been integrated into their genomic DNA, introducing the genetically modified embryonic stem cells into blastocysts, and implanting the blastocysts containing the genetically modified embryonic stem cells into the uterus to complete pregnancy.

[0270] The target nucleotide sequence can be prepared using recombinant DNA technology and introduced into cells / embryos via viral transduction, microinjection, electroporation, or other methods. Cells / embryos / animals containing the target nucleotide sequence can be identified using appropriate screening methods, such as Southern blotting or PCR.

[0271] The main obstacle to using monoclonal antibodies produced from immunized animals in humans lies in their xenogeneic origin. The host will mount an immune response to non-host antibodies, leading to clearance and potentially causing adverse side effects. Various methods have been employed to reduce or eliminate their immunogenicity, such as producing chimeric antibodies containing human Fc regions, and humanization, which involves modifying the variable region of the antibody to resemble a human antibody sequence.

[0272] Recently, transgenic technology has been applied, in which the endogenous immunoglobulin gene loci of animals are replaced by human homologous genes. Monoclonal antibodies produced from such mice using traditional hybridoma technology are fully human antibodies. Human Ig transgenic mouse strains such as Xenomouse (Abgenix; Green et al., Nat. Genet. (1994) 7:13-21; Green, J Immunol Methods (1999) 231(1-2):11-23), UltiMAb (Mederex; Lonberg and Huszar, Int Rev Immunol (1995) 13:65-93; Lonberg, Nat Biotechnol (2005) 23:1117-1125), and Velocimmune (Regeneron; Murphy, PNAS (2014) 111(14): 5153-5158) have produced a variety of human monoclonal antibodies with acceptable safety and efficacy approvals. Utilizing such antibody discovery platforms overcomes a major obstacle by reducing the immunogenicity of the produced antibodies while preserving mouse immunity. These mice were engineered to retain a strong B-cell response, and repeated immunization with human antigens resulted in a strong secondary immune response. They also have the ability to develop a diverse library of mAbs.

[0273] 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.

[0274] In some embodiments, the animal's genome encodes sequences of the human immunoglobulin VH region and / or VL region. In some embodiments, the animal's genome encodes sequences of the human immunoglobulin Fc region. In some embodiments, the animal's genome encodes sequences of the human immunoglobulin VH region, VL region, and / or Fc region.

[0275] Animals containing endogenous human immunoglobulin genes or gene segments can be used to produce antibodies containing fully human Fv (VH and VL regions). Transgenic mice encoding human immunoglobulin genes / gene segments were described in Lu et al., J Biomed Sci. 2020; 27: 1, and Brüggemann et al., Arch Immunol Ther Exp (Warsz). (2015) 63(2):101-108, including Xenomouse (Abgenix; Green et al., Nat. Genet. (1994) 7:13-21; Green, J Immunol Methods (1999) 231(1-2):11-23), UltiMAb (Mederex; Lonberg and Huszar, Int Rev Immunol (1995) 13:65-93; Lonberg, Nat Biotechnol (2005) 23:1117-1125), TransChromo TM Mice (Ishida et al., Cloning Stem Cells. (2002) 4:91-102) and Velocimmune ® (Regeneron Corporation; Murphy, PNAS (2014) 111(14): 5153-5158).

[0276] In some embodiments, the animals disclosed herein comprise endogenous nucleotide sequences encoding human immunoglobulin V, D, and / or J genes or segments thereof.

[0277] In some embodiments, the animals disclosed herein comprise endogenous nucleotide sequences encoding human immunoglobulin genes or gene segments encoded by animal genomes described in the following literature: Lu et al., J Biomed Sci. 2020; 27: 1; Brüggemann et al., Arch Immunol Ther Exp (Warsz). (2015) 63(2):101-108; Green et al., Nat. Genet. (1994) 7:13-21; Green, J Immunol Methods (1999) 231(1-2):11-23; Lonberg and Huszar, Int Rev Immunol (1995) 13:65-93; Lonberg, Nat Biotechnol (2005) 23:1117-1125; Ishida et al., Cloning Stem Cells. (2002). 4:91-102 or Murphy, PNAS (2014) 111(14): 5153-5158), and US 7,135,287 B1, US 7,105,348 B2 or US 2006 / 059575 A1, all of which are incorporated herein by reference in their entirety.

[0278] In some embodiments, the antibodies produced by the animal immune system of this disclosure comprise fully human VH and / or VL region sequences. In some embodiments, the antibodies produced by the animal immune system of this disclosure comprise fully human Fc region sequences. In some embodiments, the antibodies produced by the animal immune system of this disclosure comprise fully human VH, VL, and / or Fc region sequences. In some embodiments, the antibodies produced by the animal immune system of this disclosure comprise fully human amino acid sequences.

[0279] In some embodiments, the animal as described in this disclosure has been immunized with a first peptide / polypeptide 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 an initial immune response against the target amino acid sequence.

[0280] In some embodiments, the animals described in this disclosure are individuals / subjects of specific strains of mice. In some embodiments, the animals are C57BL / 6 mice, BALB / c mice, A / J mice, CD1 mice, ICR mice, 129S2 / SvPas mice, or FVB / N mice. The mouse strains mentioned in the preceding sentences are described in *The Jackson Laboratory Handbook on Genetically Standardized Mice*, 6th edition, October 2009, Jackson Laboratory, edited by Kevin Flurkey and Joanne M. Currer.

[0281] In some embodiments, the animal is a genetically engineered mouse (i.e., a transgenic mouse).

[0282] In some embodiments, the animal is a genetically engineered mouse whose endogenous immunoglobulin locus has been replaced with a human homolog. In some embodiments, the animal is a Xenomouse mouse, UltiMAb mouse, TransChromo mouse, or Velocimmune mouse. In some embodiments, the animal is Lu et al., J Biomed Sci. 2020; 27: 1; Brüggemann et al., Arch Immunol Ther Exp (Warsz). (2015) 63(2):101-108; Green et al., Nat. Genet. (1994) 7:13-21; Green, J Immunol Methods (1999) 231(1-2):11-23; Lonberg and Huszar, Int Rev Immunol (1995) 13:65-93; Lonberg, Nat Biotechnol (2005) 23:1117-1125; Ishida et al., Cloning Stem Cells. (2002) 4:91-102; Murphy, PNAS (2014) 111(14): 5153-5158; US Patent The mice described in US 7,135,287 B1, US 7,105,348 B2, or US 2006 / 059575 A1.

[0283] In some embodiments, the animal is a genetically engineered mouse with extended lifespan (i.e., compared to a mouse of the same kind lacking such genetic modification). Mice with genetic modifications that result in extended lifespan are described in Ladiges et al., Aging Cell. (2009) 8(4):346-52 (the entire contents of which are incorporated herein by reference); see Table 1 therein for details. In some embodiments, the animal is the mouse described in Table 1 of Ladiges et al., Aging Cell. (2009) 8(4):346-52. In some embodiments, the animal is an Ames Dwarf mouse, an αMUPA Tg mouse, or a p66shc mouse. - / - Mice, GHr / BP - / - Mice, Ghrhr lit / lit Mice, Snell Dwarf mice, Igf1r + / - Mice, FIRKO mice, Klotho Tg mice, Mit CAT Tg mice, MT Tg heart mice, UCP2 Tg brain mice, PappA - / - Mice, AC5 - / - Mice, Surf1 - / - Mice, PEPCK Tg muscle mice, Irs1 - / - Mice, Irs2 + / - Mice, Irs2 + / - Brain mice or IGF-1 Tg heart mice.

[0284] 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 a similar mouse lacking such genetic modification). In some embodiments, the animal is a mouse as described by Rithidech et al., Blood Cells Mol Dis. (1999) 25(1):38-45.

[0285] In some embodiments, the animal is a mouse with immune cell tolerance deficiency, such as a genetically engineered mouse with immune cell tolerance deficiency. In some embodiments, the animal is the mouse described in Khattri et al., J Immunol. (2001) 167(11):6312-6320.

[0286] In some embodiments, the animal is a chronically immune-activated mouse, such as a chronically immune-activated genetically engineered mouse. In some embodiments, the animal is a mouse as described in Subramanian et al., Proc Natl Acad Sci US A. (2006) 103(26):9970-9975.

[0287] In some embodiments, the animal is a mouse with an autoimmune dysregulation or hyperimmune phenotype, such as a genetically engineered mouse. In some embodiments, the animal is a mouse containing genetic variations that spontaneously generate an autoimmune dysregulation or hyperimmune phenotype. In some embodiments, the animal is a mouse with an autoimmune dysregulation or hyperimmune phenotype induced by chemical or peptide / peptide treatment. In some embodiments, the mouse is a NOD mouse, NZB / W F1 mouse, MRL mouse, BXSB mouse, LPR mouse, GLD mouse, Motheaten mouse, Scurfy mouse, Baff Tg mouse, Bcl2 Tg mouse, or Bim mouse. - / - Mice, C1qa - / - Mouse, C4 - / - Mice, Cd19Tg mice, Cd19 Cre -Traf3 fl / fl Mice, Cd22 - / - Mice, Cd40lTg 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, Ox40 Cre -Pten fl / fl Mice, p65P13K Tg mice, Pd1 - / - Mice, Pkbα Tg mice, Prkcd - / - Mice, Pten + / - Mice, Taci - / - Mouse or Tyro3 - / - Axl - / - Mertk - / - Mice.

[0288] 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 in Rajan et al., J Immunol (1996) 157 (2): 941-949. In some embodiments, the animal is a mouse with collagen-induced arthritis in a DBA / 1 background, as described in Courtenay et al., 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 in Van der Fits et al., J Immunol (2009) 182 (9): 5836-45.

[0289] 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 antigens than their counterparts lacking such genetic modifications. Genetically engineered hyperimmune mice include DiversimAb mice (Abveris) and DivergimAb mice (Abveris). In some embodiments, the animal is a DiversimAb mouse or a DivergimAb mouse.

[0290] The inventors have advantageously discovered that using hyperimmune mice to prepare the animals of the present invention helps to produce antibodies with high affinity and titer.

[0291] Therefore, in some embodiments, the animal is a hyperimmune mouse. The term "hyperimmune mouse" (also referred to as "autoimmune mouse") as used herein can refer to a mouse with a background or phenotype of hyperimmunity, autoimmune dysregulation, or a high immune response, and / or a strain of mice with hyperimmunity, autoimmune dysregulation, or a high immune response.

[0292] In some embodiments, the hyperimmune mouse is a genetically engineered mouse.

[0293] In some embodiments, the hyperimmune mouse may have one or more of the following characteristics: Compared with mice without a background or phenotype of hyperimmunity, autoimmune dysregulation, or a high immune response, they exhibit a higher immune response (or a strong / overactive adaptive response), characterized, for example, by an expanded initial B cell pool, enhanced primary or secondary immune responses after antigen immunization, or increased B cell activation after antigen immunization.

[0294] Compared with mice without hyperimmunity, autoimmune dysregulation, or a background or phenotype of high immune response, tolerance to antigens (self or exogenous) is altered, for example, characterized by increased retention of self-reactive B cells, uncontrolled stimulation and proliferation of self-reactive B cells, or negative selection for loss of self-reactivity resulting from somatic hypermutation.

[0295] B cells and T cells develop normally or nearly normally.

[0296] The lifespan is adapted to the cycle of antibody discovery activities.

[0297] A strong T-cell-dependent antibody response.

[0298] Expanded germinal centers and B cell populations.

[0299] It can produce autoantibodies that can switch classes.

[0300] High serum antibody titers can be generated when using autoepitaxes for immunization, such as epitopes or antigens that have high homology (>80%) with the self.

[0301] Immunization with weak immunogenic antigens can produce high serum antibody titers, meaning that such antigens cannot elicit a strong immune response in mice without hyperimmune, autoimmune dysregulation, or a background or phenotype of high immune response.

[0302] Therefore, in some embodiments, when immunized with self-epitopes or weakly immunogenic antigens, the animals (e.g., hyperimmunized mice) are able to produce strong antibody titers.

[0303] Hyperimmune mice can be generated using one or more of the following methods: Mutations that affect B cell activation, proliferation, and survival, such as altered BCR and co-receptor signaling or loss of Fas-FasL-dependent apoptosis (as described in mice by Miyamoto et al., Nature (2002) 416, 865-869 and Groom et al., J Clin Invest. (2002) 109(1):59-68).

[0304] For example, by negatively regulating cytokine signaling used for APC recruitment and migration (such as CCL2), or by altering antigen presentation processes through mutations in antigen processing genes (such as TAP1 or LMP2).

[0305] Impact T reg Cells and T FHMutations that activate and affect cell function, such as those that alter TCR signaling or lead to FOXP3-mediated loss of differentiation (as described in Zahorsky-Reeves and Wilkinson, European Journal of Immunology (2001) 31(1) 196-204).

[0306] Spontaneously acquired gene mutations that lead to an autoimmune phenotype, such as in NZB / W F1 mice.

[0307] 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 transgenic mice, Bcl2 transgenic mice, and Bim mice. - / - Mice, C1qa - / - Mouse, C4 - / - Mice, Cd19Tg mice, Cd19 Cre -Traf3 fl / fl Mice, Cd22 - / - Mice, Cd40lTg 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, Ox40 Cre -Pten fl / fl Mice, p65P13K Tg mice, Pd1 - / - Mice, Pkbα Tg mice, Prkcd - / - Mice, Pten + / - Mice, Taci - / - Mouse or Tyro3 - / - Axl - / - Mertk - / - Mice.

[0308] In some embodiments, the hyperimmunized mouse is an NZB / W F1 mouse. The NZB / W F1 mouse (also referred to herein as “NZBWF1” ​​or “NZBWF1 / J” mouse) is an F1 hybrid of New Zealand Black (NZB) and New Zealand White (NZW), described in Dubois et al., JAMA (1966) 195(4):285-289 and Bagavant et al., Autoimmun Rev. (2020) 19(2): 102686.

[0309] In some embodiments, the hyperimmune mouse comprises an endogenous nucleotide sequence for inducible knockout of CD40. In some embodiments, the mouse is an NZBWF1 / J mouse. In some embodiments, the mouse is a hyperimmune CD40 mouse. flox / flox Cd79a + / CreERT2 Mice. In some embodiments, the mice are NZBWF1 / J CD40. flox / flox ;Cd79a + / CreERT2 Mice.

[0310] In some embodiments, the animal is a humanized mouse. The humanized mouse has been transplanted with human cells or tissues. The humanized mouse may also be a transgenic mouse.

[0311] In some embodiments, the humanized mice are described in Chen and Murawsky, Front Immunol (2018), Vol. 9, and Rüker, F., Wozniak-Knopp G., eds., Introduction to Antibody Engineering, in Ma, B., Osborn, M. (2021), Transgenic Animals for the Generation of Human Antibodies, Life Science Learning Materials, Springer, Cham. https: / / doi.org / 10.1007 / 978-3-030-54630-4_5.

[0312] In some embodiments, the animal is a humanized hyperimmune mouse.

[0313] Production of antigen-binding molecules Various aspects of this disclosure relate to methods for inducing the production of antigen-binding molecules capable of binding to target proteins.

[0314] Various aspects of this disclosure relate to the generation of antigen-binding molecules, for example, for subsequent purification. Various aspects of this disclosure relate to the generation of cell populations that produce antigen-binding molecules.

[0315] As described herein, within the context of various aspects and embodiments of this disclosure, "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 described herein 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.

[0316] Cells capable of generating antigen-binding molecules / producing antigen-binding molecules for use in applications such as therapy, research, imaging, and / or diagnostics. The methods disclosed herein can be used to produce antigen-binding molecules having specific target properties relevant to therapeutic, research, imaging, and / or diagnostic applications.

[0317] In short, the method involves introducing a substance into an animal that can be recognized as foreign (i.e., non-host) by the animal's immune system, thereby causing the animal to selectively produce antibodies capable of binding to the substance. The substance may contain an antigen or may be processed into an antigen. The immune system's ability to produce antibodies that specifically bind to antigens can be used to generate antibodies for detecting target molecules in various research, diagnostic, imaging, therapeutic, and preventative applications.

[0318] Methods for producing antibodies are well known in the art, for example, as described in *Antibodies: A Laboratory Manual*, 2nd edition, 2014, by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, the entire contents of which are incorporated herein by reference. In particular, Chapter 6 details the methods for producing antibodies by immunizing animals.

