Methods for antibody production

Abstract

The present disclosure relates to the production of antigen-binding molecules. Animals used in the production of antigen-binding molecules are disclosed, and methods, nucleic acids and cells. Also disclosed are animals comprising sequences providing for inducible inhibition of immune responses.

Description

[0001] Methods for Antibody Production

[0002] This application claims priority from US 63 / 513385 filed 13 July 2023, US 63 / 609417 filed 13 December 2023 and US 63 / 645312 filed 10 May 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the fields of cellular and molecular biology, and immunology. More specifically, the present disclosure relates to the production of antigen-binding molecules, in particular in the context of therapeutic, prophylactic, diagnostic, imaging and research applications.

[0005] Background

[0006] Animal immunization is widely used for the development of monoclonal antibodies for therapeutic and diagnostic use. Traditional mouse hybridoma techniques involve producing antibody-secreting hybridoma cell lines by fusing splenocytes harvested from immunized mice with immortalized myeloma cells. While mice are the most commonly used species for the production of antibodies by animal immunization, other species of animal are also used, such as rabbit and goat.

[0007] A key limitation of classical full protein target immunization approaches to antibody development is that they can only produce antibodies against the target with limited control over the site of binding ( / .e. the target epitope on the protein of interest), with the antibody response primarily directed to immunodominant epitopes. The alternative strategy of small antigen immunization, representing a desired binding region of the target protein, allows more control over the site of antibody binding, but at the expense of increased risk that the antibody will not bind to the protein in vivo (j.e. , that it will not recognise the protein in its “native” form), e.g. due to the antigenic sequence being unavailable, or presented differently, in the native, folded protein. As a result, there is a need for extensive screening to find antibodies that have high binding affinity to the target in vivo, with no guarantee of success.

[0008] Existing approaches to antibody production by animal immunization do not provide for the efficient and reliable production of monoclonal antibodies having desired functional properties of interest in relation to their target antigen.

[0009] Summary

[0010] In a first aspect, the present disclosure provides an animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

[0011] In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in mounting a primary humoral immune response. In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in B cell maturation.

[0012] In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or both genes selected from IGHM and IGHD. In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of IGHM and / or IGHD.

[0013] In some embodiments, the animal comprises an endogenous nucleotide sequence providing for recombinase-mediated disruption of expression of one or more genes involved in mounting a primary humoral immune response.

[0014] In some embodiments, the endogenous nucleotide sequence comprises target sequences for a recombinase flanking all or part of the nucleotide sequence of a gene involved in mounting a primary humoral immune response.

[0015] In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible expression or activity of the recombinase.

[0016] In some embodiments, the endogenous nucleotide sequence providing for inducible expression or activity of the recombinase encodes a conditional system for controlling expression or activity of the recombinase.

[0017] In some embodiments, the target sequences for a recombinase are loxP sequences, and the recombinase is a Cre recombinase.

[0018] In some embodiments, the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

[0019] In some embodiments, the animal is a mouse, a rat or a rabbit.

[0020] In some embodiments, the endogenous nucleotide sequence comprises an endogenous nucleotide sequence encoding target sequences for a recombinase flanking one or more exons of IGHM and IGHD.

[0021] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4. In some embodiments, the animal comprises an endogenous nucleotide sequence encoding a conditional system for controlling expression and / or activity of a Cre recombinase.

[0022] In some embodiments, expression of the Cre recombinase is under the control of a promoter driving expression in B cell lineage cells.

[0023] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6.

[0024] The present disclosure also provides a method for producing an antigen-binding molecule, comprising administering a peptide / polypeptide, or nucleic acid encoding a peptide / polypeptide, to an animal according to the present disclosure.

[0025] In some embodiments, the method comprises:

[0026] (i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0027] (ii) treating the animal to inhibit its ability to mount a primary immune response; and

[0028] (iii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

[0029] In some embodiments, the method comprises:

[0030] (i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0031] (ii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest; and

[0032] (iii) treating the animal to inhibit its ability to mount a primary immune response.

[0033] In some embodiments, treating the animal to inhibit its ability to mount a primary immune response comprises administering an agent for inducing expression or activity of the recombinase.

[0034] Also provided is a method for producing an antigen-binding molecule, wherein the method comprises:

[0035] (i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0036] (ii) treating the animal to inhibit its ability to mount a primary immune response; and

[0037] (iii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest. In some embodiments, the methods comprise treating the animal to inhibit its ability to mount a primary immune response before, during and / or after the administration of the second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide.

[0038] In some embodiments, the methods comprise treating the animal to inhibit its ability to mount a primary immune response comprises administering an agent that inhibits the expression of, or inhibits the activity of the product of, one or more genes involved in mounting a primary humoral immune response. In some embodiments, treating the animal to inhibit its ability to mount a primary immune response comprises administering an agent that reduces the number / proportion of naive B cells in the animal, reduces the number / proportion of IgM and / or IgD-expressing cells in the animal, and / or inhibits B cell maturation. In some embodiments, the agent inhibits the expression of, or inhibits the activity of the product of, one or more genes selected from IGHM and IGHD.

[0039] In some embodiments, the agent inhibits the activity of IgM and / or IgD. In some embodiments, the agent is selected from, or comprises, an antibody, antigen-binding molecule, polypeptide, decoy receptor, aptamer, sequestering agent or small molecule.

[0040] In some embodiments, the agent inhibits the expression of IGHM and / or IGHD. In some embodiments, e agent is selected from, or comprises, RNAi, siRNA, an antisense nucleic acid, an antisense oligonucleotide, or a gene editing system.

[0041] 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, a rat or a rabbit.

[0042] In some embodiments, the methods further comprise generating a hybridoma producing the antigenbinding molecule capable of binding to a protein / protein complex of interest.

[0043] In some embodiments, the methods further comprise isolating one or more antigen-binding molecules capable of binding to a protein comprising the amino acid sequence of interest.

[0044] In some embodiments, the methods further comprise formulating the antigen-binding molecule capable of binding to a protein comprising the amino acid sequence of interest to a pharmaceutical composition.

[0045] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

[0046] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in mounting a primary humoral immune response. In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in B cell maturation.

[0047] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or both genes selected from IGHM and IGHD.

[0048] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence providing for recombinase-mediated disruption of expression of one or more genes involved in mounting a primary humoral immune response.

[0049] The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, comprising a nucleotide sequence encoding all or part of the nucleotide sequence of a gene involved in mounting a primary humoral immune response, flanked by target sequences for a recombinase.

[0050] In some embodiments, the nucleic acid / plurality of nucleic acids further comprises a nucleotide sequence encoding a conditional system for controlling expression or activity of the recombinase.

[0051] In some embodiments, the target sequences for a recombinase are loxP sequences, and the recombinase is a Cre recombinase.

[0052] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence comprising target sequences for a recombinase flanking one or more exons of IGHM and IGHD.

[0053] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4.

[0054] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence encoding a conditional system for controlling expression and / or activity of a Cre recombinase.

[0055] In some embodiments, expression of the Cre recombinase is under the control of a promoter driving expression in B cell lineage cells.

[0056] In some embodiments, the nucleic acid, or a plurality of nucleic acids, comprises a nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6. The present disclosure also provides a vector, or a plurality of vectors, comprising the nucleic acid or plurality according to the present disclosure.

[0057] The present disclosure also provides a cell comprising the nucleic acid or plurality of nucleic acids or the vector or plurality of vectors according to the present disclosure. In some embodiments, the cell comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.

[0058] 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.

[0059] In some embodiments, the animal is a hyperimmune mouse or a mouse having a hyperimmune phenotype.

[0060] In some embodiments, the animal is a humanized mouse.

[0061] Description

[0062] The present disclosure is broadly concerned with the production of antibodies (particularly IgG antibodies, and preferably ultimately monoclonal IgG antibodies) possessing certain desirable functional properties, which are the product of binding to a region of a target protein / protein complex of interest.

[0063] This can be achieved by (i) immunizing an animal with a first peptide / polypeptide comprising an amino acid sequence of interest ( / .e. (a) an amino acid sequence of a protein / protein complex of interest, or (b) an amino acid sequence which is similar to the amino acid sequence of (a)) to elicit a primary immune response directed against the amino acid sequence of interest, (ii) inhibiting the animal’s ability to mount a primary immune response, and subsequently (iii) immunizing the animal with a second peptide / polypeptide comprising the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

[0064] In some embodiments, this can be achieved by (i) immunizing an animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response with a first peptide / polypeptide comprising an amino acid sequence of interest ( / .e. (a) an amino acid sequence of a protein / protein complex of interest, or (b) an amino acid sequence which is similar to the amino acid sequence of (a)) to elicit a primary immune response directed against the amino acid sequence of interest, (ii) inhibiting the animal’s ability to mount a primary immune response, and subsequently (iii) immunizing the animal with a second peptide / polypeptide comprising the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

[0065] Inhibiting the ability of the animal to mount a primary immune response favors a secondary immune response to the amino acid sequence of interest on administration of the second peptide / polypeptide. Secondary immune responses are associated with the production of antibodies (in particular IgG antibodies) which bind to their target with high affinity.

[0066] Inhibiting a primary immune response and / or promoting a secondary immune response

[0067] Aspects and embodiments of the present disclosure relate to the inhibition of a primary immune response (e.g. a primary humoral immune response) in an animal, e.g. using an agent. In some embodiments the animal comprises an endogenous nucleotide seguence providing for inducible inhibition of a primary immune response.