[0319] In particular, various aspects of the methods disclosed herein relate to the generation of monoclonal antibodies. Such methods may include isolating cells from a subject that generate antigen-binding molecules. Such methods may include generating monoclonal hybridomas from cells isolated from a subject, wherein the hybridomas generate a single type (i.e., antibodies with single specificity).

[0320] Methods for producing antibodies involve introducing an antigen into an animal to induce antibody production, the antibody of which can be recovered from the animal.

[0321] Various aspects and embodiments of the methods disclosed herein include: (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 animals to suppress their ability to elicit a primary immune response; and (iii) Administering the animal a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide contains the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

[0322] In some embodiments, the treatment of the animal to suppress its ability to elicit a primary immune response is carried out after a period of time sufficient for the cells (or derived cells) activated / stimulated by the administration of the first peptide / peptide to complete immunoglobulin isotype conversion (i.e., conversion to cells expressing IgG, IgE, or IgA). In some embodiments, the treatment of the animal to suppress its ability to elicit a primary immune response is carried out after a period of time sufficient for the cells activated / stimulated by the administration of the first peptide / peptide (or derived cells) to differentiate into plasma B cells and / or memory B cells.

[0323] In some embodiments, a method for producing antigen-binding molecules includes one or more of the following steps: Prepare / formulate peptides / polypeptides / nucleic acids / cells for introduction into animals; Introducing peptides / polypeptides / nucleic acids / cells into animals; Detection and / or monitoring of the production of antigen-binding molecules in animals; Detect and / or monitor the production of cells in animals that express / contain antigen-binding molecules; Collect antigen-binding molecules produced by animals; Isolation / purification of antigen-binding molecules produced by animals; Collect cells from animals that produce antigen-binding molecules; Isolate / purify cells that produce antigen-binding molecules from animals; 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.

[0324] 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 collected from the animal's blood (e.g., PBMCs obtained from blood) or from an animal's organ (e.g., spleen). In some embodiments, the isolated cells that produce antigen-binding molecules are cultured in vitro. In some embodiments, the method includes culturing cells isolated from a subject in vitro.

[0325] 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 may be recovered from, for example, animal blood, plasma, serum, or ascites fluid.

[0326] In some embodiments, antigen-binding molecules can be isolated from cells obtained from animals. In some embodiments, the cells are B cells. In some embodiments, antigen-binding molecules can be isolated from the cell culture supernatant of B cells cultured in vitro.

[0327] In some embodiments, the antigen-binding molecule is obtained from a hybridoma capable of producing an antigen-binding molecule that can bind 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 prepared as described in this 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.

[0328] Methods for isolating (i.e. purifying) antigen-binding molecules from samples containing antigen-binding molecules (e.g., cells, cell lysates, cell culture media, blood, plasma, serum, ascites) are well known to those skilled in the art and are described in detail in *Antigen-binding molecules: A Laboratory Manual*, 2nd edition, 2014, Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated above by reference), particularly 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.

[0329] The isolated or purified antigen-binding molecule used in this article refers to a composition containing an antibody, wherein at least 80%, 90%, 95%, 99%, or 100% (by weight, or by weight of the protein component in the composition) is an antigen-binding molecule component of the composition.

[0330] In some embodiments, the methods of this disclosure employ single B-cell cloning techniques. Single B-cell cloning techniques for producing monoclonal antibodies are described in Carbonetti et al., J Immunol Methods (2017) 448: 66-73 and Lei et al., Front Microbiol (2019) 10:672, both of which are incorporated herein by reference in their entirety. Such methods typically involve culturing B cells obtained from an animal as monoclonal cells in vitro, for example, in the presence of factors that promote B-cell proliferation and / or antibody production by B cells. B cells can be obtained from animal blood (e.g., PBMC populations derived from animal blood) or from animal organs (e.g., spleen). B cells sorted using FACS or other cell sorting techniques can be cultured as single cells. B cells expressing antibodies with the target properties are sequenced to determine the amino acid sequence of the antibody and / or the nucleic acid sequence encoding the antibody.

[0331] Methods for generating hybridomas are well known to those skilled in the art, for example, as described in *Antibodies: A Laboratory Manual*, 2nd edition, 2014, Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated above by reference), particularly Chapter 7. In short, an animal is immunized as described in this disclosure to stimulate an adaptive immune response, followed by the isolation of B lymphocytes from that animal and their fusion with a suitable myeloma cell line to generate a hybridoma.

[0332] Prior to hybridoma preparation, the yield of antigen-binding molecules (e.g., antigen-binding molecule titer) produced by the subject is determined as described herein. If the titer is too low, one or more enhancement steps may be performed as described herein, and the yield of antigen-binding molecules may be monitored (e.g., by repeated blood sampling) until a sufficiently high titer is achieved. In some embodiments, the method includes screening test animals for hybridoma production based on the yield or titer of antigen-binding molecules. The yield or titer of the antigen-binding molecules can be detected in blood, plasma, serum, or ascites samples obtained from said animals.

[0333] In some embodiments, subjects and / or cells may be screened based on a method for producing antigen-binding molecules, said method being based on detecting the generation of antigen-binding molecules that can bind to target proteins.

[0334] In some embodiments, collecting cells that produce antigen-binding molecules includes collecting the spleen and / or lymph nodes of a test animal. In some embodiments, preparing a hybridoma includes fusing antigen-binding molecule-producing cells (e.g., B cells) obtained from a subject with myeloma cells. In some embodiments, fusing antigen-binding molecule-producing cells (e.g., B cells) obtained from a subject with myeloma cells includes co-centrifugation in polyethylene glycol (PEG). In some embodiments, preparing a hybridoma includes culturing cells in a selective medium (e.g., a medium containing hypoxanthine-aminopterin-thymidine) for screening.

[0335] As described herein, the production of antibodies that bind to the target protein and / or other peptides / peptides can be tested, for example, by immunoprecipitation, immunoblotting, or in vitro binding assays (such as flow cytometry, ELISA, etc.). In some embodiments, antibody yield or antibody titer can be determined in the cell culture supernatant of in vitro cultured hybridomas.

[0336] This disclosure also provides an antigen-binding molecule capable of binding to a target protein, wherein the antigen-binding molecule is obtained or is available by the methods for producing antigen-binding molecules described herein.

[0337] 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., tablet). Liquid formulations may be formulated for administration to selected areas of a human or animal body via injection or catheter. This disclosure also provides a pharmaceutical composition prepared by methods as described herein.

[0338] Antigen-binding molecules produced by the methods disclosed herein can be mass-produced using methods known to those skilled in the art.

[0339] Hybridomas can be expanded in vitro using standard cell culture methods, or in vivo, such as in the ascites fluid of a host animal. In some embodiments, the methods of this disclosure include expanding hybridomas by in vitro cell culture. In some embodiments, the methods include expanding hybridomas in vivo by injecting the hybridoma into a host animal.

[0340] In some embodiments, antigen-binding molecules may be prepared using recombinant DNA techniques known to those skilled in the art. For example, a polynucleotide encoding the antibody may be obtained from B cells or hybridoma cells that produce the antibody by reverse transcription PCR (RT-PCR) using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and the sequence of the polynucleotide may be determined. The isolated polynucleotides encoding the heavy and light chains may be cloned into a suitable expression vector, and when the vector is transfected into host cells (such as E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulin proteins), monoclonal antibodies may be produced.

[0341] Target amino acid sequence As used herein, “target amino acid sequence” means: (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)).

[0342] As used herein, an amino acid sequence “similar” to a reference amino acid sequence refers to an amino acid sequence that shares certain characteristics with a reference amino acid sequence. In some embodiments, an amino acid sequence “similar” to a reference amino acid sequence means that the protein containing that amino acid sequence is a subtype, variant, or homolog of the protein containing the reference amino acid sequence. In some embodiments, the sequence identity of an amino acid sequence “similar” to a reference amino acid sequence (e.g., the amino acid sequence of a target protein) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the reference amino acid sequence. In some embodiments, the 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.

[0343] Conversely, an amino acid sequence "dissimilar to" a reference amino acid sequence refers to an amino acid sequence with less than 100% sequence identity to 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, an amino acid sequence "dissimilar to" a 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.

[0344] Comparing a given amino acid sequence (i.e., the query sequence) with a reference amino acid sequence can be achieved by sequence alignment and comparing amino acids at corresponding positions. In some embodiments, sequence alignment is performed within regions of 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 both the query and reference sequences.

[0345] 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 together 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 linked together. The length of a polypeptide is typically greater than about 50 amino acids.

[0346] References to peptides and polypeptides in this article also include complexes containing such peptides / polypeptides, which can be homologous or heterologous polymeric complexes containing two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) peptides / polypeptides (e.g., formed through non-covalent interactions).

[0347] The target protein can be any protein. For example, the target protein can be a protein relevant to diagnosis, prognosis, imaging, or treatment. In some embodiments, the target protein can be a candidate therapeutic target for an antigen-binding molecule. As used herein, “target protein” means “one or more target proteins.” That is, an antibody capable of binding a protein may bind to more than one target protein. The reference to “target protein” in this document also includes target protein complexes, which can be homologous or heterologous polymeric complexes (e.g., formed through non-covalent interactions) comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) polypeptides.

[0348] In some embodiments, the target protein is a protein whose expression / activity or upregulation of 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 cells in a pathogen / infectious agent, cell, or tissue that are expected to be destroyed or eliminated. In some embodiments, the target protein is expressed by a pathogen / infectious agent, cell, or tissue cell that is expected to elicit a humoral immune response. In some embodiments, the target protein is associated with cancer, infectious disease, or 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, infectious agents, cells infected by infectious agents, or autoimmune effector cells (i.e., effector cells of autoimmune pathology). In some embodiments, the target protein is a variant of a protein associated with a disease / condition (e.g., cancer-related and / or autoimmune disease-related).

[0349] In some embodiments, the antigen-binding molecule generated according to the method described herein can recognize a protein associated with the target protein.

[0350] "A protein associated with a target protein" refers to a protein that shares certain characteristics with a reference target protein. For example, the methods disclosed herein can be used to induce antigen-binding molecules capable of recognizing variants of proteins that are pathogens / infectious agents. That is, the methods can be used to induce broadly neutralizing antigen-binding molecules. In some embodiments, the protein associated with a target protein comprises the target amino acid sequence. In some embodiments, the protein associated with a target protein comprises the amino acid sequence of the target protein.

[0351] Related proteins include isotypes, fragments, variants, or homologs of the target protein (including proteins that are members of the same protein family, such as paralogs and orthologs). An isotype, fragment, variant, or homolog of a specific target protein is optionally characterized as having an amino acid sequence identity of at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with the amino acid sequence of the reference protein.

[0352] As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in the target sequence that are identical to those in the reference sequence after sequence alignment (if necessary, vacancies are introduced 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 known to those skilled in the art, such as using publicly available computer software, such as ClustalOmega (Söding, J., 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer, 2005, BMC Bioinformatics, 6(298)) and MAFFT software (Katoh and Standley, 2013, Molecular Biology and Evolution, 30(4) 772-780). When using such software, it is preferable to use the default parameters, such as open shot penalty and extension penalty.

[0353] In some embodiments, the target amino acid sequence may be a shared sequence or a majority sequence in corresponding regions of multiple related proteins. In some embodiments, the target amino acid sequence may be a shared sequence in regions corresponding to the amino acid sequence of the target protein in isotypes, homologs, or variants of two or more target proteins.

[0354] 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., a B cell and / or T cell-mediated immune response). 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.

[0355] In some embodiments, the target amino acid sequence is a continuous amino acid sequence of the target protein, or a similar sequence thereof. In some embodiments, such as in instances where the target amino acid sequence folds to form a discontinuous epitope, the target amino acid sequence is a discontinuous amino acid sequence of the target protein, or a similar sequence thereof.

[0356] 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, when folded, provides a discontinuous epitope. In some embodiments, the target amino acid sequence is a discontinuous amino acid sequence that collectively forms the discontinuous epitope.

[0357] In some embodiments, the target amino acid sequence is a continuous amino acid sequence of the target protein, or a similar sequence thereof. In some embodiments, the target amino acid sequence is a discontinuous amino acid sequence of the target protein, or a similar sequence thereof. In some embodiments, the target amino acid sequence is a discontinuous amino acid sequence of the target protein complex, or a similar sequence thereof. In some embodiments, the target amino acid sequence is a discontinuous amino acid sequence composed of the amino acid sequences of two or more polypeptides in the target protein complex, or a similar sequence thereof.

[0358] In some embodiments, the target amino acid sequence has a length of 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 has a length of 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 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 length. In some embodiments, the target amino acid sequence is 20 to 100, 20 to 50, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 amino acids in length. In some embodiments, the target amino acid sequence is 5 to 30 amino acids in length.

[0359] When the target amino acid sequence provides or is predicted to provide a discontinuous epitope, the target amino acid sequence may also refer to the continuous amino acid sequence that folds to form the discontinuous epitope, or to the discontinuous amino acid sequence that constitutes the discontinuous epitope.

[0360] 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 comprise, for example, 2, 3, 4, 5, 6, or 7 discontinuous amino acid sequences. Each such discontinuous sequence can contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids, but is preferably less than, for example, 30, 25, 20, or 15 amino acids.

[0361] Technicians can identify the antigenic sequence of a target protein using methods known in the art. For some proteins, known or predicted antigenic amino acid sequences are stored 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 identified through literature searches, such as using the internet.

[0362] The amino acid sequence that can form a B-cell epitope and thus potentially elicit an effective antibody can be predicted from a query sequence using a variety of methods. Technicians can predict whether an amino acid sequence is antigenic by comparing it to known or predicted antigenic sequences (e.g., against other proteins) and / or based on the characteristics of that amino acid sequence. See, for example, El-Manzalawy and Honavavar, Immunome Res, 2010, 6(Suppl 2): ​​S2, the entire contents of which are incorporated herein by reference. Such methods take into account, for example, the hydrophilicity, flexibility, accessibility, tortuosity, exposed surface area, polarity, and antigenicity of the amino acid sequence. Whether a peptide / peptide contains a T-cell epitope can be determined, for example, by methods described in Desai et al., 2014, Methods Mol Biol 1184:333-364.

[0363] 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 antigenic sequences includes EMBOSS:antigenic, BepiPred, IEDB Analysis Resource, SVMTriP, SCRATCH, ElliPro, COBEPro, BEPro, PEPITO, and DiscoTope.

[0364] The antigenic sequence of a target protein can also be identified experimentally by determining whether an amino acid or amino acid sequence is antigenic. For example, those skilled in the art can determine whether a given amino acid sequence is antigenic by immunizing a subject (e.g., a mammal) with a peptide containing that amino acid sequence and determining whether an adaptive immune response is elicited. In the methods disclosed herein, one or more of the above methods can be used alone or in combination to identify the antigenic amino acid sequence of a target protein.

[0365] When assessing whether an amino acid sequence is similar to a reference amino acid sequence of a target protein that provides or is predicted to provide a discontinuous epitope, the amino acid sequence can be compared with the continuous amino acid sequence that folds to form the discontinuous epitope, or with the discontinuous amino acid sequence that together constitutes the discontinuous epitope.

[0366] 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.

[0367] It should be understood that the first peptide / polypeptide encompasses peptides / polypeptides that contain or consist of the following sequences: (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)

[0368] In some embodiments, the first peptide / peptide comprises a target amino acid sequence and also comprises other amino acids. In some embodiments, the first peptide / peptide comprises a target amino acid sequence and additionally comprises 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, or 1-50 amino acids at one or both ends (i.e., the N-terminus or C-terminus) of the target amino acid sequence.

[0369] In some embodiments, where the target amino acid sequence is derived from a protein, these additional amino acids correspond to amino acids at corresponding positions in the target amino acid sequence. For example, if the target amino acid sequence corresponds to amino acids 20 to 30 of the target protein amino acid sequence, and the first peptide / polypeptide comprises the target amino acid sequence and 5 additional amino acids located at the N-terminus of the target amino acid sequence, then these 5 additional amino acids may correspond to positions 15 to 19 of the target protein amino acid sequence.

[0370] The first peptide / polypeptide may contain more than one peptide chain. For example, the first peptide / polypeptide may be a peptide / polypeptide complex, such as containing 2, 3, 4, 5, or 6 or more peptides / polypeptides. The peptides / polypeptides may be coupled to each other or may be administered as a composition (e.g., a mixture) of two or more uncoupled peptides / polypeptides.

[0371] In some embodiments, the first peptide / peptide is provided in a form conjugated with a carrier protein. As used herein, a "carrier protein" refers to a protein used to elicit an immune response against the peptide / peptide it is conjugated with (i.e., the peptide / peptide it serves as a "carrier"). Due to their size and complexity, carrier proteins can induce an immune response including against the conjugated peptide / peptide. Many proteins can be used as carriers and can be selected based on immunogenicity, solubility, and the availability of useful functional groups that can be used to conjugate the target peptide / peptide. Carrier proteins are well known in the field of immunology, for example, as described in Thermo Scientific Pierce Antibody Production and Purification Technical Handbook, Second Edition (2010), Thermo Scientific, Inc., USA (1601975 09 / 10), the entire contents of which are incorporated herein by reference.

[0372] In some embodiments, the carrier protein is selected from keyhole cyanin (KLH), brown shell hemocyanin (CCH; also known as blue carrier protein), bovine serum albumin (BSA), cationic BSA (cBSA), hepatitis B core antigen (HBc), thyroglobulin, and ovalbumin (OVA). In some embodiments, the carrier protein is selected from KLH, BSA, HBc, and OVA.

[0373] Peptides / peptides can be coupled to carrier proteins using methods well known to those skilled in the art, including amine-thiol crosslinking (e.g., using succinimide-6-((β-maleimide propionamido)hexanoate)SMPH), EDC coupling (carboxyl and amino crosslinking), maleimide coupling (thiol crosslinking), and glutaraldehyde coupling (amine-amine crosslinking) (see Thermo Scientific Pierce Antibody Production and Purification Technical Handbook, 2nd Edition (2010), Thermo Scientific, USA (1601975 09 / 10)).

[0374] 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 at least one 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, for example, present in a mixture or coupled with another. “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 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides. "First peptide / polypeptide" or "nucleic acid encoding first peptide / polypeptide" can be a mixture of 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 nucleic acids encoding peptides / polypeptides.

[0375] As described in this article, two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially.

[0376] Second peptide / polypeptide The methods disclosed herein include administering a second peptide / polypeptide, or a nucleic acid encoding the 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.

[0377] In some embodiments, the second peptide / polypeptide is the same as the first peptide / polypeptide. In some embodiments, the second peptide / polypeptide is different from the first peptide / polypeptide.

[0378] It should be understood that a peptide / peptide "different" from a reference peptide / peptide comprises an amino acid sequence with less than 100% sequence identity to the reference peptide / peptide. In some embodiments, the second peptide / peptide has an amino acid sequence with less than 100% sequence identity to the first peptide / peptide, for example, less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%. In some embodiments, the second peptide / peptide has an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the first peptide / peptide.

[0379] It should be understood that the second peptide / peptide encompasses peptides / peptides containing the following sequences: (a) the target amino acid sequence contained in the first peptide / peptide, or (b) an amino acid sequence similar to the target amino acid sequence contained in the first peptide / peptide.

[0380] It should also 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) the amino acid sequence similar to the amino acid sequence of (i).

[0381] In some embodiments, the second peptide / peptide comprises a target amino acid sequence and also comprises other amino acids. In some embodiments, the second peptide / peptide additionally comprises at least 5, 10, 15, 20, 40, 50, 80, 100, 200, or 300 amino acids at one or both ends (i.e., the N-terminus or C-terminus) of the target amino acid sequence. In some embodiments, the second peptide / peptide additionally comprises at least 1-5, 1-10, 1-15, 1-20, 1-40, 1-50, 1-80, 1-100, 1-200, or 1-300 amino acids at one or both ends (i.e., the N-terminus or C-terminus) of the target amino acid sequence.

[0382] The second peptide / polypeptide may contain more than one peptide / polypeptide chain. For example, the second peptide / polypeptide may be a peptide / polypeptide complex, such as containing 2, 3, 4, 5, or 6 or more peptides / polypeptides. The peptides / polypeptides may be coupled to each other or may be applied as a composition (e.g., a mixture) of two or more uncoupled peptides / polypeptides.

[0383] The peptides / polypeptides and nucleic acids described herein can be administered in the form of cells containing / expressing the peptide / polypeptide / nucleic acid, or in the form of synthetic formulations containing the peptide / polypeptide / nucleic acid.

[0384] In some embodiments, the second peptide / polypeptide may further comprise one or more amino acids located at one or both ends of the second peptide / polypeptide. In some embodiments, the second peptide / polypeptide may further comprise 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 or both ends, or comprise 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4 or 1-3 amino acids.

[0385] In some embodiments, the second peptide / polypeptide can induce the generation of an antigen-binding molecule that can bind to a target protein as well as isotypes, variants or homologs of the target protein.

[0386] 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 at least one 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, present in a mixture or coupled with another. “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 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids encoding 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides. "Second peptide / polypeptide" or "nucleic acid encoding second peptide / polypeptide" can be a mixture of 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 nucleic acids encoding peptides / polypeptides.

[0387] As described in this article, two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially.

[0388] Nucleic acids encoding peptides / polypeptides As described herein, the peptides / polypeptides of this disclosure can be expressed by a nucleic acid encoding the peptide / polypeptide. This nucleic acid can be or is contained in a vector. In some embodiments, the nucleic acid can be DNA encoding the peptide / polypeptide described herein.

[0389] Nucleic acids / vectors can be administered to subjects to express peptides / polypeptides as described herein. The nucleic acids / vectors may be present in cells, and these cells may be administered to animals. The nucleic acids / vectors may be integrated into the genome of a cell, and these cells may be administered to animals. The nucleic acids / vectors can be used for recombinant expression of peptides / polypeptides as described herein.

[0390] As used herein, a “vector” is a nucleic acid molecule used as a means of transferring exogenous nucleic acids into cells. This vector can be a vector that expresses nucleic acids in cells. Such vectors may include a promoter sequence operatively linked to a nucleotide sequence encoding the sequence to be expressed. Vectors may also include a stop codon and an expression enhancer. Peptides or polypeptides can be expressed from vectors as described in this disclosure using any suitable vector, promoter, enhancer, and stop codon known in the art. The term “operatively linked” includes the covalently linking of a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) such that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thus forming an expression cassette). Thus, if the regulatory sequence can influence the transcription of the nucleic acid sequence, then 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 murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0391] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements required for protein expression in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, such as a vector containing a cytomegalovirus (CMV) promoter or an SV40 promoter to drive protein expression.

[0392] Methods for immunizing subjects with nucleic acids encoding target peptides / peptides to elicit antibody responses are described, for example, in Aurisicchio et al., (2012) J Cell Physiol 227: 3381-3388, the entire contents of which are incorporated herein by reference.

[0393] In some embodiments of the methods disclosed herein, nucleic acids encoding peptides / polypeptides as described herein are administered to animals. In such embodiments, the peptide or polypeptide is expressed in the animal following immunization.

[0394] application Various aspects of this disclosure relate to administering peptides / polypeptides or nucleic acids encoding peptides / polypeptides to animals. Various aspects of this disclosure also include administering agents to animals for inducing inhibition of the animal's ability to elicit a primary immune response and / or promoting the animal's ability to elicit a secondary immune response.

[0395] The step of “administering” a peptide / polypeptide / nucleic acid to an animal includes administering the peptide / polypeptide / nucleic acid to the animal once or multiple times. In some embodiments, “administering” a peptide / polypeptide / nucleic acid includes administering the peptide / polypeptide / nucleic acid to the animal in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses.

[0396] For example, in some embodiments, the method includes administering a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) to an animal in two separate doses, as defined herein, and administering a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) to the animal in two separate doses.

[0397] In some embodiments, the method includes administering a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) to an animal in 3, 4, 5, or 6 doses, and administering a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) to the animal in 1, 2, 3, or 4 doses.

[0398] In some embodiments, the method includes administering a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) to an animal in two separate administrations and administering a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) to the animal in one separate administration.

[0399] Similarly, the step of “administering” an agent that causes suppression of the primary immune response and / or promotion of the secondary immune response includes administering the agent to the animal once or multiple times. In some embodiments, “administering” the agent includes administering the agent to the animal in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses.

[0400] In some embodiments, the method includes administering a reagent as defined herein to the animal in 1, 2, 3, or 4 separate doses.

[0401] In some embodiments, in a dosing step involving multiple administrations, the time interval between each individual administration is at least 24 hours, 36 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 7 days, 10 days, or 12 days. In some embodiments, in a dosing step involving multiple administrations, the time interval between each individual administration is approximately 5-30 days, 7-20 days, for example, approximately 10-16 days. In some embodiments, in a dosing step involving multiple administrations, the time interval between each individual administration is approximately 2-30 days, 5-20 days, for example, approximately 6-8 days.

[0402] Dosing according to the dosing procedure described herein may use the same materials or different materials, provided that these materials meet the requirements for materials required for dosing according to this dosing procedure.

[0403] For example, administering a first peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as defined herein may include administering the same peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) simultaneously or sequentially, or administering two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different peptides / polypeptides, each of which independently satisfies the requirements of a first peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein. Similarly, administering a second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as defined herein may include administering the same peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) simultaneously or sequentially, or administering two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different peptides / polypeptides, each of which independently satisfies the requirements of a second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein.

[0404] A single dose as described herein can be administered sequentially. That is, in some embodiments, a peptide / polypeptide / nucleic acid is first administered to the subject, followed by a separate dose at a given time interval.

[0405] In some embodiments, the peptide / polypeptide may be administered in the form of a reagent comprising or expressing the peptide / polypeptide. In some embodiments, the peptide / polypeptide may be administered in the form of a reagent comprising a nucleic acid encoding the peptide / polypeptide. In some embodiments, the nucleic acid may be administered in the form of a reagent comprising the nucleic acid.

[0406] 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 particle.

[0407] In some specific embodiments, the peptide / polypeptide may be administered in the form of a cell containing or expressing the peptide / polypeptide. In some embodiments, the peptide / polypeptide may be administered in the form of a cell containing a nucleic acid encoding the peptide / polypeptide. In some embodiments, the nucleic acid may be administered in the form of a cell containing the nucleic acid.

[0408] The cells expressing the peptide / polypeptide can express the peptide / polypeptide endogenously. That is, the peptide / polypeptide can be encoded and / or expressed by the cell's nucleic acids before any nucleic acid encoding the peptide / polypeptide is introduced into the cell. The cells expressing the peptide / polypeptide can also express the peptide / polypeptide exogenously. That is, the peptide / polypeptide can be encoded and / or expressed by nucleic acids already introduced into the cell. In some embodiments, the cells expressing the peptide / polypeptide may have been modified or engineered to express or overexpress the peptide / polypeptide.

[0409] In some embodiments, the applied peptide / polypeptide / nucleic acid / cell / reagent is isolated or purified. As used herein, isolated or purified peptide / polypeptide / nucleic acid / cell / reagent refers to a composition in which at least 80%, 90%, 95%, 99%, or 100% (by weight) is a peptide / polypeptide / nucleic acid / cell / reagent component.

[0410] In some embodiments, the peptide / polypeptide / nucleic acid is administered in the form of a cell or a protein-containing extract.

[0411] Peptides / polypeptides / nucleic acids / cells / reagents can be administered to subjects in any suitable manner, such as the methods described in *Antibodies: A Laboratory Manual*, 2nd edition, 2014, by Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated above by reference), particularly Chapter 6.

[0412] The materials administered to animals can be appropriately formulated based on the material itself, the route of administration, the animal species, and the expected response.

[0413] For example, peptides, polypeptides (optionally coupled with carrier proteins such as KLH, BSA, or OVA), cells, and reagents can be diluted with sterile saline and mixed with adjuvants (e.g., complete or incomplete Freund's adjuvant, aluminum salts (e.g., aluminum sulfate, aluminum phosphate, aluminum hydroxide), CpG, or adjuvants described in Lee and Nguyen, Immune Netw. (2015) 15(2):51-57, the entire contents of which are incorporated herein by reference) to form stable emulsions. Nucleic acids (optionally provided in the form of liposome-nucleic acid complexes) can be injected as saline solution, introduced as an aqueous solution via pneumatic (jet) injection, or introduced via a gene gun coated on gold beads.

[0414] When introducing the material into an animal via injection, any suitable injection site can be used. For example, the injection can be intraperitoneal, intravascular (e.g., intravenous or intraarterial), intradermal, subcutaneous, intramuscular, intraosseous, intrasheath, epidural, intracardiac, intra-articular, intracavitary, and intravitreal. Preferred injection routes according to the method of this disclosure are intraperitoneal and intravascular (e.g., intravenous or intraarterial).

[0415] The appropriate amount or number of cells for the peptide / polypeptide / nucleic acid / reagent used for individual application can be readily determined by a technician, for example, by referring to *Antibodies: A Laboratory Manual*, 2nd edition, 2014, by Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated above by reference). The appropriate volume and concentration of the reagent used for application can also be readily determined by a technician.

[0416] In some embodiments, peptides / polypeptides / nucleic acids / cells may be formulated in different forms for different administration and / or different administration steps. For example, in some embodiments, different carrier proteins or adjuvants may be used in different administration and / or different administration steps. In some embodiments, one or more adjuvants may be used in one or more administration and / or administration steps according to the methods of this disclosure, and different adjuvants or no adjuvant may be used in other one or more administration and / or administration steps.

[0417] For example, in one embodiment of the method as described in this disclosure, the step of administering a first peptide / polypeptide to a subject may include administering a first peptide / polypeptide formulated with, for example, complete Freund's adjuvant and CpG to the subject, while the step of administering a second peptide / polypeptide to a subject may include administering a second peptide / polypeptide formulated with, for example, complete Freund's adjuvant and CpG to the subject.

[0418] The peptides / peptides / nucleic acids / cells / reagents described herein can be administered to animals in amounts suitable for evoking the desired response.

[0419] For example, administering a peptide / peptide as defined herein to an animal may include administering 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., total amount, or per administration). In some embodiments, administering a peptide / peptide as defined herein to an animal may include administering 5-500 μg, 10-200 μg, 20-80 μg, or about 50 μg of peptide / peptide (e.g., total amount, or per administration).

[0420] For example, administering a reagent as defined herein to an animal may include administering 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 reagent (e.g., total amount, or amount administered per dose). In some embodiments, administering a reagent as defined herein to an animal may include administering 5-500 μg, 10-200 μg, 20-80 μg, or about 50 μg of the reagent (e.g., total amount, or amount administered per dose).

[0421] Administering an agent as defined herein to an animal may include administering an agent 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 (e.g., total dose or per administration). In some embodiments, administering an agent as defined herein to an animal may include administering an agent at a concentration of 1-25 mg / kg, 2-20 mg / kg, 5-15 mg / kg, or about 10 mg / kg (e.g., total dose or per administration).

[0422] The administration steps of the method disclosed herein may be performed sequentially. That is, in some embodiments, the administration steps as described herein are performed first, and then a separate administration step is performed after a given time interval.

[0423] The time interval between sequentially performed dosing steps is preferably a time interval suitable for the animal to achieve the desired response. For example, in embodiments of the method disclosed herein, after administration of a first peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein, a period of time sufficient for the animal to generate one or more antigen-binding molecules against the first peptide / polypeptide is allowed before administration of a second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein.

[0424] In some embodiments, the time interval between sequentially performed dosing steps is one of the following: at least 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 time interval between sequentially performed dosing steps is about 3 to 21 days, for example, about 5 to 18 days, 7 to 16 days, or 12 to 16 days. In some embodiments, the time interval between sequentially performed dosing steps is about 14 days. In some embodiments, the time interval between sequentially performed dosing steps is about 23 days. In some embodiments, the time interval between sequentially performed dosing steps is about 30 days.

[0425] In embodiments where the dosing procedure involves multiple independent administrations of material to an animal, the time interval described herein can be the interval between the last administration of a dosing procedure and the first administration of a subsequent dosing procedure.