[0068] The aim is to favor a secondary humoral immune response to the amino acid seguence of interest as presented by the second peptide / polypeptide over a primary humoral immune response to regions of the second peptide / polypeptide which are not present in the first peptide / polypeptide.

[0069] That is, it is intended to suppress a primary humoral immune response to regions of the second peptide / polypeptide other than the amino acid seguence of interest, and / or enhance a secondary humoral immune response to the amino acid seguence of interest as presented in the second peptide / polypeptide.

[0070] To be clear, it is not intended to inhibit a primary humoral immune response to the first peptide / polypeptide. In particular, it is not intended to prevent immunoglobulin isotype switching by, or to deplete, cells activated / stimulated to proliferate by administration of the first peptide / polypeptide, or cells derived from such cells ( / .e. the progeny of cells activated / stimulated to proliferate by administration of the first peptide / polypeptide).

[0071] In some embodiments, inhibition of the ability to mount a primary humoral immune response does not inhibit the development of IgG-, IgA- and / or IgE-expressing cells from cells activated / stimulated to proliferate by administration of the first peptide / polypeptide. In some embodiments, the development of plasma B cells and / or memory B cells from cells activated / stimulated to proliferate by administration of the first peptide / polypeptide is not inhibited.

[0072] In some embodiments, inhibition of the ability to mount a primary humoral immune response does not deplete IgG-, IgA- and / or IgE-expressing cells (e.g. IgG-, IgA- and / or IgE- expressing cells produced by immunoglobulin isotype switching from cells activated / stimulated to proliferate by administration of the first peptide / polypeptide). In some embodiments, plasma B cells and / or memory B cells (e.g. plasma B cells and / or memory B cells produced by immunoglobulin isotype switching from cells activated / stimulated to proliferate by administration of the first peptide / polypeptide) are not depleted.

[0073] Affinity maturation of memory B cells producing antigen-binding molecules capable of recognizing the amino acid sequence of interest is preferably favored over the generation of a primary humoral immune response to regions other than the amino acid sequence of interest following introduction of the second peptide / polypeptide. In such embodiments, the agent may suppress the primary immune response to sequences in the second peptide / polypeptide which are not present in the first peptide / polypeptide.

[0074] Primary and secondary immune responses are components of the adaptive immune response. The adaptive immune response is described e.g. in Janeway’s Immunobiology 9thEdn.; Murphy et al., 2017 (Garland Science, Taylor & Francis), in particular at part IV.

[0075] The primary immune response refers to a response of the adaptive immune system of a subject following an initial exposure to an antigen.

[0076] Triggering of the primary immune response generally involves antigen uptake and processing and presentation on MHC Class II molecules by antigen presenting cells (APCs) such as dendritic cells. In the presence of appropriate costimulation, naive T cells comprising a T cell receptor (TCR) specific for an MHC class Ikpeptide complex become activated and are stimulated to proliferate.

[0077] Antigen also binds to cognate B cell receptors (BCRs) expressed on the surface of naive B cells which express IgM and IgD. The bound antigen is internalized, processed and presented in the context of MHC class Ikpeptide complex on the surface of the B cell. Effector follicular T helper cells (TFH) comprising a TCR specific for the MHC class 11 :peptide complex presented by the B cell are stimulated to produce cytokines such as IL-4 and IL-21 , which induce B cell proliferation and differentiation into plasma B cells and memory B cells.

[0078] Antibodies initially produced by the primary immune response are predominantly IgM isotype antibodies, and bind to the target antigen with low affinity.

[0079] The secondary immune response refers to a response of the adaptive immune system of a subject following exposure to an antigen to which the subject has already produced a primary immune response.

[0080] Upon subsequent exposure, antigen binds to the BCR of memory B cells ( / .e. memory B cells generated by the primary immune response on initial exposure to the antigen), which internalise, process and present to, and thus activate, memory TFH cells.

[0081] Upon antigen-binding, memory B cells in the germinal centers undergo V region somatic hypermutation, resulting in the production of closely related B cell clones having BCRs with different affinities for the antigen. B cells with high affinity BCRs recognise antigen presented by follicular dendritic cells, and process and present antigen to TFH cells, which promote the survival of the B cells. Activated B cells maturing in the germinal centers also undergo immunoglobulin class switching to produce IgG, IgA or IgE antibodies. B cells expressing high-affinity BCRs differentiate to mature plasma B cells, which produce large amounts of high-affinity antibody to the antigen. As used herein, a “humoral immune response” refers to an immune response involving the production of antibodies from B cells.

[0082] A primary humoral immune response may be characterized by: activation of a naive B cell ( / .e. stimulation of a naive B cell to proliferate, differentiate and / or undergo immunoglobulin class switching), production of IL-4 and / or IL-21 by a B cell, immunoglobulin class switching of a IgM- and / or IgD-expressing cell to an IgG-, IgE-, or IgA-expressing cell, differentiation of a naive B cell to a plasma B cell, differentiation of a naive B cell to a memory B cell, and / or production of antibodies (e.g. IgM antibodies) which bind to their target antigen with low affinity.

[0083] B cell development is described e.g. in Pieper et al., J Allergy Clin Immunol (2013) 131 (4):959-71 , which is hereby incorporated by reference in its entirety. Characteristics of B cells are described in e.g. Carsetti et al., Cytometry A. 2022;101 (2):131 -139, which is hereby incorporated by reference in its entirety.

[0084] As used herein, a “naive” B cell refers to a mature B cell which has not encountered the antigen for which the BCR of the B cell is specific. A naive B cell may also be referred to as a mature naive B cell, or a mature B cell. A naive B cell may be characterized by expression of one or more of the following (e.g. at the cell surface): MHC Class II, IgM and IgD. A naive B cell may be characterized by lack of expression (e.g. at the cell surface) of CD27.

[0085] As used herein, a “plasma” B cell refers to a B cell which expresses large amounts of soluble antibody. A plasma B cell may be characterized by expression of one or more of the following (e.g. at the cell surface): CD27, CD38, CD138, CD78, CD126, CXCR4 and BCMA. A plasma B cell may be characterized by lack of expression (e.g. at the cell surface): of CD20 and / or CD24.

[0086] As used herein, a “memory” B cell refers to a B cell formed in a germinal center following a primary immune response. A memory B cell may be characterized by expression of one or more of the following (e.g. at the cell surface): CD19, CD20, CD21 , CD24, CD27, CD95, CD148, MHC Class II and TACL

[0087] Reference to a given gene or protein in the present specification includes isoforms, fragments, variants or homologues of the gene / protein, from any species. It will be appreciated that in aspects and embodiments relating to a particular animal, the gene or protein is the appropriate homologue encoded by the genome of the animal. For example, where the animal is a mouse, the gene / protein may be the mouse homologue of the relevant gene / protein. In a further example of a transgenic mouse encoding the human homologue of the relevant gene / protein, the gene may be the human homologue of the relevant gene / protein.

[0088] A secondary humoral immune response may be characterized by: activation of a memory B cell ( / .e. stimulation of a memory B cell to proliferate and / or differentiate), somatic hypermutation of a memory B cell, differentiation of a memory B cell to a plasma B cell, and / or production of antibodies (e.g. IgG antibodies) which bind to their target antigen with high affinity. Some aspects and embodiments of the present disclosure are concerned with agents providing for inhibition of a primary humoral immune response in an animal. Some aspects and embodiments of the present disclosure are concerned with animals comprising endogenous nucleotide sequences providing for inducible inhibition of a primary humoral immune response in the animal. It will be appreciated that the effect of such inhibition can be to favor / promote a secondary humoral immune response in the animal.

[0089] Also provided by the present disclosure are agents, e.g. as described herein, for use in the methods disclosed herein, i.e. for inhibition of a primary humoral immune response in an animal. Also provided are the use of said agents, i.e. for inhibition of a primary humoral immune response in an animal.

[0090] Herein, “inhibition" may also be referred to as “antagonism”. Agents capable of inhibiting a response / expression / an activity may be referred to as “inhibitors" or “antagonists” of the relevant response / expression / activity.

[0091] In some embodiments, inhibition of a primary humoral immune response in accordance with the present disclosure comprises one or more of: inhibiting the expression of a gene involved in mounting a primary humoral immune response, inhibiting the activity of the product of a gene involved in mounting a primary humoral immune response, reducing the number / proportion of naive B cells, and / or reducing the number / proportion of IgM and / or IgD-expressing cells.

[0092] As used herein, “expression” may refer to gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be analyzed by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, or reporter-based methods.

[0093] As used herein, a factor / activity / cell type which is “involved in” a given response / process (e.g. mounting an immune response, immunoglobulin isotype switching, etc.) refers to a factor / activity / cell type which is implicated in the relevant response / process. The factor / activity / cell type preferably contributes positively to (i.e. promotes, potentiates) the relevant response / process, and may e.g. be required for the relevant response / process, meaning that the response / process does not occur in the absence of the factor / activity / cell type.

[0094] Aspects and embodiments of the present disclosure are concerned with inhibiting the expression (i.e. gene or protein expression) of one or more genes involved in mounting a primary humoral immune response, and / or inhibiting the activity of the product of one or more genes involved in mounting a primary humoral immune response. In preferred embodiments, the one or more genes are not involved in, or required for, mounting a secondary humoral immune response. In some embodiments, the one or more genes involved in mounting a primary humoral immune response are IGHM and / or IGHD.