[0426] In some embodiments, the administration steps of the methods disclosed herein may be performed simultaneously. That is, in some embodiments, one administration step as described herein is performed simultaneously with another administration step as described herein, or immediately before / after it.

[0427] In certain embodiments, the methods of this disclosure involve simultaneously administering an agent capable of inhibiting a primary immune response and / or promoting a secondary immune response, and a second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein, to an animal.

[0428] The time interval between concurrent dosing steps is preferably less than 72 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours or 6 hours.

[0429] In some embodiments, when the dosing steps are performed simultaneously, the reagents for each dosing step are formulated together and administered to the animal as a single reagent. In some embodiments, the reagents for each dosing step are formulated as separate reagents and administered to the animal.

[0430] The administration steps described herein, performed sequentially or simultaneously, can be administered to animals via the same or different routes.

[0431] In some embodiments, an agent for suppressing the primary immune response and / or promoting the secondary immune response in animals is administered before, at the same time (e.g., simultaneously), and / or after administration of the second peptide / peptide. In some embodiments, the agent for suppressing the primary immune response and / or promoting the secondary immune response in animals is administered before and / or after administration of the second peptide / peptide, for example, by one or more administrations up to 5 days before administration of the second peptide / peptide, and / or by one or more administrations up to 10 days after administration of the second peptide / peptide.

[0432] In some embodiments, an agent for suppressing the primary immune response and / or promoting the secondary immune response in animals is administered 3 days and / or 1 day before administration of the second peptide / peptide, and 1 day, 3 days and / or 6 days after administration of the second peptide / peptide.

[0433] In some embodiments, one, three, and / or six days after administration of the second peptide / peptide, an agent for suppressing the primary immune response and / or promoting the secondary immune response in the animal is administered.

[0434] In aspects of the present invention where an agent capable of suppressing the primary immune response and / or promoting the secondary immune response in an animal is administered, it should be understood that the agent is administered to suppress the primary immune response against the second segment / peptide and / or promote the secondary immune response.

[0435] Similarly, it should be understood that administering to animals a first peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein, administering an agent that can induce suppression of the primary immune response and / or promotion of the secondary immune response, and administering a second peptide / polypeptide (or nucleic acid encoding the peptide / polypeptide) as described herein, can substantially not suppress the primary immune response against the first peptide / polypeptide, but suppress the primary immune response against the second peptide / polypeptide, and / or promote the secondary immune response.

[0436] In some embodiments, the method includes one or more further administration steps as described herein.

[0437] For the purposes stated below, the administration procedure is as follows: (a) Administering a first peptide / polypeptide, or a nucleic acid encoding the first peptide / polypeptide, to an animal as described herein, wherein the first peptide / polypeptide comprises a target amino acid sequence; (b) Administering to the animals an agent used to suppress (e.g., induce suppression) the primary immune response and / or promote the secondary immune response; and (c) Administering to the animal a second peptide / polypeptide, or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide comprises the 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 (e.g., larger than) the first peptide / polypeptide.

[0438] In some embodiments, the methods of this disclosure include a combination of one or more of the following administration steps as defined 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) + (b).

[0439] In some embodiments, the administration steps are as follows: (a) Administering a first peptide / polypeptide, or a nucleic acid encoding the first peptide / polypeptide, to an animal as described herein, wherein the first peptide / polypeptide comprises a target amino acid sequence; (b) administering to the animal a second peptide / polypeptide, or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide comprises the 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); and (c) Administer to the animal an agent for suppressing (e.g., inducing suppression) the primary immune response and / or promoting the secondary immune response.

[0440] In some embodiments, the administration steps are as follows: (a) Administering a first peptide / polypeptide, or a nucleic acid encoding the first peptide / polypeptide, to an animal as described herein, wherein the first peptide / polypeptide comprises a target amino acid sequence; (b) Administering to the animal an agent for suppressing (e.g., inducing suppression) the primary immune response and / or promoting the secondary immune response; (c) Administering to the animal a second peptide / polypeptide, or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide comprises the 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); and (d) Administer to the animal an agent for suppressing (e.g., inducing suppression) the primary immune response and / or promoting the secondary immune response.

[0441] In some embodiments, the method further includes an enhancement step. An enhancement step may include administering a peptide / polypeptide / nucleic acid as described herein to an animal without the use of a carrier or adjuvant.

[0442] Enhancement steps are well-known to those skilled in the art in immunology. Enhancement steps may be included in methods for generating antigen-binding molecules, for example, to increase the titer before isolating the antigen-binding molecules or before harvesting cells for hybridoma generation.

[0443] Methods for generating immunity against proteins / pathogens may also include enhancement steps, such as to induce a recall response.

[0444] In some embodiments, a boosting step is performed before isolating antigen-binding molecules. In some embodiments, a boosting step is performed before harvesting B lymphocytes for hybridoma generation. In some embodiments, a boosting step is performed at least 12 hours to 5 days, about 1-4 days, or about 2-3 days before harvesting B lymphocytes for hybridoma generation.

[0445] In some embodiments, the enhancement step includes administering to the animal a peptide / polypeptide / nucleic acid that has already been administered to the animal, for example, in a dosing step prior to (a) or (c) above.

[0446] In some embodiments, the method includes performing reinforcement steps more than once (e.g., 2, 3, 4, 5, or 6 times). The intervals between multiple reinforcement steps may be at least 12 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours. In some embodiments, the intervals between reinforcement steps may be approximately 12-48 hours, for example, approximately 24 hours.

[0447] The enhancement step may include introducing the peptide / polypeptide / nucleic acid via injection. In some embodiments, particularly those related to methods for producing antigen-binding molecules, the enhancement step may include injecting the peptide / polypeptide / nucleic acid into the peritoneal cavity of an animal.

[0448] Assess the production of antigen-binding molecules The methods disclosed herein may further include monitoring / evaluating the immune response of animals after receiving 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.

[0449] 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.

[0450] In some embodiments, the method includes detecting the presence of an antigen-binding molecule having one or more target functional properties, or an immune cell / population of immune cells capable of producing such an 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.). Such functional properties of the antigen-binding molecule can be analyzed using appropriate experiments targeting such functions of the target protein / protein complex.

[0451] 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 to detect the production of antigen-binding molecules and the presence of cells with the target characteristics (e.g., the production of antigen-binding molecules capable of recognizing the target antigen).

[0452] The analysis of detecting and quantifying the generation of antigen-binding molecules is well known to those skilled in the art and is described, in, for example, *Antibodies: A Laboratory Manual*, 2nd edition, 2014, by Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated above by reference), particularly Chapter 15. For instance, after one or more administration steps as described in this disclosure, blood, plasma, serum, or ascites samples may be collected from a subject and analyzed by ELISA or flow cytometry. As used herein, “plasma” refers to the liquid component of blood, free of cells, and may be the liquid portion of blood obtained after the removal of blood cells. As used herein, “serum” refers to plasma free of 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.

[0453] Immunoassays can be used to detect the production of antigen-binding molecules. Immunoassays can be used to determine whether a sample obtained from a subject contains antigen-binding molecules capable of binding a given peptide or polypeptide.

[0454] In some embodiments, the method includes quantifying antigen-binding molecules in a sample, for example by determining antibody titers. Antibody titer is a measure of the amount of antibodies produced by a subject that can recognize (i.e. bind) a given antigen. In assays such as immunoassays, antibody titer is expressed as the reciprocal of the highest dilution of a test sample (e.g., a serum sample) that would make the antigen test positive.

[0455] As used herein, “binding” of an antigen-binding molecule refers to a specific interaction between the molecule and its homologous antigen. “Specific interaction” refers to a non-specific interaction between an antibody and an antigen. The binding of an antigen-binding molecule to an antigen is mediated by non-covalent interactions such as van der Waals forces, electrostatic interactions, hydrogen bonds, and hydrophobic interactions. Specifically, this interaction occurs 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 is contacted by an antigen-binding molecule. Specifically, an epitope is a portion of an antigen that is bound to an antigen-binding molecule. Epitopes are provided by an antigenic amino acid sequence. Epitopes can be linear, consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). Alternatively, epitopes can be conformational, consisting of discontinuous amino acid sequences within the amino acid sequence of any antigen. These discontinuous amino acids may be located in different regions of a peptide / polypeptide, and may be in close proximity when the antigen folds (e.g., folds into its native structure).

[0456] 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, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance (SPR, see, for example, Hearty et al., Methods Mol Biol (2012) 907:411-442), or biolayer interference techniques (see Lad et al. (2015), J Biomol Screen 20(4): 498-507), and flow cytometry. Such analyses can measure 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 non-covalent complex formed between the protein / domain and the antigen-binding molecule.

[0457] Furthermore, specific regions of the binding chaperone of antigen-binding molecules can be analyzed using methods well-known in the art, including X-ray cocrystallization analysis of antibody-antigen complexes, mass spectrometry hydrogen-deuterium exchange analysis, cryo-electron microscopy, peptide scanning, and mutation localization. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, and Abbott et al., Immunology (2014) 142: 526-535, the entire contents of which are incorporated herein by reference.

[0458] Other products disclosed herein This disclosure also provides one or more nucleic acids comprising the nucleotide sequences described herein.

[0459] In some embodiments, the nucleic acid / multiple nucleic acids are purified or isolated, for example, separated from other nucleic acids or naturally occurring biological materials. In some embodiments, the nucleic acid / multiple nucleic acids comprise or consist of DNA and / or RNA.

[0460] It should be understood that in some embodiments, the various nucleic acids described in this disclosure may comprise one or more nucleotide sequences as described in this disclosure (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In such embodiments, the various nucleotide sequences may each independently conform to any embodiment of the nucleotide sequences described herein.

[0461] This disclosure also provides one or more vectors that contain nucleic acids / multiple nucleic acids as described in this disclosure.

[0462] The nucleic acid / multiple nucleic acids may be contained in one or more vectors. As used herein, a "vector" is a nucleic acid molecule used to transfer exogenous nucleic acids into cells. A vector may be a vector for expressing nucleic acids in cells (i.e., an expression vector). A vector may include a promoter sequence operatively linked to a nucleotide sequence encoding the sequence to be expressed. A vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used.

[0463] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retroviral vectors such as murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0464] In some embodiments, the vector may be a eukaryotic vector, such as a vector containing elements for expression in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, such as a vector containing a cytomegalovirus (CMV) promoter or an SV40 promoter.

[0465] This disclosure 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 be a non-human animal cell, such as a mouse, rat, hamster, camel, guinea pig, rabbit, goat, chicken, primate (such as a non-human primate, such as a monkey), sheep, donkey, cattle, cat, dog, pig, or horse cell. In some embodiments, the cell is a mammalian cell (such as a non-human mammalian cell). In some embodiments, the cell is derived from rodent species (e.g., species of the genera *Mice*, *Rats*, or *Guinea Pigs*) or lagomorpha species (e.g., species of the family *Leporidae*). In some embodiments, the cell is a mouse cell.

[0466] In some embodiments, the cell is a pluripotent cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is an embryonic stem cell.

[0467] This disclosure also provides an embryo comprising the cells described herein. This disclosure further provides a blastocyst comprising the cells described herein.

[0468] Such nucleic acids, vectors, cells, embryos, and blastocysts can be used to produce animals as described in this disclosure. Therefore, this disclosure also provides an animal produced by implanting a blastocyst containing cells as described in this disclosure into the uterus.

[0469] Preparation of peptides / polypeptides The peptides and polypeptides used in the methods described herein can be prepared according to methods known to those skilled in the art.

[0470] Peptides can be prepared by chemical synthesis (e.g., liquid-phase or solid-phase synthesis). For example, peptides / peptides can be synthesized using methods described, such as those in Chandrudu et al., Molecules (2013), 18: 4373-4388 (the entire contents of which are incorporated herein by reference). Alternatively, peptides / peptides can be produced by recombinant expression. Molecular biology techniques suitable for the recombinant production of peptides / peptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manua, 4th ed., Cold Spring Harbor Laboratory Press, 2012, and Nat Methods. (2008); 5(2): 135-146, the entire contents of which are incorporated herein by reference.

[0471] Recombinant production as described in this disclosure can be performed using any cell suitable for expressing the peptide / polypeptide. The cell can be a prokaryotic or eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, such as an archaea or bacterial cell. In some embodiments, the bacteria can be Gram-negative bacteria, such as Enterobacteriaceae bacteria, like *Escherichia coli*. In some embodiments, the cell is a eukaryotic cell, such as yeast cells, plant cells, insect cells, or mammalian cells, such as CHO cells, HEK cells (e.g., HEK293), HeLa cells, or COS cells. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptide.

[0472] In some cases, the cells are not prokaryotic because some prokaryotic cells cannot perform the same folding or post-translational modifications as eukaryotic cells. Furthermore, eukaryotic cells can achieve extremely high expression levels, and proteins can be purified from eukaryotic cells more easily using appropriate tags. Specific plasmids can also be used to enhance the secretion of peptides / polypeptides in the culture medium.

[0473] In some embodiments, the polypeptide can be prepared by cell-free protein synthesis (CFPS), for example using the system described in Zemella et al., Chembiochem (2015) 16(17): 2420-2431, the entire contents of which are incorporated herein by reference.

[0474] Production may involve culturing or fermenting eukaryotic cells modified to express the target peptide / peptide. Cultivation or fermentation can be carried out in a bioreactor equipped with appropriate nutrients, air / oxygen, and / or growth factor supplies. Secreted proteins can be collected by separating the culture medium / fermentation broth from the cells, extracting the protein components, and isolating individual proteins to isolate the secreted peptide / peptide. Cultivation, fermentation, and isolation techniques are well known to those skilled in the art and described, for example, in Green and Sambrook, *Molecular Cloning: A Laboratory Manual* (4th edition; incorporated herein by reference above).

[0475] A bioreactor comprises one or more containers for culturing cells. Culturing in a bioreactor can be continuous, with reactants continuously flowing into the reactor and cultured cells continuously flowing out. Alternatively, culturing can be batch-processed. The bioreactor monitors and controls environmental conditions such as pH, oxygen, inflow and outflow rates, and agitation within the containers to provide optimal conditions for the cultured cells.

[0476] After culturing cells expressing the target peptide / peptide, the target peptide / peptide can be isolated. Any suitable method for isolating proteins from cells known in the art can be used. To isolate the peptide, it may be necessary to isolate the cells from the nutrient medium. If the peptide / peptide is secreted by cells, the cells can be isolated from the medium containing the secreted target peptide / peptide by centrifugation. If the target peptide / peptide aggregates within the cells, protein isolation may involve separating the cells from the cell culture medium by centrifugation, treating the cell pellet with lysis buffer, and disrupting the cells by sonication, rapid freeze-thaw, or osmotic lysis.

[0477] The target peptide / peptide may then need to be isolated from the supernatant or culture medium containing 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 can precipitate in precipitants of different concentrations (e.g., ammonium sulfate). For example, water-soluble proteins will be extracted in low-concentration precipitants. Therefore, proteins with different solubilities can be distinguished by adding precipitants of varying concentrations. Dialysis can then be used to remove ammonium sulfate from the separated proteins.

[0478] Other methods for distinguishing different proteins are also known in the art, such as ion exchange chromatography and size exclusion chromatography. These methods can be used as alternatives to precipitation methods or can be performed after precipitation.

[0479] Once the target peptide / peptide is isolated from the culture, it may be desirable or necessary to concentrate the peptide / peptide. Various methods for protein concentration are known in the art, such as ultrafiltration or lyophilization.

[0480] Sequence identity To determine the percentage of identity between two or more amino acid or nucleic acid sequences, pairwise or multiple sequence alignment can be performed using various methods known to those skilled in the art, such as publicly available computer software like ClustalOmega (Söding, J., 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al., 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer, 2005, BMC Bioinformatics, 6(298)), and MAFFT software (Katoh and Standley, 2013, Molecular Biology and Evolution, 30(4) 772-780). When using such software, it is preferable to use default parameters, such as vacancy penalties and extension penalties.

[0481] sequence *** This disclosure includes combinations of the described aspects and preferred features, unless such combinations are expressly not permitted or expressly avoided.

[0482] The features disclosed in the foregoing specification, subsequent claims, or drawings may be represented in their specific form, or in the manner of implementing the disclosed function, or in the form of a method or approach for obtaining the disclosed result, as the case may be. Such features may be used alone or in any combination of such features to implement various forms of the invention.

[0483] To avoid any doubt, any theoretical explanations provided herein are intended to enhance the reader's understanding. The inventors do not intend to be bound by any of these theoretical explanations.

[0484] The chapter titles used in this document are for organizational purposes only and should not be construed as limiting the topics described.

[0485] In this specification, including the following claims, unless the context otherwise requires, the words “comprising” and “including” and variations thereof such as “comprising”, “containing” and “including” should be understood to mean including the stated element or step or group of elements or steps, but not excluding any other element or step or group of elements or steps.

[0486] If the nucleic acid sequence is disclosed in this paper, its reverse complementarity can also be explicitly considered. Furthermore, when the nucleic acid sequence encoding a polypeptide is disclosed in this paper, equivalent polypeptide coding sequences arising from the degeneracy of the genetic code can also be explicitly considered.

[0487] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. A range may be expressed herein as “about” a particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from that particular value and / or to another particular value. Similarly, when a numerical value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value constitutes another embodiment. The term 'about' in relation to numerical values ​​is optional, for example, indicating + / - 10%.

[0488] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, and the products can also be present in vitro, ex vivo, or in vivo. The term “in vitro” is intended to include experiments performed with materials, biological substances, cells, and / or tissues under laboratory or culture conditions; the term “in vivo” is intended to include experiments and manipulations performed with intact multicellular organisms. In some embodiments, the methods performed in vivo can be performed on non-human animals. “Ex vivo” refers to operations that are present or performed outside of an organism (e.g., outside of a human or animal) and may involve tissues (e.g., intact organs) or cells taken from that organism.

[0489] For information on standard molecular biology techniques, see Sambrook, J., Russell, DW, Molecular Cloning, A Laboratory Manual, 3rd edition, 2001, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York.

[0490] 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 references herein are incorporated herein by reference in their entirety. Although the invention has been described in conjunction with exemplary embodiments described below, many equivalent modifications and variations will be apparent to those skilled in the art upon obtaining this disclosure. Therefore, the exemplary embodiments of the invention described above should be considered illustrative rather than restrictive. Various changes may be made to the embodiments without departing from the spirit and scope of the invention. Attached Figure Description

[0491] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings.

[0492] Figure 1A and 1B (1A) The schematic diagram shows the mouse genome target site, target vector, target allele (after integration of the target vector insert fragment) and constitutive knock-in allele used to construct Cd79a-CreERT2 knock-in mice (as described in Example 8.1); (1B) The schematic diagram shows the target vector, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0493] Figure 2A and 2B (2A) The schematic diagram shows the mouse genome target site, target vector, target allele (after integration of the target vector insert fragment), conditional knockout allele, and Cre recombination knockout allele used to construct Cd40 CKO mice (as described in Example 8.2); (2B) The schematic diagram shows the target vector, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0494] Figure 3 Immunization, administration, and sampling timelines for evaluating experiments that use anti-CD40L antibodies to block initial B cell stimulation to suppress the primary immune response.

[0495] Figure 4(4A) The graphs show the immune response to the antigen. 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 at days 7, 14, and 26; n=6 at days 31, 35, and 40; and n=2 at day 50. *p<0.05, Holm-Sidak corrected multiple unpaired t-test.

[0496] Figure 5 The graph shows the percentage of B cells with CreERT2-mediated ZsGreen allele recombination detected in B cells isolated from bone marrow, spleen, and lymph nodes, and the lox-Stop-lox ZsGreen allele homozygosity (Gt(ROSA)26Sor) shown on specified days after treatment with tamoxifen at 100 mg / kg or 200 mg / kg body weight. tm6(CAG -ZsGreen1)Hze And Cd79a CreERT2 The percentage of non-B cells containing CreERT2-mediated ZsGreen allele recombination in the tissues of heterozygous mice.

[0497] Figure 6 The chart shows the homozygous lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sor) at specified days after treatment with 100 mg / kg body weight tamoxifen or solvent control (corn oil), respectively. tm6(CAG-ZsGreen1)Hze And Cd79a CreERT2 In heterozygous mice, the percentage of B cells with CreERT2-mediated ZsGreen allele recombination was detected in B cells isolated from bone marrow, spleen, and lymph nodes.

[0498] Figure 7A and 7B The diagram and images illustrate the Cd40 concentration 48 hours after treatment with 200 mg / kg body weight of tamoxifen. flox / flox Cd79a + / CreERT2 In mouse B cells, the in vivo CreERT2-mediated deletion of Cd40 exons 2-5. (7A) The schematic diagram shows the structure of the Cd40flox locus before (top) and after (bottom) tamoxifen-induced, CreERT2-mediated deletion of Cd40 exons 2-5. The figure indicates the control used for PCR amplification and Cd40. KO Positions of upstream and downstream primers for the amplified fragment. (7B) Image of the PCR reaction product. Using Cd40 after 48 hours of treatment with 200 mg / kg body weight tamoxifen (TAM) or solvent control (corn oil).flox / flox Cd79a + / CreERT2 Genomic DNA was extracted from cells isolated from the bone marrow of mice and then amplified using Cd40. KO The primers for the (left) and control (right) fragments were used for PCR, followed by separation by agarose gel electrophoresis. The figure indicates the control and Cd40 fragments. KO The expected location of the amplified fragment.

[0499] Figure 8 Histograms and bar graphs show the concentrations of Cd40, determined by flow cytometry, at 48 hours after treatment with 200 mg / kg body weight tamoxifen or corn oil (solvent control). flox / flox Cd79a + / CreERT2 Cd45r obtained from mouse spleen and lymph nodes + The proportion of cells expressing Cd40.

[0500] Figure 9A and 9B The diagrams and graphs illustrate the reduction of lox-Stop-loxZsGreen allele homozygosity (Gt(ROSA)26Sor) by repeated administration of tamoxifen. tm6(CAG-ZsGreen1)Hze And Cd79a CreERT2 Maintenance of CreERT2-mediated ZsGreen allele recombination in B cells isolated from the bone marrow, spleen, and lymph nodes of heterozygous mice. (9A) Schematic diagram showing the timing of administration of tamoxifen and solvent control (corn oil) to mice in groups 1 and 2. (9B) 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 mice in groups 1 and 2 at specified days after administration of the first dose of tamoxifen.

[0501] Figure 10 The diagram illustrates the process of directing Cd40... flox / flox Cd79a + / CreERT2 In mice, administration of tamoxifen was used to maintain the timing of B cell-specific, CreERT2-mediated deletion of Cd40 exons 2–5.

[0502] Figure 11 The bar chart shows the concentrations of Cd40, determined by NGS sequencing technology. flox / flox Cd79a + / CreERT2 Diversity in the use of VH and VL genes in B cells was observed in samples collected from (CD40 cKO) mice and BALB / c mice.

[0503] Figure 12The figures show the serum binding profiles and titers of full-length or epitope-deleted proteins in BALB / c mice that had been boosted once or twice with or without anti-CD40 antigens. Analysis was performed by flow cytometry. MFI = mean fluorescence intensity.

[0504] Figures 13A to 13C (13A) The figure shows the effects of tamoxifen-induced gene deletion on BALB / c mice and Cd40 mice. flox / flox Cd79a + / CreERT2 IgG in the spleen and lymph nodes of (CD40 cKO) mice + Quantitative results of B cells. (13B) The figure shows the B220 in the lymph nodes, spleen and bone marrow of BALB / c mice and CD40 cKO mice after tamoxifen-induced gene deletion. + Quantitative results of B cells. (13C) The graph shows IgG in the spleen and lymph nodes of BALB / c mice and CD40 cKO mice after tamoxifen-induced gene deletion. + Quantitative results of B cells.

[0505] Figures 14A to 14C (14A) Image of PCR reaction products. BALB / c or Cd40 was used on day 10 after the last treatment with 200 mg / kg body weight of tamoxifen (dosing regimen of 5 doses of tamoxifen over a 40-day course). flox / flox Cd79a + / CreERT2 Genomic DNA was extracted from cells isolated from the spleen and ears of (CD40 cKO) mice, and PCR was performed, followed by separation by agarose gel electrophoresis. Primers used amplified an 815 bp wild-type (wt) amplicon, a 713 bp flux (fl) amplicon (both detectable in the absence of CreERT2-mediated CD40flox locus recombination), and a 467 bp CD40 cKO amplicon (detectable after CreERT2-mediated CD40flox locus recombination). (14B and 14C) CD40 protein expression in B cells of lymphoid tissues (spleen and lymph nodes) from tamoxifen-induced transgenic (CD40 cKO) and wild-type mice was compared by flow cytometry and quantitative analysis.

[0506] Figure 15 An immunization strategy for generating specific targeting epitopes against each target protein. "Antigen 1" refers to a peptide containing the amino acid sequence of the targeting epitope within the target protein. "Antigen 2" refers to the full-length target protein containing that targeting epitope.

[0507] Figures 16A to 16D(15A) The graph shows the binding of antibodies generated in the serum of CD40 cKO mice and wild-type mice, as determined by ELISA, to target protein 1, which may have been in its full length or lacking the target epitope as defined in Example 17. (15B) FACS analysis of the binding profiles of antibodies against target protein 1, as defined in Example 17, present in the serum of CD40 cKO mice and wild-type mice. (15C) The graph shows the binding of antibodies generated in the serum of CD40 cKO mice and wild-type mice, as determined by ELISA, to the target or non-target domains of target protein 2, as defined in Example 16. (15D) FACS analysis of the binding profiles of antibodies against target protein 2, as defined in Example 17, present in the serum of CD40 cKO mice and wild-type mice.

[0508] Figure 17 Flow cytometry analysis showed the binding of four representative recombinant antibodies to the full-length extracellular domain (ECD) or epitope deletion (ΔEp) extracellular domain of target protein 1 as defined in Example 17.

[0509] Figures 18A to 18D The chart shows the four Cd40 values ​​as determined by ELISA. flox / flox Cd79a + / CreERT2 Dose-response curves of representative antibody clones obtained from mice binding to the full-length extracellular domain of the target protein 3 as defined in Example 17.

[0510] Figure 19 The graph shows the antibody binding in serum of BALB / c or NZBWF1 mice after immunization with recombinant human DLL3-Fc-tagged protein, as determined by ELISA. Serum was collected on day 38 post-immunization.

[0511] Figure 20 The graph shows the antibody binding in serum of BALB / c or NZBWF1 mice after immunization with two doses of a 13-mer peptide conjugated with KLH, as determined by ELISA. Serum was collected on day 25 post-immunization.

[0512] Example Example 1: Transgenic mice with induced CD40 gene knockout Transgenic mice with the CD40 gene homologous to the CD40 gene were prepared.

[0513] In short, mouse embryonic stem cells were modified using CRISPR / Cas9-mediated gene editing technology (as described by Lee et al., *Drug Discovery Today: Disease Models*, Vol. 20, 2016: pp. 13-20) to include loxP target sequences flanking the CD40 gene exon and to encode CreERT under the control of a promoter used for expression in B-cell lineage cells. The modified embryonic stem cells were then injected into blastocysts, which were subsequently implanted into the uterus to complete pregnancy, thereby producing transgenic mice.

[0514] In the obtained transgenic mice, CD40 expression in knockout mice can be induced by administration of tamoxifen.

[0515] Example 2: Transgenic mice encoding a human immunoglobulin gene that can be inducibly knocked out the CD40 gene Transgenic mice encoding a human immunoglobulin gene that is a homolog of the inducible knockout CD40 gene in mice were produced. In short, using CRISPR / Cas9-mediated gene editing technology (as described by Lee et al., *Drug Discovery Today: Disease Models*, Vol. 20, 2016: pp. 13-20), embryonic stem cells derived from transgenic mice encoding human immunoglobulin genes were modified to include loxP target sequences flanking exons of the CD40 gene and to encode CreERT under the control of a promoter used for expression in B-cell lineage cells. The modified embryonic stem cells were then injected into blastocysts, which were subsequently implanted into the uterus to complete pregnancy, thereby producing transgenic mice.

[0516] The resulting transgenic mice are able to produce antibodies with fully human variable region (VH and VL) sequences, and CD40 expression in mice can be induced by administration of tamoxifen.

[0517] Example 3: Antibody production using transgenic mice with induced CD40 gene knockout Mice prepared as described in Examples 1 and 2 were used to produce antibodies capable of binding the target amino acid sequence in their native conformation.

[0518] 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 expressed and purified from Chinese hamster ovary (CHO) cells or HEK293 cells via recombinant expression.

[0519] The peptide was conjugated to KLH and hepatitis B core antigen (HBc) carriers for immunization. Succinimidyl 6-((β-maleimide propionamido)hexanoate) (SMPH) was used as the linker between the peptide and the protein carrier. Successful conjugation to the protein carrier was confirmed by SDS-PAGE analysis.

[0520] Mice were first immunized with 50 μg of peptide (which presents a small fragment of the extracellular domain of the target protein) [first injection].

[0521] After the first injection of peptide-based immunization: (a) A group of mice were administered tamoxifen (by intraperitoneal injection) to induce CD40 knockout; and (b) Another group of mice did not receive the treatment to induce CD40 knockout.

[0522] Subsequently, both groups of mice were immunized with 50 μg of a polypeptide that presents the full-length amino acid sequence of the extracellular domain of the target protein [second injection].

[0523] After the second injection, mice were immunized twice more with the same peptide or polypeptide as the second injection [third and fourth injections].

[0524] After the third and fourth injections, mice were given one to three booster injections using the same peptides or polypeptides used in the second immunization [booster injections].

[0525] Injection sites include the armpits, groin, feet, back, and abdomen.

[0526] Different injections use different formulations, as follows: First injection: Peptide + Full Freund's adjuvant Second injection: ECD + incomplete Freund's adjuvant Third and fourth injections: ECD + incomplete Freund's adjuvant Enhanced injection: ECD (with or without adjuvant) Example 4: Analysis of antibody production by ELISA Following the final booster injection, serum was collected from the mice, and the binding of the antibodies to the following substances was analyzed: (1) Peptides, and (2) Extracellular domain (ECD) of the target protein.

[0527] The analysis was performed using enzyme-linked immunosorbent assay (ELISA).

[0528] In short, the ELISA plates were coated overnight at 4°C with peptides or ECD (1 μg / ml dissolved in PBS). After coating, the ELISA plates were washed with washing buffer (0.05% Tween 20 added to 1x PBS), then blocked with 1% BSA for 1 hour at room temperature, followed by washing three times with washing buffer.

[0529] Mouse serum was collected, serially diluted, and added to the wells of an ELISA plate. The plate was incubated at room temperature for 1 hour. After washing with washing buffer, horseradish peroxidase (HPR)-conjugated antibody was added to the wells and incubated at room temperature for 1 hour.

[0530] Subsequently, the ELISA plate was treated with TMB substrate solution at room temperature for 10 minutes for color development. The color development was terminated by adding 2M sulfuric acid (H2SO4), and the absorbance value was read at 450 nm within 30 minutes after termination.

[0531] Example 5: Preparation of hybridoma Hybridomas were prepared, and antibody production generated by the hybridomas was analyzed as follows.

[0532] Fusion (Day 1): Mice were dissected under sterile conditions to obtain spleens and lymph nodes, and single-cell suspensions of cells from these tissues were prepared.

[0533] Cells are fused with myeloma cells using polyethylene glycol (PEG) fusion or electrofusion methods.

[0534] For PEG fusion, the ClonaCell-HY Hybridoma Cloning Kit was used, and cells were fused according to the manufacturer's instructions (Stemcell Technologies, Canada). The fused cells were cultured overnight in ClonaCell-HY Medium C (Stemcell Technologies, Canada) at 37°C with 5% CO2. The next day, the fused cells were collected by centrifugation, resuspended in 10 ml of ClonaCell-HY Medium C, and then gently mixed with 90 ml of semi-solid methylcellulose-based ClonaCell-HY Medium D (Stemcell Technologies, Canada) containing HAT. The mixture was then seeded into 96-well plates. The cells were grown at 37°C with 5% CO2. After 7–10 days, the supernatant was screened by enzyme-linked immunosorbent assay (ELISA) to identify single hybridoma clones and screen for antibody-producing hybridomas.