[0095] Inhibition of gene or protein expression of a given gene may comprise e.g. inhibiting transcription of the gene, inhibiting post-transcriptional processing (e.g. splicing) of RNA transcribed from the gene, reducing the stability of RNA transcribed from the gene, promoting degradation of RNA transcribed from the gene, inhibiting translation of RNA transcribed from the gene into protein, inhibiting post-translational processing of a polypeptide encoded by the gene, reducing the stability of a polypeptide encoded by the gene, or promoting degradation of a polypeptide encoded by the gene.

[0096] Gene expression can be analyzed by means well known to the skilled person. The level of RNA encoding a given gene can be determined e.g. by techniques such as RT-qPCR. Protein expression can also be determined by means well known to the skilled person. The level of a given protein / isoform thereof can be determined e.g. by antibody-based methods including western blot, immunohisto / cytochemistry, flow cytometry, ELISA, etc.

[0097] Inhibition of gene or protein expression of a given gene may be to less than 1 times, e.g. one of <0.99 times, 20.95 times, 20.9 times, 20.85 times, 20.8 times, 20.75 times, 20.7 times, 20.65 times, 20.6 times, 20.55 times, 20.5 times, 20.45 times, 20.4 times, 20.35 times, 20.3 times, 20.25 times, 20.2 times, 20.15 times, 20.1 times, 20.05 times, or 20.01 times the level of expression observed in the uninhibited state. In some embodiments, inhibition of gene or protein expression inhibits greater than 5%, e.g. one of 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% of the expression observed in the uninhibited state.

[0098] Inhibition of gene or protein expression may be achieved e.g. by altering / disrupting the nucleotide sequence of the gene, or altering / disrupting nucleotide sequence required for expression of the gene (e.g. a regulatory sequence governing expression of the gene). In some embodiments, inhibiting gene or protein expression may comprise altering a nucleotide sequence, e.g. by substitution, deletion or insertion of one or more nucleotides. For example, in particular aspects and embodiments the present disclosure contemplates inhibiting gene or protein expression by deletion of all or part of the nucleotide sequence of the relevant gene.

[0099] In some embodiments, altering / disrupting the nucleotide sequence, may comprise e.g. altering / removing a regulatory sequence (e.g. a promoter, an enhancer) for transcription of the gene, introducing a premature stop codon in the sequence transcribed from the gene, altering the nucleotide sequence to encode a truncated and / or non-functional gene product, or altering the nucleotide sequence to encode a gene product which is misfolded and / or degraded. Altering / disrupting the nucleotide sequence inhibit / prevent gene or protein expression from a gene may be referred to as gene ‘knockout’.

[0100] Nucleotides sequences may be disrupted e.g. by homologous recombination, or by target nucleic acid modification using site-specific nucleases (SSNs, also referred to herein as ‘gene editing systems’).

[0101] Modification by homologous recombination may involve the exchange of nucleic acid sequence through crossover events guided by homologous sequences, and is reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4:Unit 4.29 and Vasquez et al., PNAS (2001) 98(15): 8403-8410 both of which are hereby incorporated by reference in their entirety. The homologous sequences flank all or part of the nucleotide sequence to be disrupted. Recombination may be catalysed by a recombinase. For example, in particular aspects and embodiments the present disclosure contemplates disruption of a nucleotide sequence by homologous recombination between loxP sequences, catalysed by a Cre recombinase.

[0102] Disruption of a nucleotide sequence by homologous recombination can be achieved in an animal e.g. as described hereinabove in relation to Cre-LoxP, F\p-FRT and Dre-rox systems.

[0103] Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. (2016) 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequences of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB. SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems.

[0104] ZFN systems are reviewed e.g. in Umov et al., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence. TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “Nl” binds to A, “NG” binds to T and “NN” or “NK” binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501 .). CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the single-guide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0105] As used herein, a “product of” a gene may e.g. refer to a nucleic acid transcribed from the gene, or a peptide / polypeptide (including peptides / polypeptides modified with chemical moieties, e.g. carbohydrate and / or lipid moieties) produced by translation of a nucleic acid transcribed from the gene (and where appropriate, further post-translational processing).

[0106] Inhibition of the activity of the product of a given gene may comprise e.g. inhibiting gene or protein expression of the gene (e.g. as described hereinabove), or inhibiting one or more activities of the product of the gene. An activity of a product of a gene may e.g. be catalytic activity, binding (e.g. protein-protein interaction, such as ligand-receptor binding, multimerization, etc.), signalling, transport, storage, structural support etc.

[0107] Inhibition of an activity of a product of a given gene may be to less than 1 times, e.g. one of 50.99 times, 50.95 times, 50.9 times, 50.85 times, 50.8 times, 50.75 times, 50.7 times, 50.65 times, 50.6 times, 50.55 times, 50.5 times, 50.45 times, 50.4 times, 50.35 times, 50.3 times, 50.25 times, 50.2 times, 50.15 times, 50.1 times, 50.05 times, or 50.01 times the level of the activity observed in the uninhibited state. In some embodiments, inhibition an activity of a product of a given gene inhibits greater than 5%, e.g. one of S>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% of the activity observed in the uninhibited state.

[0108] In some embodiments the agent is capable of decreasing the expression of a target gene or protein ( / .e. a gene or protein involved in mounting a primary immune response), and / or is capable of decreasing activity of said target gene or protein. In some cases, the agent inhibits, degrades, silences, knocks down, reduces or otherwise decreases expression and / or activity of a gene or protein involved in mounting a primary immune response.

[0109] The agent may possess one or more of the following properties in relation to a target gene or protein ( / .e. a gene or protein involved in mounting a primary immune response): acts to inhibit expression of the gene and / or protein; interferes with transcription of the gene; interferes with translation of mRNA encoding the protein; degrades mRNA encoding the protein; binds to the protein; sequesters the protein; competes for binding of the protein; and / or blocks activity of the protein.

[0110] In some embodiments the agent is capable of inhibiting the expression of, or inhibiting the activity of the product of, genes IGHM and / or IGHD. Such agents may be used to treat an animal as described herein. In some embodiments, the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of a primary immune response, as described herein. In some embodiments the animal does not comprise an endogenous nucleotide sequence providing for inducible inhibition of a primary immune response.

[0111] In some embodiments, the agent is an antibody or antigen-binding molecule (both referred to herein as “antigen-binding molecule”) e.g. an anti-IgM or anti-lgD antibody. In some cases, the antigen-binding molecule is specific for a protein involved in mounting a primary immune response, for example selected from IgM and / or IgD. In some cases, the antigen-binding molecule displays specific binding to a protein involved in mounting a primary immune response, for example selected from IgM and / or IgD. In some cases, the antigen-binding molecule displays specific binding to IgM or IgD. In some cases, the antigenbinding molecule is an anti-IgM or anti-lgD antigen-binding molecule.

[0112] The antigen-binding molecule may be an antagonist antigen-binding molecule that inhibits or reduces a biological activity of a target protein involved in mounting a primary immune response, such as IgM or IgD.

[0113] The antigen-binding molecule may bind to a particular region of interest of a target protein involved in mounting a primary immune response, such as IgM or IgD. The antigen-binding region of an antigenbinding molecule may bind to a linear epitope of a target protein involved in mounting a primary immune response, such as IgM or IgD, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). In some embodiments, the antigen-binding region molecule may bind to a conformational epitope of a target protein involved in mounting a primary immune response, such as IgM or IgD, consisting of a discontinuous sequence of amino acids of the amino acid sequence.

[0114] The antigen-binding molecule may be a multispecific antigen-binding molecule. By “multispecific” it is meant that the antigen-binding molecule displays specific binding 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, e.g. comprising non-identical VHs and VLs). Multispecific antigenbinding molecules may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety.

[0115] In some embodiments the antigen-binding molecule binds to a target protein involved in mounting a primary immune response, such as IgM or IgD, and another target (e.g. an antigen other than the target protein), and so is at least bispecific. The term “bispecific” means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.

[0116] The ability of a given polypeptide to bind specifically to a given molecule or another given peptide / polypeptide can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol 2012, 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., J Biomol Screen. 2015, 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay. Binding affinity may be expressed in terms of dissociation constant (KD).

[0117] The region of a peptide / polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibodyantigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety.

[0118] In some embodiments the antigen-binding molecule inhibits the interaction between two binding partners. The ability of an antigen-binding molecule to inhibit interaction between two binding partners can be determined by analysis of the downstream functional consequences of such interaction in an appropriate assay e.g. by detecting the production of protein from a reaction using ELISA, Western blotting or electrophoresis methods.

[0119] A person skilled in the art will be able to produce suitable antigen binding molecules using e.g. techniques as described herein or those known in the art, see e.g. Chiu and Gilliland, Curr Opin Struct Biol. 2016, 38:163-173, Jakobovits A, Curr Opin Biotechnol. 1995 Oct;6(5):561-6, and Bruggemann M et al., Arch Immunol Ther Exp (Warsz). 2015; 63(2): 101-108. One suitable technique is phage display technology, see e.g. 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 may also be produced by techniques such as chemical synthesis (see e.g. Chandrudu et al., Molecules (2013), 18: 4373-4388), recombinant expression such as the techniques set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135- 146, or cell-free-protein synthesis (CFPS; see e.g., Zemella et al. Chembiochem (2015) 16(17): 2420- 2431), all of which are hereby incorporated by reference in their entirety. The antigen-binding molecule may be monoclonal, i.e. a homogenous population of antibodies specifically targeting a single epitope on an antigen. Monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). Suitable polyclonal antibodies can also be prepared using methods well known in the art.

[0120] The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Antigen-binding fragments of antibodies, such as Fab and Fab2 fragments may also be used / provided as can genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognized by early protease digestion experiments. Further confirmation was found by "humanization" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855).

[0121] Antibodies and antigen-binding fragments according to the present disclosure comprise the complementarity-determining regions (CDRs) of an antibody which is capable of binding to the relevant target molecule, i.e. one or more proteins involved in mounting a primary humoral immune response as described herein.

[0122] The agent may be nucleic acid-based or comprise nucleic acid elements. The agent may promote silencing of gene expression via RNA-mediated interference (RNAi) or antisense degradation mechanisms, e.g. via RNase H.

[0123] In some embodiments the agent is, or comprises, an antisense nucleic acid. An "antisense nucleic acid" as referred to herein is a nucleic acid (e.g. DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid (e.g. an mRNA translatable into a protein, such as an FHR protein) and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA) or altering transcript splicing (e.g. by a single stranded morpholino oligo). Antisense nucleic acids may be single stranded, e.g. gapmers, or may be double stranded e.g. siRNA. Antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid (e.g. target mRNA) via Watson-Crick base pairing. In some cases the antisense nucleic acids specifically bind to the target nucleic acid. In some cases, the antisense nucleic acid hybridizes to the target nucleic acid sequence (e.g. mRNA) under stringent hybridization conditions. In some cases, the antisense nucleic acid hybridizes to the target nucleic acid (e.g. mRNA) under moderately stringent hybridization conditions.

[0124] The nucleotide sequence of an antisense nucleic acid is sufficiently complementary to the target nucleic acid of interest such that it binds or hybridises to the target nucleic acid. Thus, if a skilled person knows the sequence of the target nucleic acid, it is easy and routine to design a suitable antisense nucleic acid that will hybridize to the target to achieve the desired effect.

[0125] The target RNA may be an mRNA that encodes for a protein involved in mounting a primary immune response, e.g. IgM and / or IgD.

[0126] In some cases the agent is capable of promoting RNA interference (RNAi). RNAi uses small doublestranded RNA molecules to cause degradation of target mRNA. Non-limiting examples of antisense nucleic acids for use as agents according to the present invention include siRNAs (including their derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA), micro RNAs (miRNA, including their long primary transcripts (pri-miRNAs) and partially processed 60-70 base pair hairpin transcripts (pre-miRNAs)), saRNAs (small activating RNAs) and small nucleolar RNAs (snoRNA) or certain of their derivatives or pre-cursors. Antisense nucleic acid molecules may stimulate RNA interference (RNAi). siRNA nucleic acids are -21-25 nucleotides in length and comprise a guide strand which hybridizes with the target mRNA, plus a complementary passenger strand (e.g., each complementary sequence of the double stranded siRNA is 21-25 nucleotides in length, and the double stranded siRNA is about 21-25 base pairs in length). They promote degradation of the target mRNA via RISC. Structure and function of siRNAs are well known in the art and are described in e.g. Kim and Rossi, Biotechniques. 2008 Apr; 44(5): 613-616. Suitable siRNA molecules for use in the methods of the present invention may be designed by schemes 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 for making siRNA molecules can be found in the websites of several commercial vendors such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine. The sequence of any potential siRNA candidate generally can be checked for any possible matches to other nucleic acid sequences or polymorphisms of nucleic acid sequence using the BLAST alignment program (see the National Library of Medicine internet website). Typically, a number of siRNAs are generated and screened to obtain an effective drug candidate, see, U.S. Pat. No. 7,078,196. siRNAs can be expressed from a vector and / or produced chemically or synthetically. Synthetic RNAi can be obtained from commercial sources, for example, Invitrogen (Carlsbad, Calif.). RNAi vectors can also be obtained from commercial sources, for example, Invitrogen. microRNAs (miRNAs) also regulate gene expression via RISC. They are initially expressed as long primary transcripts (pri-miRNAs), which are processed within the nucleus into 60-70 nucleotide hairpins (pre-miRNAs), which are further processed in the cytoplasm into small double stranded nucleic acids that interact with RISC and target mRNA. miRNAs comprise “seed sequences” that are essential for binding to target mRNA. “Seed sequences” usually comprise six nucleotides and are situated at positions 2-7 at the miRNA 5’ end.

[0127] In some embodiments the agent comprises a double stranded nucleic acid molecule in which one strand is wholly or partially complementary to, or hybridizes with, an mRNA sequence encoding a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD. In some embodiments the agent comprises a siRNA molecule comprising a guide strand complementary to, or that hybridizes with, a portion of an mRNA sequence that encodes all or part of a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD. In some embodiments the agent comprises a miRNA molecule (ph-, pre- or mature miRNA) comprising a seed sequence capable of hybridizing to a portion of an mRNA sequence that encodes all or part of a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD. In some cases the agent is a single stranded antisense oligonucleotide (ASO). ASOs modify expression of a target RNA, either by altering splicing or by recruiting RNase H to degrade the target RNA. RNase H recognises DNA:RNA hybrids formed when the ASO binds to the target RNA. ASOs tend to be 18-30 base pairs in length. Many ASOs are designed as chimeras, comprising a mix of bases with different chemistries, or as gapmers, comprising a central DNA portion surrounded by ‘wings’ of modified bases. ASOs are described in e.g. Scoles et al., Neurol Genet. 2019 Apr; 5(2): e323.

[0128] Antisense nucleic acids may comprise naturally occurring nucleotides or modifications such as e.g. phosphorothioate linkages, phosphorodiamidate linkages, methoxyethyl nucleotide modifications e.g. 2- MOE, ‘locked’ nucleic acids e.g. LNAs, peptide nucleic acids (PNAs), and / or 5’ -methylcytosine modifications.

[0129] In some embodiments the agent comprises an antisense oligonucleotide that is capable of hybridizing to a portion of an mRNA sequence that encodes all or part of a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD.

[0130] Antisense nucleic acids described herein may comprise or consist of nucleotide sequences having 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 to their target nucleic acid. Complementarity may be calculated over the whole length of the antisense nucleic acids and / or over all or part of the target nucleic acid to which the antisense nucleic acid binds.

[0131] The nucleic acid molecule may be an aptamer. The term "aptamer" as used herein refers to oligonucleotides (e.g. short oligonucleotides or deoxyribonucleotides), that bind (e.g. with high affinity and specificity) to proteins, peptides, and small molecules. Aptamers typically have defined secondary or tertiary structure owing to their propensity to form complementary base pairs and, thus, are often able to fold into diverse and intricate molecular structures. The three-dimensional structures are essential for aptamer binding affinity and specificity, and specific three-dimensional interactions drives the formation of aptamer-target complexes. Aptamers can be selected in vitro from very large libraries of randomized sequences by the process of systemic evolution of ligands by exponential enrichment (SELEX as 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 off-rate modified aptamers) (Gold L et al. (2010) Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12):e15004). SOMAmers are short, single stranded deoxyoligonucleotides with protein-like properties thanks to functional groups that mimic amino acid side chains. Applying the SELEX and the SOMAmer technology includes for instance adding functional groups that mimic amino acid side chains to expand the aptamer's chemical diversity. As a result, high affinity aptamers for a target may be enriched and identified. Aptamers may be DNA or RNA molecules and may be single stranded or double stranded. The aptamer may comprise chemically modified nucleic acids, for example in which the sugar and / or phosphate and / or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.

[0132] Aptamers may be synthesized by methods which are well known to the skilled person. For example, aptamers may be chemically synthesized, e.g. on a solid support. Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detrity lated , then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer (e.g., see Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, S. L.; Lyer, R. P. (1992). Tetrahedron 48 (12): 2223).

[0133] Aptamers may be peptides selected or engineered to bind specific target molecules. Peptide aptamers and methods for their generation and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101 , which is hereby incorporated by reference in its entirety. Peptide aptamers may optionally have a minimum length of one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Peptide aptamers may optionally have a maximum length of one of 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. Suitable peptide aptamers may optionally have a length of one of 2-30, 2-25, 2-20, 5-30, 5-25 or 5-20 amino acids.

[0134] Aptamers may have KD’S in the nM or pM range, e.g. less than one of 500nM, 100nM, 50nM, 10nM, 1 nM, 500pM, 100pM.

[0135] An aptamer or SOMAmer suitable for use as described herein may bind to a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD. An aptamer or SOMAmer suitable for use as described herein may display specific binding for a protein involved in mounting a primary immune response, as described herein, e.g., IgM or IgD. The aptamer may inhibit the function of a protein involved in mounting a primary immune response, e.g., IgM or IgD, for example blocking its binding to its cognate binding partner or ligand.

[0136] An agent may be a sequestering agent, e.g., of protein involved in mounting a primary immune response as described herein. The agent may be a protein molecule.