[0535] Alternatively, for electrofusion, use the NEPA21 Super Electroporator and follow the manufacturer's operating procedure (Nepagene) to fuse cells. Fusion cells are incubated overnight at 37°C in 5% CO2 in ClonaCell-HY Medium C (Stemcell Technologies, Canada). The next day, collect fusion cells by centrifugation, resuspend in 1 ml of ClonaCell-HY Medium C, and then gently mix with 90 ml of semi-solid methylcellulose-based ClonaCell-HY Medium D (Stemcell Technologies, Canada) containing HAT components and 500 µg of FITC-labeled anti-mouse antibody (Jackson Immunoresearch). Cells are then seeded into 8 to 16 6-well plates. Cells are incubated at 37°C in 5% CO2 for 7 days to allow clone formation. Clones are scanned for FITC fluorescence using a Clonepix (Fortebio) device and picked, then transferred to 96-well plates containing AOF medium. After culturing the selected clones for 5 days, the supernatant was screened by enzyme-linked immunosorbent assay (ELISA).

[0536] Second round of ELISA: On day 13, the cell culture supernatant in each well of the 96-well plate was subjected to a second round of ELISA detection.

[0537] Amplification / Cryopreservation: On day 14, cells from wells showing positive results in the second round of ELISA were transferred to 24-well plates for culture and amplification. When the cultures were nearly confluent, the cells were collected and cryopreserved.

[0538] Example 6: Peptide-binding antibodies and ECD-binding antibodies and hybridomas Compared to mice that did not knock out the CD40 gene before the second injection (group (b) mice), mice immunized with a protocol that induced CD40 gene knockout by administering tamoxifen to the mice before the second injection (group (a) mice – see Example 3) produced higher titers of antibodies capable of binding to the extracellular domain of the target protein.

[0539] No difference was observed between mice in groups (a) and (b) in terms of the titer of antibodies that can bind to peptides.

[0540] In group (a), the proportion of mice with antibodies in their serum that can bind to peptides and the extracellular domains of target proteins was higher than that in group (b).

[0541] In group (a), the proportion of mice that produced hybridomas capable of generating antibodies that bind to peptides and the extracellular domains of target proteins was higher than in group (b).

[0542] Example 7: Hybridoma producing antibodies that bind to the peptide and ECD Cells from wells that tested positive by ELISA were diluted to approximately one cell per well in a multi-well plate and cultured in vitro for 1–2 weeks.

[0543] Subsequently, as described in Example 4, the binding of the cell culture supernatant in the wells to the following substances was analyzed by ELISA: (1) The target protein of the recombinant Fc tag; (2) Optional, Fc alone; and (3) Peptides used to prepare antibodies.

[0544] Cells from wells that produce antibodies that can bind only to (1) and (3) are diluted into multi-well plates with approximately one cell per well and cultured in vitro for 1–2 weeks.

[0545] Subsequently, the cell culture supernatant in the wells was analyzed using the ELISA method described above. The cells in these wells were considered monoclonal cells.

[0546] Compared to mice that did not knock out the CD40 gene before the second injection (group (b) mice), mice immunized with a protocol that induced CD40 gene knockout by administering tamoxifen to the mice before the second injection (group (a) mice – see Example 3) had a higher success rate in generating hybridomas capable of producing monoclonal antibodies that can bind peptides and contain the extracellular domains of those peptides.

[0547] Cells from the wells are transferred to T25 culture flasks for culture and expansion. After 3-4 weeks, the cells are cryopreserved in liquid nitrogen or used for ascites production.

[0548] Example 8: Exemplary transgenic mice 8.1 Constitutive Cd79a-CreERT2 knock-in mice A mouse containing an endogenous nucleotide sequence for constitutive knock-in of CreERT2 at the Cd79a site was prepared.

[0549] A targeting vector was designed based on the mouse Cd79a transcript (NCBI Ref: NM_007655.4). The insertion fragment of this targeting vector is as follows: Figure 1A As shown in the diagram, it includes (from 5' to 3'): A short homologous arm of about 3 kb contains exon 1 of Cd79a with a mutated translation start codon; A puromycin resistance gene, PuroR, is flanked by FRT sites (used to achieve Flp recombinase-mediated excision). The first 7 nucleotides of exon 2 of Cd79a, followed by the Kozak sequence and the open reading frame of CreERT2; The 3' UTR of Cd79a and the human growth hormone polyadenylate signal sequence (hGHpA; to prevent transcriptional readthrough); A long homologous arm of approximately 6 kb, comprising exons 3 to 5 of Cd79a and Arhgef1; and Nucleic acid encoding thymidine kinase.

[0550] See the schematic diagram of the target vector. Figure 1B The nucleotide sequence of the targeting vector is shown in SEQ ID NO:1.

[0551] The targeting vector was transfected into Balb / c embryonic stem cell lines. 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 Flp recombinase-mediated PuroR excision, thereby obtaining mature knock-in alleles.

[0552] Embryonic stem cells containing knock-in alleles were injected via microinjection into blastocysts of Balb / c mice (e.g., Sumiyama et al., PLoS One (2018) 13(9):e0203056), and the blastocysts were then implanted into female Balb / c mice to induce pregnancy, thereby producing transgenic mice.

[0553] Cd79a-CreERT2 mice constitutively express CreERT2 in B-cell lineage cells under the control of the Cd79a promoter.

[0554] 8.2 Conditional Cd40 knockout mice A mouse containing an endogenous nucleotide sequence for inducible knockout of Cd40 was prepared.

[0555] A targeting vector was designed based on the mouse Cd40 transcript (NCBI Ref: NM_011611.2). The insertion fragment of this targeting vector is as follows: Figure 2A As shown in the diagram, it includes (from 5' to 3'): A short homologous arm of approximately 3 kb; The region flanked by loxP sites contains: a puromycin resistance gene PuroR, flanked by FRT sites (for Flp recombinase-mediated excision), and exons 2 to 5 of Cd40; A homologous arm approximately 6 kb long; and Nucleic acid encoding thymidine kinase.

[0556] See the schematic diagram of the target vector. Figure 2B Its nucleotide sequence is shown in SEQ ID NO:2.

[0557] The targeting vector was transfected into Balb / c embryonic stem cell lines. 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 Flp recombinase-mediated PuroR excision, thereby obtaining mature conditional knockout alleles.

[0558] It is anticipated that Cre recombinase-mediated exon 2-5 excision of Cd40 will delete approximately half of the amino acid sequence of the target protein, introduce frameshift mutations in downstream exons 6-9, and introduce an early stop codon in exon 6. The nucleic acid transcript encoded by exon 2-5 after Cre recombinase-mediated excision is expected to be nonfunctional, and nonsense-mediated RNA degradation may also occur.

[0559] Embryonic stem cells containing conditionally knocked-out alleles were injected via microinjection into blastocysts of Balb / c mice (as described in Sumiyama et al., PLoS One (2018) 13(9):e0203056), and the blastocysts were then implanted into female Balb / c mice to induce pregnancy, thereby producing transgenic mice.

[0560] Cd40 CKO mice were used to conditionally knock out Cd40 expression in Cre.

[0561] Cd40 CKO mice were crossed with Cd79a-CreERT2 knock-in mice described in Example 8.1 to obtain Cd40 mice. flox Allele homozygous and Cd79a CreERT2 Allelic heterozygous mice were obtained, thus achieving knockout of Cd40 expression in B cells after tamoxifen treatment. The resulting transgenic mice had the following genotype: Cd40 flox / flox Cd79a + / CreERT2 .

[0562] Example 9: Inhibition of primary immune response by blocking CD40-CD40L signaling Experiments were conducted to detect the ability of anti-CD40L antibody treatment to limit the primary response of B cells to a secondary antigen.

[0563] This anti-CD40L antibody binds to CD40L expressed on activated T cells. Treatment with anti-CD40L blocks the interaction between the CD40 receptor on B cells and the CD40L ligand on activated T cells. It was found that immediate anti-CD40L treatment following antigen immunization inhibits the initiation of adaptive humoral immunity by blocking the co-stimulation of naive B cells by activated T cells, thereby preventing the formation of functional germinal centers.

[0564] The timeline of immunization, drug administration, and sampling is as follows: Figure 3 As shown.

[0565] On day 1, 8-9 week old mice were immunized with the primary antigen EGFR-hFc (50 μg dissolved in complete Freund's adjuvant) to induce a primary response against EGFR. On day 7, mice were again immunized with EGFR-hFc (50 μg dissolved in incomplete Freund's adjuvant) to enhance the primary response against EGFR. On day 30, mice were immunized a third time with the same antigen (EGFR-hFc, 50 μg dissolved in incomplete Freund's adjuvant) or a new antigen (CD33-mFc, 50 μg dissolved in incomplete Freund's adjuvant), or with an equal volume of PBS dissolved in incomplete Freund's adjuvant.

[0566] Immunized mice were intraperitoneally injected with three doses of 10 mg / kg anti-CD40L antibody (anti-mouse CD154; MR-1 clone) or isotype antibody (IgGaK). These three doses were administered on days 1, 3, and 6 after the third immunization (days 31, 33, and 36 of the entire experimental timeline).

[0567] Serum was collected from mice on days 7, 14, 26, and 31 (to assess the primary response to EGFR) and days 36, 40, and 50 (to assess the secondary response to EGFR and the primary response to CD33). In ELISA assays, the binding of serum antibodies to EGFR-His and CD33-His proteins was assessed, followed by detection with anti-mouse IgG-HRP secondary antibody and a chromogenic reaction using the substrate 3,3',5,5'-tetramethylbenzidine. Samples were initially diluted 1:100 and then serially diluted 3-fold to obtain 11 dilution gradients for ELISA assays. Response to the antigen was expressed as the dilution factor at which 50% maximum binding was achieved.

[0568] result Figure 4A shows the immune response observed after two immunizations with EGFR-hFc to generate an initial immune response, followed by a third immunization with PBS (left panel), EGFR-hFc (middle panel), or CD33-mFc (right panel), and then treatment with anti-CD40L antibody.

[0569] Mice treated with anti-CD40L antibody showed a secondary response to EGFR (the primary antibody), but a very limited immune response to CD33 (the secondary antibody).

[0570] A secondary immune response was only observed after the third administration of EGFR (top center of the image). The maximum effect of anti-CD40L treatment on the secondary EGFR response was observed on day 40: the EGFR response in anti-CD40L-treated mice decreased to 75% of that in isotype-treated mice. On day 50, the secondary EGFR response in anti-CD40L-treated mice recovered to 91% of that in controls. A persistent effect of anti-CD40L treatment on the primary CD33 response was observed on both days 40 and 50.

[0571] Mice treated with anti-CD40L showed a 5% response to CD33 compared to the control on day 40 and a 2% response compared to the control on day 50 (bottom right figure). On day 50, anti-CD40L treatment significantly suppressed the initial response (p=0.02, Holm-Sidak corrected multiple unpaired t-test).

[0572] Therefore, short-term treatment with anti-CD40L antibodies can suppress the primary response to neoantigens without significantly affecting the secondary response. The effect of anti-CD40L treatment on the primary response can last up to 14 days after injection.

[0573] Example 10: Carrying Cd79a CreERT2 Characterization of CreERT2 activity in constitutively knock-in allele transgenic mice Using lox-Stop-lox ZsGreen allele homozygosity (Gt(ROSA)26Sor tm6(CAG-ZsGreen1)Hze And Cd79a CreERT2 Heterozygous mice were used to assess the induction of B cell-specific cre / lox recombination in vivo. In these mice, tamoxifen-induced CreERT2 expression is expected to lead to B cell-specific ZsGreen expression.

[0574] In summary, 6-8 week old mice were intraperitoneally injected with a single dose of tamoxifen (100 mg / kg or 200 mg / kg body weight). Mice were sacrificed on days 1, 2, 4, and 5 post-treatment for analysis. Cells isolated from bone marrow, spleen, lymph nodes, and blood were stained using Zombie NIR reagent and anti-mouse Cd45r antibody and evaluated by flow cytometry to identify Cd45r. + B cells and Cd45r - The proportion of non-B cells exhibiting ZsGreen fluorescence.

[0575] The results are as follows Figure 5 As shown. Cd45r in all lymphatic tissues + ZsGreen fluorescence generated by Cre-mediated recombination was specifically detected in B cells. Total Cd45r was also observed in bone marrow, spleen, lymph nodes, and blood. + ZsGreen fluorescence was detected in 60-90% of B cells, while Cd45r fluorescence was detected in less than 5% of these tissues. - Non-B cells exhibited ZsGreen fluorescence. Effective B cell-specific Cre-mediated recombination was achieved within 2 days of treatment with tamoxifen in a dose-dependent manner. These data suggest that cells carrying single Cd79a cells... CreERT2 Allelic transgenic mice can achieve effective, tamoxifen-induced, B-cell-specific Cre / lox-mediated gene knockout in a dose-dependent manner.

[0576] In further experiments, the dynamic changes of Cre / lox-mediated gene knockout in mice were evaluated.

[0577] In short, 6-8 week old mice were intraperitoneally injected with a single dose of tamoxifen (100 mg / kg body weight) or corn oil (solvent control). Mice were sacrificed on days 1, 2, 6, 11, and 17 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 identify Cd45r. + B cells and Cd45r - The proportion of non-B cells exhibiting ZsGreen fluorescence.

[0578] The results are as follows Figure 6 As shown. Tamoxifen can effectively induce Cd45r in the spleen, bone marrow, lymph nodes, and blood. + B cells undergo Cre-mediated recombination to produce ZsGreen fluorescence. In all tissue types, recombinant Cd45r cells exhibiting ZsGreen fluorescence were detected at day 2. +The proportion of B cells was highest. Recombinant Cd45r was observed in the spleen, lymph nodes, and blood by day 17 post-treatment. + The total number of B cells decreased slightly (approximately 10%). Recombinant Cd45r in the bone marrow... + The proportion of B cells decreased rapidly starting two days after tamoxifen injection. By day 17, B cells in the bone marrow were predominantly ZsGreen-negative, non-recombinant cells. These data suggest that cells carrying a single Cd79a... CreERT2 Allele-derived transgenic mice can achieve efficient, tamoxifen-induced, B-cell-specific, Cre / lox-mediated gene knockout. Although all lymphoid tissues showed a high response to tamoxifen-induced CreERT2 activity, and a high proportion of recombinant B cells was maintained in peripheral lymphoid tissues more than 10 days after tamoxifen treatment, the proportion of recombinant cells in bone marrow cells decreased more rapidly.

[0579] Example 11: Cd40 flox / flox Cd79a + / CreERT2 Characterization of CreERT2-induced Cd40 knockout in transgenic mice Cd40 was evaluated flox / flox Cd79a + / CreERT2 Tamoxifen-induced Cd40 deletion in mice (described in Example 8). Briefly, 6-week-old sexually active mice were intraperitoneally injected with a single dose of tamoxifen (200 mg / kg body weight) or corn oil (solvent control). Mice were sacrificed 48 hours after treatment for analysis. Genomic DNA was isolated from bone marrow cells and used for genotyping PCR. Two sets of primers were used to detect Cd40. KO Deletion of exons 2-5 in alleles, and presence of [something] in Cd40. flox and Cd40 KO Control sites in the alleles. The first set of primers was used to amplify a 473 bp control fragment, regardless of the presence of CreERT2-mediated Cd40. flox In the locus recombination (control), this fragment was detectable. The second set of primers was used to amplify a 269 bp fragment, which only occurs during CreERT2-mediated Cd40 recombinant events. flox The locus can only be detected after recombination.

[0580] The results are as follows Figure 7B As shown. The control site was intact and detected in both tamoxifen and solvent-treated mice. Cd40 KO The amplified allele fragments were detected only in cells isolated from mice treated with tamoxifen. These data suggest that in Cd40 flox / flox Cd79a + / CreERT2In mice, the deletion of fluxed exons 2-5 of Cd40 was successfully achieved by treatment with tamoxifen.

[0581] In further experiments, the expression of Cd40 in B cells of peripheral lymphoid tissue was analyzed to assess the role of Cd40 at the cellular level. flox / flox Cd79a + / CreERT2 Tamoxifen-induced Cd40 knockout in mice.