[0137] The agent may be a small molecule. For example, the small molecule may bind to a protein involved in mounting a primary immune response as described herein and prevent / reduce its usual function and / or prevent / reduce its interaction with a cognate binding partner. The small molecule may prevent / reduce correct folding of the target protein. An agent may be a decoy receptor. In some embodiments, a decoy receptor refers to a peptide or polypeptide capable of binding a protein involved in mounting a primary immune response as described herein. The receptor may be a receptor, including fragments and derivatives thereof, for a protein involved in mounting a primary immune response as described herein. A decoy receptor may be able to recognise and bind a specific ligand but may not be able to signal or activate a subsequent response. A decoy receptor may bind a protein involved in mounting a primary immune response as described herein to form a complex. A decoy receptor may act as an inhibitor of a protein involved in mounting a primary immune response as described herein by preventing / reducing the ability or availability of the proteins to bind to their receptor(s). A decoy receptor may act as an inhibitor of a protein involved in mounting a primary immune response by binding to a binding partner of said protein, e.g. in the region that would usually be bound by a binding partner, and preventing interaction between said protein and one or more binding partners.

[0138] A decoy receptor may be soluble (not membrane bound), or may be membrane bound e.g. expressed on a cell surface. Decoy receptors may be presented and / or administered on a surface of a nanocarrier, for example, a nanoparticle, liposome, bead, polymer, metal particle, dendrimer, nanotube or micro-sized silica rods, see e.g. Wilczewska AZ et al., Pharmacol Rep. 2012, 64(5):1020-1037.

[0139] Methods for detecting whether a decoy receptor competes for binding for a target protein may be for example SPR (see e.g. Hearty et al., Methods Mol Biol 2012, 907:411-442), competition ELISA assay or solid phase binding assays. Other suitable methods will be known in the art.

[0140] Agents that decrease the amount of a protein involved in mounting a primary immune response and / or decrease expression of a gene encoding a protein involved in mounting a primary immune response may fall into more than one of the categories above. For example, an antigen binding molecule or decoy receptor may also be a sequestering agent.

[0141] Any of the agents described herein may be optionally isolated and / or substantially purified.

[0142] Reducing the number / proportion of naive B cells or IqM and / or IqD-expressinq cells

[0143] Aspects and embodiments of the present disclosure are concerned with reducing the number / proportion of naive B cells or reducing the number / proportion of IgM and / or IgD-expressing cells in an animal. It will be appreciated that naive B cells express IgM and / or IgD.

[0144] The purpose is to deplete / remove the population of cells that would otherwise be available for mounting a primary humoral immune response following subsequent immunization of the animal. That is, the number / proportion of naive B cells, or IgM and / or IgD-expressing cells, is reduced to reduce / prevent the ability of the animal to mount a primary humoral immune response to an antigen following subsequent challenge. It is not intended to deplete / remove cells activated / stimulated to proliferate by administration of the first peptide / poly peptide, or cells derived from such cells ( / .e. the progeny of cells activated / stimulated to proliferate by administration of the first peptide / polypeptide).

[0145] In some embodiments, treatment of the animal to reduce the number / proportion of naive B cells or IgM and / or IgD-expressing cells is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have undergone immunoglobulin isotype switching ( / .e. to IgG-, IgE-, or I gA-ex pressing cells). In some embodiments, treatment of the animal to reduce the number / proportion of naive B cells or IgM and / or IgD-expressing cells is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have differentiated into plasma B cells and / or memory B cells.

[0146] In some embodiments, treatment of the animal to inducibly reduce the number / proportion of naive B cells or IgM and / or IgD-expressing cells is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have undergone immunoglobulin isotype switching ( / .e. to IgG-, IgE-, or IgA-expressing cells). In some embodiments, treatment of the animal to inducibly reduce the number / proportion of naive B cells or IgM and / or IgD-expressing cells is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have differentiated into plasma B cells and / or memory B cells.

[0147] In some embodiments, reducing the number / proportion of naive B cells or reducing the number / proportion of IgM and / or IgD-expressing cells does not comprise reducing the number / proportion of IgG-, IgE-, or IgA-expressing cells. In some embodiments, reducing the number / proportion of naive B cells or reducing the number / proportion of IgM and / or IgD-expressing cells does not comprise reducing the number / proportion of plasma B cells and / or memory B cells.

[0148] A reduction in the number / proportion of naive B cells or a reduction in the number / proportion of IgM and / or IgD-expressing cells can be achieved by inhibition of the development of such cells. Inhibition of the development of naive B cells or IgM and / or IgD-expressing cells may be achieved by inhibiting the expression or activity of one or more factors involved in the development / maturation of a precursor cell to the relevant cell type. Inhibiting the development of naive B cells or IgM and / or IgD-expressing cells may comprise inhibiting the maturation of a precursor cell to a naive B cell or an IgM and / or IgD-expressing cell.

[0149] As used herein, a “precursor cell to a naive B cell” refers to a cell type upstream of a naive B cell in course of B cell development. In some embodiments, a precursor cell to a naive B cell may be selected from: a stem cell, a pro-B cell, an early pro-B cell, a late pro-B cell, a pre-B cell, a large pre-B cell, a small pre-B cell or an immature B cell. A stem cell may be a hematopoietic stem cell, and may e.g. be characterized by expression (e.g. surface expression) of CD34, and / or lack of expression (e.g. surface expression) of CD10. An early pro-B cell may be characterized by expression (e.g. surface expression) of CD10, CD43, CD45 and / or MHC class II. A late pro-B cell may be characterized by expression (e.g. surface expression) of CD19, CD43, CD45 and / or MHC class II. A large pre-B cell may be characterized by expression (e.g. surface expression) of pre-BCR, CD19, CD43, CD45 and / or MHC class II. A small pre-B cell may be characterized by expression (e.g. surface expression) of pre-BCR, CD19, CD45 and / or MHC class II. An immature B cell may be characterized by expression (e.g. surface expression) of CD10, CD19, CD20, CD24, CD38, CD45, IgM and / or MHC class II, and / or lack of expression (e.g. surface expression) of CD27.

[0150] A reduction in the number / proportion of naive B cells or a reduction in the number / proportion of IgM and / or IgD-expressing cells can also be achieved by inhibition of the expression of one or more factors expressed by such cells. For example, a reduction in the number / proportion of naive B cells or IgM and / or IgD-expressing cells might be achieved by inhibition of the expression of IGHM and / or IGHD.

[0151] A reduction in the number / proportion of naive B cells or a reduction in the number / proportion of IgM and / or IgD-expressing cells may be achieved by inhibition of the activity of one or more factors expressed by such cells. For example, a reduction in the number / proportion of naive B cells or IgM and / or IgD- expressing cells might be achieved by inhibition of the activity of IgM and / or IgD.

[0152] Inhibiting immunoglobulin isotype switching

[0153] Aspects and embodiments of the present disclosure are concerned with inhibiting immunoglobulin isotype switching. The general purpose is to remove the ability of the animal to mount an IgG response to regions of the second peptide / polypeptide which are dissimilar to regions of the first peptide / polypeptide following subseguent immunization.

[0154] Some aspects and embodiments of the present disclosure comprise inhibiting the expression (gene or protein expression) of a gene involved in immunoglobulin isotype switching and / or inhibiting the activity of the product of a gene involved in immunoglobulin isotype switching.

[0155] Immunoglobulin class switching is also referred to as “isotype switching” and “class switch recombination”, and is reviewed e.g. in Stavnezer and Schrader, J Immunol. (2014) 193(11): 5370-5378, which is hereby incorporated by reference in its entirety.

[0156] Mature naive B cells express both IgM and IgD. Activation by binding of antigen causes the cells to proliferate, and if they encounter the appropriate factors (e.g. IL-4) they are triggered to undergo class switch recombination to switch from expressing IgM and IgD to expression of IgG, IgE, or IgA. During class switching, the constant region of the immunoglobulin heavy chain changes but the variable regions, and therefore antigenic specificity, stay the same.

[0157] Immunoglobulin class switching involves replacement of the p and 5 heavy chain constant (CH) regions of the expressed Ig with y, E or a CH regions, and occurs by deletional recombination between two different switch (S) regions. Class switch recombination (CSR) is instigated by activation-induced cytidine deaminase (AICDA), which converts cytosines in S regions to uracils. The uracils are subsequently removed by two DNA repair pathways, resulting in mutations, single-strand DNA breaks, and the doublestrand breaks required for CSR.

[0158] Aspects and embodiments of the present disclosure concern inhibition of immunoglobulin class switching of IgM- and / or IgD-expressing B cells to IgG-, IgE-, and / or I gA-ex pressing cells. In particular embodiments, the present disclosure concerns inhibition of immunoglobulin class switching of IgM- and / or IgD-expressing B cells to IgG-expressing B cells.

[0159] It is not intended to inhibit immunoglobulin isotype switching by cells activated / stimulated to proliferate by administration of the first peptide / polypeptide, or cells derived from such cells ( / .e. the progeny of cells activated / stimulated to proliferate by administration of the first peptide / polypeptide).

[0160] In some embodiments, treatment of the animal to inhibit, e.g. inducibly inhibit, immunoglobulin isotype switching is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have undergone immunoglobulin isotype switching ( / .e. to IgG-, IgE-, or I gA-ex pressing cells). In some embodiments, treatment of the animal to inhibit, e.g. inducibly inhibit, immunoglobulin isotype switching is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have differentiated into plasma B cells and / or memory B cells.

[0161] In some embodiments, inhibiting immunoglobulin isotype switching does not comprise inhibiting immunoglobulin isotype switching by cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells).