[0582] In short, administer Cd40 to 6-week-old infants flox / flox Cd79a + / CreERT2 Mice were intraperitoneally injected with a single dose of tamoxifen (200 mg / kg body weight) or corn oil (solvent control). Mice were sacrificed 48 hours after tamoxifen treatment for analysis. Cells isolated from the spleen and lymph nodes were stained with Zombie NIR reagent, anti-mouse Cd45r antibody, and anti-mouse Cd40 antibody, and Cd40 was measured by flow cytometry. + Cd45r + B cells and Cd40 - Cd45r + The proportion of B cells.

[0583] The results are as follows Figure 8 As shown. Cd45 isolated from the spleen and lymph nodes of tamoxifen-treated mice. + In B cells, a high proportion (>75%) is Cd40. - In contrast, Cd45 isolated from the spleen and lymph nodes of solvent-treated mice... + In B cells, <7% were Cd40. - These data indicate that tamoxifen treatment can effectively induce Cd40. flox / flox Cd79a + / CreERT2 Cd40 knockout in mice prevents B cells from expressing Cd40.

[0584] Example 12: Maintaining high levels of CreERT2-induced gene knockout The ability of repeated administration of tamoxifen to maintain high levels of CreERT2-mediated fluxed locus knockout was investigated.

[0585] In short, the lox-Stop-lox ZsGreen allele homozygous (Gt(ROSA)26Sor) is directed towards 6-8 week old lox-Stop-lox ZsGreen allele homozygous (Gt(ROSA)26Sor) tm6 (CAG-ZsGreen1)Hze Mice were injected intraperitoneally with 200 mg / kg body weight of tamoxifen. The animals were then divided into two groups (e.g.,...). Figure 9A(As shown). In group 1, mice received four additional injections of tamoxifen (100 mg / kg body weight), with each injection spaced 5 to 9 days apart. In group 2, mice were injected with corn oil (solvent control) at the same timeline. Mice were sacrificed on days 3, 10, 17, and 32 following 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 identify Cd45r cells exhibiting ZsGreen fluorescence. + The proportion of B cells.

[0586] The results are as follows Figure 9B As shown, throughout the experiment, mice receiving only a single, initial dose of tamoxifen experienced a reduction of approximately 90% in the number of recombinant B cells in their bone marrow and approximately 25% in the number of recombinant B cells in their spleen and lymph nodes. In contrast, in group 1 mice (repeatedly injected with tamoxifen every 5–9 days), a high proportion of recombinant B cells was maintained in all lymphoid tissues throughout the experiment. These data indicate that a high proportion of CreERT2-mediated fluxed locus knockout B cells can be maintained long-term through regular, repeated administration of tamoxifen.

[0587] In further experiments, the effects of Cd40 were investigated. flox / flox Cd79a + / CreERT2 In mouse B cells, repeated administration of tamoxifen maintained a high level of Cd40 knockout.

[0588] In short, administer Cd40 to 6-week-old infants flox / flox Cd79a + / CreERT2 Mice were intraperitoneally injected with 200 mg / kg body weight of tamoxifen. This was followed by an additional injection of 100 mg / kg body weight of tamoxifen at intervals of 5 to 10 days. Mice were sacrificed on days 3, 10, 17, and 32 after the initial tamoxifen administration for analysis. Cells isolated from the spleen and lymph nodes were stained with Zombie NIR reagent, anti-mouse Cd45r antibody, and anti-mouse Cd40 antibody, and Cd40 was measured by flow cytometry. + Cd45r + B cells and Cd40 - Cd45r + The proportion of B cells.

[0589] Cd45 isolated from the spleen, lymph nodes, and bone marrow of tamoxifen-treated mice at all time points + In B cells, a high proportion (>75%) is Cd40. -This indicates that high levels of CreERT2-mediated Cd40 expression knockout in B cells can be maintained long-term by regularly and repeatedly administering tamoxifen.

[0590] Example 13: Using transgenic mice with inducible Cd40 knockout to obtain antibodies targeting specific regions of the target protein. The inventors used transgenic mice with induced knockout of Cd40 to obtain antibodies that bind to the proximal membrane protease cleavage site in the extracellular domain of the target protein.

[0591] On days 0 and 7, 8-9 week old Cd40 antibodies were cleaved with a peptide containing a protease cleavage site (“antigen 1”, 50 μg dissolved in complete Freund’s adjuvant). flox / flox Cd79a + / CreERT2 Mice or BALB / c mice were immunized to induce a primary response. On day 26, mice were intraperitoneally injected with 200 mg / kg body weight of tamoxifen to induce Cd40 knockout in B cells. On days 30 and 44, mice were immunized with the full-length extracellular domain of the target protein (containing the protease cleavage site, "antigen 2", 50 μg dissolved in incomplete Freund's adjuvant). To maintain Cd40 knockout in B cells, three additional doses of 100 mg / kg body weight of tamoxifen were administered on days 34, 42, and 52.

[0592] Peripheral lymphoid tissue samples were collected from mice on day 28 (“first time point”, before induction of Cd40 knockout and immunization with the full-length extracellular domain) and day 63 (“second time point”, after induction of Cd40 knockout and immunization with the full-length extracellular domain) to analyze the use of the V gene in IgG+ B cells that bind to the extracellular domain of the target protein, and to analyze the binding of mouse-produced antibodies to: (i) soluble, cleaved forms of the extracellular domain (i.e., lacking protease cleavage sites), and (ii) uncleaved forms of the extracellular domain (i.e., lacking protease cleavage sites).

[0593] In summary, 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 software (which groups B cells belonging to a common lineage based on antibody gene sequences using computational methods). The antibody sequences were then compared with an internal antibody database to determine V(D)J gene usage. The 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 compared with their closest germline sequences, quantifying the number of somatic hypermutations (SHMs) based on the number of residues differing from the identified germline sequences. For simplicity, the analysis assumed no reversion mutations to germline residues.

[0594] The table below illustrates that Cd40 knockout did not prevent somatic hypermutation. In fact, Cd40 was found to... flox / flox ;Cd79a + / CreERT2 The number of somatic hypermutations in the VH and VL genes of representative antibody clones that produce antibodies that specifically bind to the protease cleavage sites of target proteins increased at the second time point (i.e., after Cd40 knockout).

[0595] The table below shows that Cd40 knockout did not prevent somatic hypermutation. *Only includes clones > 1. *Representative clones From Cd40 flox / flox Cd79a + / CreERT2 Approximately 20 of the most representative clonal antibody gene sequences obtained from mice and BALB / c mice were cloned into plasmids and expressed as chimeric mouse / human antibodies (containing mouse VH and VL regions, and human IgG1 CH1, linker, CH2 and CH3 regions (heavy chain), and human κCL (light chain)) expressed in ExpiCHO (ThermoFisher) cells, and purified by Protein A for subsequent characterization.

[0596] The ability of these antibodies to bind to protease cleavage sites was assessed by ELISA. In brief, the wells of a polypropylene plate were coated with 1 μg / ml Neutravidin (Invitrogen), incubated overnight at 4°C, washed with 1x phosphate-buffered saline (PBS) containing 1% BSA, and blocked at room temperature for 2 hours. After washing the plate twice with 1x PBS containing 0.05% Tween 20, 1 μg / ml of (i) biotinylated, His-labeled soluble extracellular domain of the target protein (without protease cleavage sites) or (ii) biotinylated, His-labeled full-length extracellular domain of the target protein (with protease cleavage sites) was added. The plate was washed three times with 1x PBS containing 0.05% Tween 20, drying between each step. Nine dilutions of the antibody (diluted 4-fold serially from 10 μg / ml with 1x PBS + 1% BSA) were added, and the plate was incubated at room temperature for 1 hour. The plate was washed three times with 1xPBS containing 0.05% Tween 20, drying between each step. HRP-conjugated goat anti-human secondary antibody (Invitrogen), prepared by diluting 1xPBS and 1% BSA at a ratio of 1:7000, was added to the plate. The plate was incubated in the dark at room temperature for 1 hour. A colorimetric reaction was performed using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA). The reaction was terminated with Invitrogen ELISA stop solution (Cat. No. #SS04), and the OD was measured at 450 nm using a BioTek PowerWave HT.

[0597] From Cd40 flox / flox Cd79a + / CreERT2 Of the antibodies derived from the most representative clones obtained from mice, 21% were found to specifically bind to the full-length extracellular domain of the target protein (containing protease cleavage sites) but not to the soluble extracellular domain (containing no protease cleavage sites). However, none of the antibodies derived from the most representative clones obtained from BALB / c mice showed this specific binding (i.e., binding only to the antigen containing protease cleavage sites and not to the other antigen).

[0598] Figure 11 The display shows that at Cd40 flox / flox Cd79a + / CreERT2 In mice, the diversity of VH and VL gene usage was maintained (i.e., relative to the usage of their wild-type controls).

[0599] These data suggest that transgenic mice with induced Cd40 knockout can be used to obtain a variety of antibodies targeting a given target protein region.

[0600] Example 14: Characterization of response to anti-CD40L antibody Experiments were conducted to investigate the effects of treating wild-type mice with one or two doses of anti-CD40L antibody in combination with immunization using the antigen. Briefly, adult BALB / c mice were first immunized on days 0 and 7 with a peptide containing a protease cleavage site (“antigen 1”, 50 μg dissolved in complete Freund’s adjuvant) to induce a primary response. On days 30 and 44, mice were immunized with the full-length extracellular domain of the protein (containing a protease cleavage site, “antigen 2”, 50 μg dissolved in incomplete Freund’s adjuvant). Mice were then intraperitoneally injected with three doses of 10 mg / kg anti-CD40L antibody (anti-mouse CD154, clone MR-1) on days 31, 33, and 36 post-immunization on day 30, and on days 45, 47, and 50 post-immunization on day 44. Serum was collected from mice on days 42 and 55 to evaluate epitope-specific responses driven by antigen 1 against protease cleavage sites and novel primary responses driven by antigen 2 against nonspecific regions of the extracellular domain of proteins. The binding profiles and titers of serum against cells expressing full-length or epitope-deficient proteins were analyzed by FACS. Briefly, CHO cells were transiently transfected with either (i) a plasmid encoding the membrane-bound form of the target protein (containing protease cleavage sites) or (ii) a plasmid encoding the extracellular domain of the target protein in a soluble form (without protease cleavage sites) and the native transmembrane domain (for membrane localization and surface expression). Untransfected CHO cells served as a negative control. Cells were collected 18 hours later and each cell type was divided into groups of 3 × 10⁻⁶ cells. 4 Each cell / well was incubated with 50 µL of serum (obtained from mice on the specified date) at 4°C for 1 hour in 3-fold serial dilutions (starting with a 1:200 dilution in PBS). Cells were then washed twice with 150 µL of FACS buffer (1xPBS + 1% FBS) and resuspended in 50 µL of Alexa Fluor 647-labeled AffiniPure goat anti-mouse IgG (Jackson Immuno, 1:1000 dilution) antibody and incubated at 4°C for 20 minutes. Cells were washed twice with FACS buffer and resuspended in DAPI solution to a final volume of 40 µL; unstained wells were resuspended in 40 µL of DAPI-free FACS buffer. Samples were subsequently analyzed on an iQue3 flow cytometer, and data were analyzed using FlowJo v10.8.1 software.

[0601] The results are as follows Figure 12As shown in the figure. These results indicate that the first dose of antiCD40L effectively blocks novel primary responses against nonspecific domains outside the target epitope (protease cleavage site), but subsequent doses of antiCD40L do not. These results suggest a need to develop genetically based approaches to control CD40 signaling in order to effectively and sustainably control novel primary responses in animals over a long period.

[0602] Example 15: Characterization of B cell traits following tamoxifen-induced conditional gene deletion The inventors used transgenic mice with induced CD40 knockout to study whether the IgG class switching response was maintained after tamoxifen-induced conditional gene deletion.

[0603] On days 0 and 7, peptides containing protease cleavage sites (“antigen 1”, 50 μg dissolved in complete Freund’s adjuvant) were used to treat 8–9 week old Cd40 cells. flox / flox Cd79a + / CreERT2 Mice (described in Example 8) or BALB / c mice were immunized to induce a primary response. On day 26, mice were intraperitoneally injected with 200 mg / kg body weight of tamoxifen to induce Cd40 knockout in B cells. On days 30 and 44, mice were immunized with the full-length extracellular domain of the target protein (containing the protease cleavage site, “antigen 2”, 50 μg dissolved in incomplete Freund's adjuvant). To maintain Cd40 knockout in B cells, mice were additionally injected with three doses of 100 mg / kg body weight of tamoxifen on days 34, 42, and 52.

[0604] Peripheral lymphoid tissue samples were collected from mice on day 28 (“first time point”, before induction of Cd40 knockout and immunization with full-length extracellular domain) and day 63 (“second time point”, after induction of Cd40 knockout and immunization with full-length extracellular domain). Cells isolated from lymph nodes, spleen, and bone marrow were stained with Zombie NIR reagent and anti-mouse CD45R, IgG1, IgG2a, and IgG2b antibodies, and IgG+ and CD45R were assessed by flow cytometry. + The proportion of B cells.

[0605] The results are as follows Figure 13A As shown, compared with wild-type BALB / c mice, the proportion of IgG+ B cells in the lymph nodes and spleen of CD40 cKO mice was decreased. Compared with wild-type, CD45R cells in the spleen of CD40 cKO mice were significantly reduced after tamoxifen-induced gene deletion. + The total number of B cells is reduced, but no reduction is observed in lymph nodes or bone marrow. Figure 13B LN = lymph node; SP = spleen; BM = bone marrow.

[0606] The above experiment was repeated once more using the second target protein. The results are as follows: Figure 13C As shown, compared with wild-type BALB / c mice, CD40 cKO mice showed a decreased proportion of IgG+ B cells in the lymph nodes and spleen.

[0607] In summary, these results indicate that interruption of CD40 signaling inhibits IgG+ B cell proliferation during the novel immune response process, and that the antibodies isolated from CD40 cKO mice originated from a smaller class-switching B cell pool.

[0608] Example 16: Other characteristics of transgenic mice with induced CD40 knockout Further evaluation was conducted on Cd40. flox / flox Cd79a + / CreERT2 Tamoxifen-induced CD40 deletion in mice (described in Example 8.2). Briefly, on days 0 and 7, 8-9 week old mice were induced to lose CD40 by a peptide containing a protease cleavage site (“antigen 1”, 50 μg dissolved in complete Freund’s adjuvant). flox / flox Cd79a + / CreERT2 Mice (described in Example 8) or BALB / c mice were immunized to induce a primary response. On day 26, mice were intraperitoneally injected with 200 mg / kg body weight of tamoxifen to induce Cd40 knockout in B cells. On days 30 and 44, mice were immunized with the full-length extracellular domain of the target protein (containing the protease cleavage site, “antigen 2”, 50 μg dissolved in incomplete Freund's adjuvant). To maintain Cd40 knockout in B cells, three additional doses of 100 mg / kg body weight of tamoxifen were administered on days 34, 42, and 52.

[0609] Mice were sacrificed 10 days after the last tamoxifen treatment for analysis. Genomic DNA was isolated from cells in the spleen and ear for genotyping PCR. The first set of primers was used to amplify either a 713 bp wild-type fragment or an 815 bp CD40 fragment. flox Amplified fragments, both of which occurred in CreERT2-mediated CD40 flox All loci can be detected during recombination. The second set of primers was used to amplify a 497 bp CD40cKO amplification fragment, which is generated during CreERT2-mediated CD40 amplification. flox Recombination at the locus can be detected.

[0610] Genotyping revealed B cell-specific, tamoxifen-induced conditional deletion of CD40 (… Figure 14AThis was demonstrated by the presence of a 467 bp band in B cells isolated from the spleen of tamoxifen-induced transgenic mice (CD40 cKO (fl / fl + tamoxifen)). The 467 bp band was absent in B cells isolated from the spleen of mice never treated with tamoxifen, and was also absent in Cd40... flox / flox Cd79a + / CreERT2 No 467 bp band was detected in the ear cells of (CD40 cKO) mice, indicating that induced knockout is tissue-specific for B cells.

[0611] Flow cytometry analysis of B cells collected from lymphoid tissue showed that, compared with wild-type mice, tamoxifen-induced transgenic mice (CD40 cKO) had 80-90% fewer CD40+ B cells in their lymphoid tissue (see [link to article]). Figure 14B and 14C ).