[0162] IgM and IgD are involved in immunoglobulin class switching. Dual depletion of both lgM+ and lgD+ cells has been shown to prevent the development of IgG after following immunization (Chentoufi et al., Cellular Immunology (2000) 205: 40-51). Thus inhibiting the expression of IGHM and / or IGHD will impair immunoglobulin class switching.

[0163] In some embodiments, inhibition of immunoglobulin class switching comprises inhibition of the expression or activity of one or more factors involved in immunoglobulin class switching. In some embodiments, inhibition of immunoglobulin class switching comprises inhibition of the expression of, or activity of the product of, one or both of IGHM and / or IGHD.

[0164] It will be appreciated that inhibition of immunoglobulin class switching can also be achieved e.g. by reducing the number / proportion of naive B cells, or reducing the number / proportion of IgM and / or IgD- expressing cells, e.g. as described hereinabove. That is, inhibition of immunoglobulin class switching can be achieved by depletion / removal of the population of cells which would otherwise undergo immunoglobulin class switching.

[0165] Aspects and embodiments of the present disclosure comprise treating an animal to inhibit a primary immune response (e.g. a primary humoral immune response), and / or promote a secondary immune response (e.g. a secondary humoral immune response).

[0166] Aspects and embodiments of the present disclosure comprise treating an animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary immune response so as to inhibit a primary immune response (e.g. a primary humoral immune response), and / or promote a secondary immune response (e.g. a secondary humoral immune response).

[0167] In some embodiments, the methods of the disclosure comprise administering an agent, e.g. as disclosed hereinabove, which induces inhibition of a primary immune response, and / or which promotes a secondary immune response, in the animal. In some embodiments, the agent induces inhibition of a primary immune response in the animal.

[0168] In some embodiments, the agent is effective to inhibit a primary immune response, and / or promote a secondary immune response, in the animal. In some embodiments the agent inhibits the expression of a gene involved in mounting a primary humoral immune response, inhibits the activity of the product of a gene involved in mounting a primary humoral immune response, reduces the number / proportion of naive B cells, reduces the number / proportion of IgM and / or IgD-expressing cells, inhibits immunoglobulin isotype switching, inhibits the expression of a gene involved in immunoglobulin isotype switching and / or inhibits the activity of the product of a gene involved in immunoglobulin isotype switching.

[0169] In some embodiments, the agent induces expression or activity of one or more factors resulting in inhibition of a primary immune response, and / or promotion of a secondary immune response.

[0170] Aspects and embodiments of the present disclosure relate to the use of such an agent in methods of producing an antigen-binding molecule, e.g. according to methods of producing an antigen-binding molecule as described herein.

[0171] In some embodiments, the agent is capable of inhibiting the expression (gene or protein expression) of a gene involved in mounting a primary humoral immune response. In some embodiments, the agent is an agent capable of inhibiting the expression (gene or protein expression) of a gene involved in immunoglobulin isotype switching.

[0172] In some embodiments, the agent is capable of altering / disrupting the nucleotide sequence of a target gene ( / .e. a gene involved in mounting a primary immune response), or altering / disrupting nucleotide sequence required for expression of the target gene (e.g. a regulatory sequence governing expression of the target gene, transcription factor). In some embodiments, the agent is capable of inducing alteration / disruption of the nucleotide sequence of a target gene ( / .e. a gene involved in mounting a primary immune response) by homologous recombination. In some embodiments, the agent is capable of increasing the expression or activity of a recombinase capable of altering / disrupting the nucleotide sequence of a target gene ( / .e. a gene involved in mounting a primary immune response).

[0173] In some embodiments the animal comprises an endogenous nucleotide sequence that encodes a tamoxifen / 4-hydoxytamoxifen-controlled system for controlling activity of the recombinase.

[0174] In aspects and embodiments employing such systems, an agent capable of inhibiting a primary immune response, and / or promoting a secondary immune response may be tamoxifen / 4-hydoxytamoxifen. Similarly, methods of the present disclosure concerning such systems may comprise administering tamoxifen / 4-hydoxytamoxifen to the animal to inhibit the ability of the animal to mount a primary immune response.

[0175] Nucleotide sequences

[0176] Aspects and embodiments of the present disclosure relate to animals comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

[0177] As used herein, a nucleotide sequence "providing for” inducible inhibition of a primary humoral immune response may encode one or more factors involved in (e.g. which are required for, or which allow for) inducible inhibition of a primary humoral immune response.

[0178] In some embodiments, the nucleotide sequence provides for site-specific recombinase-mediated inhibition of expression of one or more (e.g. one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) genes involved in mounting a primary humoral immune response.

[0179] In some embodiments, the nucleotide sequence provides for site-specific recombinase-mediated inhibition of expression of one or both of IGHM and / or IGHD.

[0180] Site-specific recombinase (SSR) systems for inhibition of gene expression are well known in the art. Such SSR systems include e.g. Cre-LoxP, F\p-FRT and Dre-rox systems, and are described e.g. in Branda and Dymecki, Developmental Cell (2004) 6(1): 7-28 and Kim et al., Lab Anim Res. (2018) 34(4): 147-159, both of which are hereby incorporated by reference in their entirety. Variants of such SSR systems and other SSR systems are also well known in the art and can similarly be employed to the ends of inhibiting expression of a target gene of interest.

[0181] Targeted disruption of a nucleotide sequence can be achieved by providing target sequences for a recombinase either side of ( / .e. upstream / 5’ of and downstream / 3’ of) all or part of the nucleotide sequence of the gene, or a nucleotide sequence required for expression of the gene. In the presence of the corresponding recombinase, the homologous recombination occurs between the target sequences, disrupting of the nucleotide sequence of the gene or the nucleotide sequence required for expression of the gene.

[0182] In the Cre-loxP system, a Cre recombinase binds to inverted repeats of loxP target sequences and promotes recombination and excision of the nucleotide sequence between the loxP target sequences. As used herein, “a Cre recombinase” refers to any peptide / polypeptide having the catalytic activity of Cre recombinase. A Cre recombinase may comprise the amino acid sequence of UniProtKB Q71TG5-1 , v1 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of UniProtKB Q71TG5-1 , v1. Cre recombinases include e.g. fusion proteins of Cre recombinase, including e.g. CreERT and CreERT2 described hereinbelow.

[0183] Whilst the above example describes the use of SSR systems for targeted excision of a nucleotide sequence, SSR systems can also be employed for targeted inversion, insertion and translocation of nucleotide sequences.

[0184] SSR-mediated gene knockout, in particular knockout using the Cre-loxP system, is described e.g. in Kim et al., Lab Anim Res. (2018) 34(4): 147-159, which is incorporated by reference herein.

[0185] It will be appreciated that alteration / disruption using a SSR system in accordance with the present disclosure inhibits / prevents gene or protein expression of the gene. In some embodiments, alteration / disruption using a SSR system inhibits / prevents the production of a product encoded by the unaltered nucleotide sequence of the gene.

[0186] In some embodiments, alteration / disruption using a SSR system reduces / prevents transcription of the gene, introduces a premature stop codon in the sequence transcribed from the gene, alters the nucleotide sequence to encode a truncated and / or non-functional gene product, or alters the nucleotide sequence to encode a gene product which is misfolded and / or degraded.

[0187] In some embodiments, the nucleotide sequence of the present disclosure encodes factors providing for site-specific recombinase-mediated disruption of expression of one or more genes involved in mounting a primary humoral immune response.

[0188] In some embodiments, the nucleotide sequence comprises target sequences for a recombinase flanking all or part of the nucleotide sequence of a gene involved in mounting a primary humoral immune response. In some embodiments, the nucleotide sequence comprises target sequences for a recombinase flanking all or part of a nucleotide sequence required for expression of the gene.

[0189] In some embodiments, the target sequences for a recombinase are loxP sequences, and the recombinase is a Cre recombinase. In some embodiments, the target sequences for a recombinase are FRT sequences, and the recombinase is Flp recombinase. In some embodiments, the target sequences for a recombinase are rox sequences, and the recombinase is Dre recombinase.

[0190] In some embodiments, the nucleotide sequence flanked by target sequences for a recombinase is a nucleotide sequence required for expression of a gene product from the gene. In some embodiments, the nucleotide sequence flanked by target sequences for a recombinase encodes all or part of one or more exons of the gene. In some embodiments, the nucleotide sequence flanked by target sequences for a recombinase encodes all or part of a regulatory sequence controlling expression of the gene, e.g. a promoter or an enhancer.

[0191] It will be appreciated that target sequences for a recombinase “flanking” a given nucleotide sequence are provided either side of the given nucleotide sequence. That is, they are provided 5’ of and 3’ to the given nucleotide sequence, in the context of the endogenous nucleotide sequence of the present disclosure. By way of illustration, an endogenous nucleotide sequence of the present disclosure may comprise the following arrangement of nucleotide sequences:

[0192] 5’-[target sequence for a recombinase]-[all or part of the nucleotide sequence of a target gene / nucleotide sequence required for expression of the target gene]-[target sequence for a recombinase]-3’

[0193] In some embodiments, a target sequence for a recombinase may be within about 5, 10, 50, 100, 250, 500 or 1000 bases of the first and / or last base of the nucleotide sequence of the target gene / nucleotide sequence required for expression of the target gene. That is, in some embodiments the final base of target sequence for a recombinase may be provided within about 5, 10, 50, 100, 250, 500 or 1000 bases of the first base of the nucleotide sequence of the target gene / nucleotide sequence required for expression of the target gene, and / or the first base of target sequence for a recombinase may be provided within about 5, 10, 50, 100, 250, 500 or 1000 bases of the final base of the nucleotide sequence of the target gene / nucleotide sequence required for expression of the target gene.

[0194] In some aspects and embodiments, the endogenous nucleotide sequence of the present disclosure further comprises a nucleotide sequence encoding the recombinase, i.e. the recombinase corresponding to the relevant target sequences for a recombinase. By way of illustration, in embodiments where the target sequences for a recombinase are loxP sequences, the endogenous nucleotide sequence may further comprise a nucleotide sequence encoding a Cre recombinase.

[0195] The endogenous nucleotide sequence may further comprise nucleotide sequence encoding regulatory nucleotide sequences (e.g. promoter and / or enhancer) for expression of the recombinase. The regulatory nucleotide sequences may be operably linked to the nucleotide sequence encoding the recombinase. In some embodiments the endogenous nucleotide sequence encodes an expression cassette for the recombinase. In aspects and embodiments of the present disclosure, the endogenous nucleotide sequence provides for inducible inhibition of a primary humoral immune response. Inhibition of a primary humoral immune response may be inducible, e.g. in response to a given chemical or physical treatment.

[0196] In embodiments employing SSR systems, SSR-mediated inhibition of gene expression may be inducible by increasing the level or activity of the relevant recombinase, e.g. by administering the recombinase and / or increasing expression of the recombinase.

[0197] In embodiments employing an endogenous nucleotide sequence comprising target sequences for a recombinase flanking all or part of the nucleotide sequence of a gene involved in mounting a primary humoral immune response, or flanking all or part of a nucleotide sequence required for expression of the gene, inhibition of expression of the relevant gene may be induced by administering the recombinase to the animal, administering nucleic acid (e.g. vector) encoding the recombinase to the animal, and / or treating the animal to increase the expression or activity of the recombinase.

[0198] It will be appreciated that increasing the level of the recombinase and / or activity of the recombinase in cells comprising the endogenous nucleotide sequence comprising target sequences for a recombinase flanking the target nucleotide sequence of interest potentiates recombination, and thus disruption of the target gene and consequent inhibition of its expression.

[0199] In some embodiments, upregulation of expression / activity of the recombinase is chemically inducible. Chemically-inducible SSR-mediated gene knockout is described e.g. in Kim etal., Lab Anim Res. (2018) 34(4): 147-159 (incorporated by reference hereinabove).

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

[0201] In aspects and embodiments employing such conditional systems, an agent capable of inhibiting a primary immune response, and / or promoting a secondary immune response may be an agent inducing expression or activity of the recombinase.

[0202] In some embodiments, the endogenous nucleotide sequence comprises a nucleotide sequence encoding a conditional expression system for controlling expression of the recombinase.

[0203] “Conditional expression” may also be referred to herein as “inducible expression”, and refers to gene / protein expression contingent on certain conditions, e.g. the presence of a particular agent. Conditional expression systems are well known in the art and are reviewed e.g. in Ryding et al. Journal of Endocrinology (2001) 171 , 1-14, which is hereby incorporated by reference in its entirety.

[0204] Conditional expression systems include systems which employ tetracycline-controlled transcriptional activation, such as Tet-On and Tet-Off systems. The Tet-On system employs nucleic acid encoding a reverse tetracycline transactivator (rtTA) protein, which is a fusion of the tetracycline repressor (TetR) protein mutated at four amino acid positions to reverse the response to tetracycline / doxycycline, and the activation domain of VP16. In the absence of tetracycline (or a derivative thereof such as doxycycline) rtTA does not bind to TetO operator sequences and the polypeptide is not expressed. In the presence of tetracycline / doxycycline, rtTA binds to TetO sequences in the TRE and activates transcription of the nucleic acid downstream of the promoter. Tet-On systems are described in Das et al., Curr Gene Ther. (2016)16(3):156-67 (hereby incorporated by reference in its entirety), and include systems using optimized rtTA variants such as the Tet-On Advanced system (which uses the rtTA variant protein rtTA2s-M2) and Tet-On 3G system.

[0205] The Tet-On Advanced system is also described in Urlinger et al. Proc. Natl. Acad. Sci. U.S.A. (2000) 97(14)7963-8 (hereby incorporated by reference in entirety), and Tet-On 3G is described in Zhou et al., Gene Ther. 13(19):1382-1390 (hereby incorporated by reference in entirety).

[0206] The Tet-Off system employs nucleic acid encoding a tetracycline transactivator (tTA) protein, which is a fusion of the tetracycline repressor (TetR) protein and the activation domain HSV protein VP16. In the absence of tetracycline (or a derivative thereof such as doxycycline) tTA binds to TetO operator sequences, which form a tetracycline-response element (TRE), located just upstream of a minimal promoter (e.g. CMV promoter). Binding of tTA to the TetO sequences in the TRE activates transcription of the nucleic acid downstream of the promoter. In the presence of tetracycline / doxycycline tTA is unable to bind TetO sequences in the TRE, and transcription of the nucleic acid downstream of the promoter is repressed. The Tet-Off system is described in Bujard et al. Proc. Natl. Acad. Sci. U.S.A. (1992) 89(12):5547-51 (hereby incorporated by reference in entirety).

[0207] Other tetracycline-controlled systems include the T-REx conditional expression system described in Yao et al., Human Gene Therapy (1998) 9(13): 1939-1950 (hereby incorporated by reference in entirety). In the T-REx system, TetR is expressed under the control of a CMV promoter, and in the absence of tetracycline / doxycycline TetR binds to two Tet operator 2 (TetO2) sequences upstream of the region of interest and repression of transcription of the region of interest. When tetracycline / doxycycline is added to the system it binds to TetR and causes its release from the TetO2 sequences, thereby releasing the region of interest from transcriptional repression.

[0208] In some embodiments the endogenous nucleotide sequence of the present disclosure comprises nucleotide sequence(s) encoding elements of a system for providing conditional expression of the recombinase. In some embodiments the endogenous nucleotide sequence encodes a tetracycline / doxycycline-controlled transcriptional activation system for controlling expression of the recombinase.

[0209] In aspects and embodiments employing such systems, an agent capable of inhibiting a primary immune response, and / or promoting a secondary immune response may be tetracycline / doxycycline. Similarly, methods of the present disclosure concerning such systems may comprise administering tetracycline / doxycycline to the animal to inhibit the ability of the animal to mount a primary immune response.

[0210] In some embodiments the endogenous nucleotide sequence encodes a conditional system for controlling activity of the recombinase.

[0211] In some embodiments, the recombinase encoded by the endogenous nucleotide sequence comprises a moiety providing for inducible regulation of recombinase activity. Regulation of recombinase activity may be achieved e.g. by influencing subcellular localization of the recombinase. That is, regulation of recombinase activity may be achieved by controlling access to target sequences for the recombinase.

[0212] Inducible recombinase activity systems are described e.g. in Kim et al., Lab Anim Res. (2018) 34(4): 147- 159 (incorporated by reference hereinabove). The tamoxifen-inducible Cre system employs a fusion protein comprising Cre recombinase fused to the estrogen receptor containing a mutated ligand binding domain (ER-LBD), known as CreER recombinase. CreER is normally localized to the cytoplasm of cells expressing the fusion protein, in a form that binds to HSP90. However, binding of synthetic steroids such tamoxifen or 4-hydoxytamoxifen disrupts interaction between HSP90 and CreER, and CreERT ( / .e. CreER-tamoxifen) translocates to the nucleus where it binds loxP target sequences and exerts recombinase activity. CreERT2 is a variant of CreER which is ~10 times more sensitive to 4-OHT in vivo.

[0213] In some embodiments, nuclear translocation of the recombinase is inducible. In some embodiments, the recombinase comprises a moiety comprising or consisting of the estrogen receptor having a mutated ligand binding domain (ER-LBD). In some embodiments, the recombinase is a Cre recombinase. In some embodiments, the Cre recombinase is CreERT, CreERT2 or a variant thereof. In preferred embodiments, the Cre recombinase is CreERT2.

[0214] In some embodiments the endogenous nucleotide sequence encodes a tamoxifen / 4-hydoxytamoxifen- controlled system for controlling activity of the recombinase.

[0215] In aspects and embodiments employing such systems, an agent capable of inhibiting a primary immune response, and / or promoting a secondary immune response may be tamoxifen / 4-hydoxytamoxifen. Similarly, methods of the present disclosure concerning such systems may comprise administering tamoxifen / 4-hydoxytamoxifen to the animal to inhibit the ability of the animal to mount a primary immune response.

[0216] Expression of the recombinase may be under the control of regulatory sequences driving expression in cells of hematopoietic origin, e.g. regulatory sequences driving expression in B cell lineage cells. Accordingly, in some embodiments the endogenous nucleotide sequence encodes a recombinase under the control of regulatory sequence(s) (e.g. a promoter) driving expression in cells of hematopoietic origin. In some embodiments the endogenous nucleotide sequence encodes a recombinase under the control of regulatory sequence(s) (e.g. a promoter) driving expression in B cell lineage cells.

[0217] In some embodiments, expression of the recombinase may be under the control of cell type- or tissuespecific regulatory sequence(s). For example, expression of the recombinase may be under the control of cell type- or tissue-specific promoter or enhancer. In this way, expression of the recombinase, and thus SSR-mediated gene knockout, may be restricted to target cells or tissues of interest. Accordingly, in some embodiments the endogenous nucleotide sequence encodes a recombinase under the control of cell type- or tissue-specific regulatory sequence(s), e.g. a cell type- or tissue-specific promoter.