[0612] In summary, these results indicate that tamoxifen-induced conditional loss of CD40 is B cell specific.

[0613] Example 17: Using transgenic mice with inducible CD40 knockout, the efficiency of antibody detection targeting epitopes is improved. Two target proteins were used to analyze the production of epitope-specific antibodies. The results are summarized below.

[0614] Immunotherapy strategies for generating target epitope-specific antibodies against each target protein (“POI”) include: Figure 15 As shown.

[0615] The table below shows the CD40 levels compared to wild-type mice. flox / flox Cd79a + / CreERT2 The percentage of target epitope-specific serum produced in (CD40 cKO) mice.

[0616] Target protein 1 (POI1) Target protein 2 (POI2) The antibody's ability to bind to protease cleavage sites was assessed using ELISA. In brief, the wells of a polypropylene plate were coated with 1 μg / ml Neutravidin (Invitrogen), incubated overnight at 4°C, washed with 1x phosphate-buffered saline (PBS) containing 1% BSA, and blocked at room temperature for 2 hours. After washing the plate twice with 1x PBS containing 0.05% Tween 20, 1 μg / ml of either (i) a biotinylated, His-labeled soluble extracellular domain of the target protein (without protease cleavage sites) or (ii) a biotinylated, His-labeled full-length extracellular domain of the target protein (containing protease cleavage sites) was added. The plate was washed three times with 1x PBS containing 0.05% Tween 20, drying between each step. Nine dilutions of the antibody (diluted 4-fold serially from 10 μg / ml with 1x PBS + 1% BSA) were added, and the plate was incubated at room temperature for 1 hour. The plate was washed three times with 1x PBS containing 0.05% Tween 20, and dried between each step. HRP-conjugated goat anti-human secondary antibody (Invitrogen), prepared by diluting 1x PBS and 1% BSA at a ratio of 1:7000, was added to the plate. The plate was incubated in the dark at room temperature for 1 hour. A colorimetric reaction was performed using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA). The reaction was terminated with Invitrogen ELISA stop solution (Cat. No. #SS04), and the OD was measured at 450 nm using a BioTek PowerWave HT.

[0617] Serum binding profiles were also analyzed using FACS. In short, CHO cells were transiently transfected with either (i) a plasmid encoding the membrane-bound form of the target protein (containing a protease cleavage site), or (ii) a plasmid encoding the extracellular domain of the target protein in a soluble form (without a protease cleavage site) and the native transmembrane domain (for membrane localization and surface expression). Untransfected CHO cells served as a negative control. After 18 hours, cells were collected and each cell type was divided into groups of 3 × 10⁻⁶ cells. 4Each cell / well was incubated with 50 µL of serum (obtained from mice on the specified date) at 4°C for 1 hour in 3-fold serial dilutions (starting with a 1:200 dilution in PBS). Cells were then washed twice with 150 µL of FACS buffer (1xPBS + 1% FBS) and resuspended in 50 µL of Alexa Fluor647-labeled AffiniPure goat anti-mouse IgG (Jackson Immuno, 1:1000 dilution) antibody and incubated at 4°C for 20 minutes. Cells were washed twice with FACS buffer and resuspended in 40 µL of DAPI solution; unstained wells were resuspended in 40 µL of DAPI-free FACS buffer. Samples were subsequently analyzed using an iQue3 flow cytometer, and data were analyzed using FlowJo v10.8.1 software.

[0618] The results for target protein 1 (POI1) are shown below. Figure 16A and 16B The results for target protein 2 (POI2) are shown in [the table below]. Figure 16C and 16D .

[0619] The table below shows the rate of epitope-specific antibody clones obtained in clonal B cell screening targeting POI1. Figure 17 The specificity of the recombinant antibody against POI1 was demonstrated by FACS.

[0620] Four CD40-derived compounds were evaluated using FACS. flox / flox Cd79a + / CreERT2 The representative recombinant antibodies from (CD40cKO) mice demonstrated their ability to bind to cells expressing the full-length target protein (including the protease cleavage site) or to cells expressing a variant of the target protein lacking that protease cleavage site. These results indicate that these antibodies are specific for targeting the protease cleavage site and do not bind to other domains in the full-length protein.

[0621] Figures 18A to 18D This demonstrates the results obtained from CD40 as determined by ELISA. flox / flox Cd79a + / CreERT2 Dose-response curves of four representative antibody clones obtained from (CD40cKO) mice binding to the full-length extracellular domain of POI1 (containing a protease cleavage site) are shown below. The EC50 values ​​of each antibody are listed in the table below. These results indicate that all four antibodies bind to the target antigen with high affinity. Example 18: NZBWF1 mice exhibit a stronger immune response to antigens highly homologous to themselves. The inventors used a highly immunoreactive mouse strain (NZBWF1) to analyze responses to antigens that are highly homologous to themselves.

[0622] 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 mouse serum was analyzed by indirect enzyme-linked immunosorbent assay (ELISA).

[0623] The results are as follows Figure 19 As shown, human DLL3 protein shares 85.8% homology with mouse DLL3 protein. Compared with BALB / c mice, NZBWF1 mice exhibit a stronger titer against this antigen.

[0624] These results indicate that NZBWF1 mice are able to respond to immunization with protein antigens and produce strong antibody titers against antigens that are highly homologous to themselves (>80%).

[0625] Example 19: NZBWF1 mice produce a stronger antibody response to weakly immunogenic antigens. The inventors used the highly immunoreactive mouse strain (NZBWF1) used in Example 18 to analyze the response to weakly immunogenic antigens.

[0626] BALB / c or NZBWF1 mice aged 6–10 weeks were immunized with two doses of a 13-mer peptide conjugated to KLH, with a 7-day interval between the two immunizations. On day 21 post-immunization, the antibody titer against the biotin-conjugated peptide in mouse serum was analyzed by indirect enzyme-linked immunosorbent assay (ELISA).

[0627] The results are as follows Figure 20 As shown, NZBWF1 mice exhibited stronger titers against this peptide antigen compared to BALB / c mice. These results indicate that NZBWF1 mice are capable of responding to immunization with small peptides and producing antibody titers against weakly immunogenic antigens.

[0628] Example 20: Exemplary Hyperimmune CD40 flox / flox Cd79a + / CreERT2 Mouse construction The method for constructing an exemplary hyperimmune mouse is as follows. Hyperimmune CD40 mice can be constructed using standard breeding strategies. flox / flox Cd79a + / CreERT2 Mice, including those carrying CD40 flox / flox and Cd79a + / CreERT2Mice with alleles (in the BALB / c background) (e.g., the mice constructed in Example 8.2) were mated with autoimmune parental strains NZB or NZW mice, and then NZB and NZW mice were mated to produce NZBWF1 offspring carrying both alleles.

[0629] In the obtained transgenic mice, CD40 expression can be knocked out by administration of tamoxifen. This results in hyperimmune CD40... flox / flox Cd79a + / CreERT2 Mice are highly immunoreactive mice capable of producing strong immune responses and can be used to generate epitope-specific antibodies. For example, these mice can produce strong antibody titers against antigens that are highly homologous (>80%) to themselves or weakly immunogenic antigens.

Claims

1. An animal comprising an endogenous nucleotide sequence for inducing suppression of the primary humoral immune response.

2. The animal as described in claim 1, characterized in that, The animal contains endogenous nucleotide sequences that are used to induce inhibition of the expression of one or more genes involved in triggering the primary humoral immune response or to inhibit the activity of their products.

3. The animal as described in claim 1 or 2, characterized in that, The animal contains endogenous nucleotide sequences that are used to inducibly inhibit the expression of one or more genes involved in immunoglobulin isotype conversion, B cell maturation, and / or plasma cell and / or memory B cell production, or to inhibit the activity of their products.

4. The animal as described in any one of claims 1-3, characterized in that, The animal contains an endogenous nucleotide sequence for inducibly inhibiting the expression of one or both genes selected from CD40 and / or CD40L, or inhibiting the activity of their products.

5. The animal as described in any one of claims 1-4, characterized in that, The animal contains an endogenous nucleotide sequence that blocks the expression of one or more genes involved in triggering the primary humoral immune response via recombinase-mediated inhibition.

6. The animal as described in any one of claims 1-5, characterized in that, The endogenous nucleotide sequence contains the target sequence of a recombinase, which is located flanking all or part of the nucleotide sequence of a gene involved in triggering the primary humoral immune response.

7. The animal as described in claim 6, characterized in that, The animal contains an endogenous nucleotide sequence that induces recombinase expression or activity.

8. The animal as described in claim 7, characterized in that, The endogenous nucleotide sequence that induces recombinase expression or activity encodes a conditional system for controlling recombinase expression or activity.

9. The animal as described in any one of claims 6-8, characterized in that, The target sequence of the recombinase is a loxP sequence, and the recombinase is a Cre recombinase.

10. The animal as claimed in any one of claims 1-9, characterized in that, The animal contains an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

11. The animal as claimed in any one of claims 1-10, characterized in that, The animal in question is a mouse, rat, or rabbit.

12. The animal as claimed in any one of claims 1-11, characterized in that, The endogenous nucleotide sequence contains the target sequence of the recombinase located flanking one or more exons of CD40 and / or one or more exons of CD40L.

13. The animal as claimed in any one of claims 1-12, characterized in that, The animal contains an endogenous nucleotide sequence that comprises or consists of a nucleotide sequence that has 60% or more identity with the nucleotide sequence of SEQ ID NO:

4.

14. The animal as claimed in any one of claims 1-13, characterized in that, The animal contains an endogenous nucleotide sequence encoding a conditional system for controlling Cre recombinase expression and / or activity.

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

16. The animal as claimed in any one of claims 1-15, characterized in that, The animal contains an endogenous nucleotide sequence that comprises or consists of a nucleotide sequence that has 60% or more identity with the nucleotide sequence of SEQ ID NO:

6.

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

18. The method as described in claim 17, characterized in that, 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 animals to suppress their ability to elicit a primary immune response; and (iii) Administering the animal a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide contains the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

19. The method as described in claim 17, characterized in that, 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 the second peptide / polypeptide, wherein the second peptide / polypeptide comprises the 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 elicit a primary immune response.

20. The method as described in claim 18 or 19, characterized in that, Treating the animal to suppress its ability to elicit a primary immune response includes administering a reagent that induces the expression or activity of the recombinase.

21. A method for producing antigen-binding molecules, characterized in that, 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 animals to suppress their ability to elicit a primary immune response; and (iii) Administering the animal a second peptide / polypeptide or a nucleic acid encoding the second peptide / polypeptide, wherein the second peptide / polypeptide contains the target amino acid sequence or an amino acid sequence similar to the target amino acid sequence.

22. The method as described in claim 21, characterized in that, The method includes 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 elicit a primary immune response.

23. The method according to any one of claims 18-22, characterized in that, Treating the animal to suppress its ability to elicit a primary immune response includes administering a reagent that inhibits the expression of one or more genes involved in eliciting a primary humoral immune response or the activity of their products.

24. The method according to any one of claims 18-23, characterized in that, Treating the animal to suppress its ability to elicit a primary immune response includes administering an agent that reduces / blocks the stimulation, differentiation, activation, maturation, and / or proliferation of naïve B cells in the animal, reduces the number / proportion of naïve B cells in the animal, and / or inhibits immunoglobulin isotype switching and / or B cell maturation.

25. The method as described in claim 23 or 24, characterized in that, The reagent inhibits the expression of one or both genes selected from CD40 and / or CD40L, or inhibits the activity of their products.

26. The method according to any one of claims 23-25, characterized in that, The reagent inhibits the activity of CD40 and / or CD40L.

27. The method according to any one of claims 23-26, characterized in that, The reagents are selected from or include antibodies, antigen-binding molecules, peptides, decoy receptors, aptamers, blocking agents, or small molecules.

28. The method according to any one of claims 23-25, characterized in that, The reagent inhibits the expression of CD40 and / or CD40L.

29. The method according to any one of claims 23-25 ​​or 28, characterized in that, The reagent is or contains RNAi, siRNA, antisense nucleic acid, antisense oligonucleotide or gene editing system.

30. The method according to any one of claims 21-29, characterized in that, The animal contains an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

31. The method according to any one of claims 21-30, characterized in that, The animal in question is a mouse, rat, or rabbit.

32. The method according to any one of claims 17-31, characterized in that, The method also includes generating a hybridoma that produces an antigen-binding molecule capable of binding to the target protein / protein complex.

33. The method according to any one of claims 17-32, characterized in that, The method further includes isolating one or more antigen-binding molecules capable of binding to proteins containing the target amino acid sequence.

34. The method according to any one of claims 17-33, characterized in that, The method further includes formulating an antigen-binding molecule capable of binding to a protein containing the target amino acid sequence into a pharmaceutical composition.

35. One or more nucleic acids comprising a nucleotide sequence for inducing suppression of the primary humoral immune response.

36. The nucleic acid or multiple nucleic acids as described in claim 35, comprising a nucleotide sequence, said nucleotide sequence being used to inducibly inhibit the expression of one or more genes involved in triggering the primary humoral immune response or to inhibit the activity of their products.

37. The nucleic acid or multiple nucleic acids as described in claim 35 or 36, comprising a nucleotide sequence, said nucleotide sequence being used to inducibly inhibit the expression of one or more genes involved in immunoglobulin isotype conversion and / or B cell maturation, or one or more genes involved in the stimulation, differentiation, activation and / or proliferation of naïve B cells, or to inhibit the activity of their products.

38. The nucleic acid or multiple nucleic acids as described in any one of claims 35-37, comprising a nucleotide sequence, said nucleotide sequence being used to inducibly inhibit the expression of one or two genes selected from CD40 and / or CD40L or to inhibit the activity of their products.

39. The nucleic acid or multiple nucleic acids as described in any one of claims 35-38, characterized in that, It contains a nucleotide sequence that blocks the expression of one or more genes involved in triggering the primary humoral immune response via recombinase-mediated blocking.

40. The nucleic acid or multiple nucleic acids as described in any one of claims 35-39, comprising a nucleotide sequence encoding all or part of a gene involved in initiating a primary humoral immune response, wherein the nucleotide sequence is flanked by target sequences of a recombinase.

41. The nucleic acid or multiple nucleic acids as described in any one of claims 35-40, further comprising a nucleotide sequence encoding a conditional system for controlling the expression or activity of recombinase.

42. The nucleic acid or multiple nucleic acids as described in any one of claims 35-41, characterized in that, The target sequence of the recombinase is a loxP sequence, and the recombinase is a Cre recombinase.

43. The nucleic acid or multiple nucleic acids as described in any one of claims 35-42, comprising a nucleotide sequence, said nucleotide sequence comprising a target sequence of a recombinase located flanking one or more CD40 exons and / or one or more CD40L exons.

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

4.

45. The nucleic acid or multiple nucleic acids as described in any one of claims 35-44, comprising a nucleotide sequence encoding a conditional system for controlling Cre recombinase expression and / or activity.

46. ​​The nucleic acid or multiple nucleic acids as described in claim 45, characterized in that, The expression of the Cre recombinase is controlled by a promoter that drives its expression in B-cell lineage cells.

47. The nucleic acid or multiple nucleic acids as described in any one of claims 35-46, comprising a nucleotide sequence, said nucleotide sequence comprising or consisting of a nucleotide sequence having 60% or more identity with the nucleotide sequence of 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 35-47.

49. A cell comprising the nucleic acid or multiple nucleic acids as described in any one of claims 35-47, or the vector or multiple vectors as described in claim 48.

50. The cell as claimed in claim 49, characterized in that, The cell contains an endogenous nucleotide sequence of a gene or gene segment encoding one or more human immunoglobulins.

51. The cell as described in claim 49 or 50, characterized in that, The cells in question are mammalian cells.

52. The cell according to any one of claims 49-51, characterized in that, The cells in question are embryonic stem cells.

53. The cell according to any one of claims 49-52, characterized in that, The cells are mouse cells, rat cells, or rabbit cells.

54. The animal as claimed in any one of claims 1-16, or the method as claimed in any one of claims 17-34, characterized in that, The animals are hyperimmune mice or mice with a hyperimmune phenotype.

55. The animal as claimed in any one of claims 1-16 or 54, or the method as claimed in any one of claims 17-34, characterized in that, The animal is a humanized mouse.

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