[0218] In some embodiments the endogenous nucleotide sequence encodes a recombinase under the control of hematopoietic cell or tissue-specific regulatory sequence(s), e.g. a hematopoietic cell or tissue-specific promoter. In some embodiments the endogenous nucleotide sequence encodes a recombinase under the control of B cell lineage-specific regulatory sequence(s), e.g. a B cell lineage-specific promoter.

[0219] In some embodiments the endogenous nucleotide sequence encodes a recombinase (e.g. a Cre recombinase, e.g. CreERT2) under the control of the CD79A promoter.

[0220] In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase (e.g. loxP sequences) flanking one or more exons of one or more genes involved in mounting a primary humoral immune response. In some embodiments, one or more genes involved in mounting a primary humoral immune response are IGHM and / or IGHD.

[0221] In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes the flanked exons of the relevant gene(s). In some embodiments, recombinase- mediated excision of the region flanked by target sequences for a recombinase removes the entire coding sequence of the relevant gene(s). In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes the promoter for transcription of the relevant gene(s). In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes / disrupts one or more splice donor and / or acceptor sites of encoded by the relevant gene(s). In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes the translation initiation codon for translation of RNA encoded by the relevant gene(s). In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase introduces a frameshift in the nucleotide sequence of the relevant gene(s). In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase has the result that the locus encodes a truncated and / or nonfunctional form of the protein(s) encoded by the relevant gene(s). In some embodiments, recombinase- mediated excision of the region flanked by target sequences for a recombinase results in non-sense- mediated degradation of the RNA transcribed from the locus. In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase (e.g. loxP sequences) flanking one or more exons of IGHM. In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase flanking one or more of exons 1 , 2, 3, 4, 5 and 6 of IGHM. In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase flanking exons 1 to 6 of IGHM. In some embodiments, recombinase- mediated excision of the region flanked by target sequences for a recombinase removes the entire coding sequence of IGHM. In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes the promoter for transcription of IGHM.

[0222] In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase (e.g. loxP sequences) flanking one or more exons of IGHD. In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase flanking one or more of exons 1 , 2 and 3 of IGHD. In some embodiments, the endogenous nucleotide sequence encodes target sequences for a recombinase flanking exons 1 to 3 of IGHD. In some embodiments, recombinase- mediated excision of the region flanked by target sequences for a recombinase removes the promoter for transcription of IGHD.

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

[0224] In some embodiments, the endogenous nucleotide sequence encodes target sequences flanking the region of the IGHM / IGHD locus shown in SEQ ID NO:3.

[0225] In some embodiments, the endogenous nucleotide sequence comprises, or consists of a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4, e.g. one of >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:4. In some embodiments, the endogenous nucleotide sequence comprises, or consists of, the nucleotide sequence of SEQ ID NO:4. In some embodiments, following recombinase-mediated excision the IGHM / IGHD locus comprises a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:5, e.g. one of >60%, >61%, >62%, >63%, >64%, >65%, >66%, >67%, >68%, >69%, >70%, >71 %, >72%, >73%, >74%,

[0226] £75%, £76%, £77%, £78%, £79%, £80%, £81%, £82%, £83%, £84%, £85%, £86%, £87%, £88%, £89%,

[0227] £90%, £91%, £92%, £93%, £94%, £95%, £96%, £97%, £98%, £99% or 100% nucleotide sequence identity to SEQ ID NO:5. In some embodiments, following recombinase-mediated excision the

[0228] IGHM / IGHD locus comprises the nucleotide sequence of SEQ ID NO:5.

[0229] In some embodiments, the endogenous nucleotide sequence encodes a recombinase, e.g. a Cre recombinase. In preferred embodiments, the Cre recombinase is CreERT2.

[0230] In some embodiments, the endogenous nucleotide sequence encodes a recombinase (e.g. a Cre recombinase, e.g. CreERT2) under the control of a conditional system for controlling expression and / or activity of the recombinase. In some embodiments, the endogenous nucleotide sequence encodes a tamoxifen / 4-hydoxytamoxifen-controlled system for controlling activity of the recombinase.

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

[0232] In some embodiments, the endogenous nucleotide sequence comprises a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6, e.g. one of £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:6. In some embodiments, endogenous nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:6.

[0233] Animals

[0234] The present disclosure relates to animals for the production of antigen-binding molecules, in which a primary immune response can be inhibited, e.g. using a method described herein.

[0235] Aspects and embodiments of the present disclosure relate to animals comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary immune response. It will be appreciated that endogenous nucleotide sequence provides for inducible inhibition of a primary humoral immune response in the animal.

[0236] In aspects and embodiments of the present disclosure, an animal may comprise an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response according to an embodiment described herein. An animal according to the present disclosure may be an individual / subject of any species of animal. In preferred embodiments, the animal is a non-human animal.

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

[0238] In some embodiments the animal is an individual / subject of a species of the order Rodentia (e.g. an induvial / subject of a species of the genus Mus, Rattus or Cavia) or Lagomorpha (e.g. an induvial / subject of a species of the family Leporidae). In some embodiments, the animal is a mouse, a rat or a rabbit.

[0239] In some embodiments, the animal is a mouse (that is, in some embodiments the animal is an individual / subject of a species of the genus Mus e.g. an individual / subject of the species Mus musculus).

[0240] In some embodiments, the animal is a rat (e.g. an individual / subject of the genus Rattus; e.g. an individual / subject of the species Rattus norvegicus or Rattus rattus).

[0241] In some embodiments, the animal is a rabbit (e.g. an individual / subject of a species of the genus Oryctolagus; e.g. an individual / subject of the species Oryctolagus cuniculus).

[0242] An animal according to the present disclosure may have a genome comprising a nucleotide sequence providing for inducible inhibition of a primary humoral immune response. The nucleotide sequence providing for inducible inhibition of a primary humoral immune response is preferably comprised in genomic DNA of the animal. That is, the nucleotide sequence may be integrated into or form part of the genomic DNA of cells of the animal.

[0243] An animal according to the present disclosure may have a genome comprising, or may comprise genomic DNA comprising, a nucleotide sequence providing for inducible inhibition of a primary humoral immune response may be said to comprise an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

[0244] An animal according to the present disclosure may comprise more than one (e.g. one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) endogenous nucleotide sequences providing for inducible inhibition of a primary humoral immune response.

[0245] An animal according to the present disclosure may comprise plural (e.g. one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) endogenous nucleotide sequences, each endogenous nucleotide sequence conforming to an embodiment of an endogenous nucleotide sequence as described herein. In such embodiments, the plural endogenous nucleotide sequences may provide for inducible inhibition of the expression or activity of different genes / products thereof. By way of illustration, an animal according to the present disclosure may comprise: an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or activity of a product of, IGHM; and / or an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or activity of a product of, IGHD.

[0246] In some embodiments, the animal comprises endogenous nucleotide sequence(s) providing for inducible inhibition of the expression of, or activity of a product of, one or more genes involved in mounting a primary humoral immune response. In some embodiments, the animal comprises endogenous nucleotide sequence(s) providing for inducible inhibition of the expression of, or activity of a product of, one or both of IGHM and / or IGHD.

[0247] In some embodiments, the animal comprises endogenous nucleotide sequence(s) providing for inducible knockout of one or more genes involved in mounting a primary humoral immune response. In some embodiments, the animal comprises endogenous nucleotide sequence(s) providing for inducible knockout of one or both of IGHM and / or IGHD.

[0248] As used herein, “inducible knockout” refers to gene knockout which is inducible e.g. in response to a given chemical or physical treatment. Inducible knockout may also be referred to as “conditional knockout”. Inducible gene knockout technology is described e.g. in Kim etal., Lab Anim Res. (2018) 34(4): 147-159. Knockout may be inducible by treatment resulting in an increase in the level of expression or activity of a factor mediating gene knockout. For example, knockout may be mediated by a site-specific recombinase (SSR) system, and may be inducible by treatment resulting in an increase in the level of expression or activity of the relevant recombinase, which may effect gene knockout through binding to target sequences for the recombinase flanking all or part of the nucleotide sequence of a target gene. Knockout may be restricted to cell type(s) or tissues(s) of interest, e.g. by placing expression of the relevant recombinase under the control of regulatory sequence(s) (e.g. a promoter or enhancer) governing expression in the cell type(s) or tissues(s) of interest.

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

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

[0251] In some embodiments, the animal comprises an endogenous nucleotide sequence encoding target sequences for a recombinase (e.g. loxP sequences) flanking one or more exons of IGHD. In some embodiments, the animal comprises an endogenous nucleotide sequence encoding target sequences for a recombinase flanking one or more of exons 1 , 2 and 3 of IGHD. In some embodiments, the animal comprises an endogenous nucleotide sequence encoding target sequences for a recombinase flanking exons 1 to 3 of IGHD. In some embodiments, recombinase-mediated excision of the region flanked by target sequences for a recombinase removes the promoter for transcription of IGHD.

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

[0253] In some embodiments, the animal comprises an endogenous nucleotide sequence encoding target sequences flanking the region of the IGHM / IGHD locus shown in SEQ ID NO:3. In some embodiments, the animal comprises an endogenous nucleotide sequence providing for excision of the region of the IGHM / IGHD locus shown in SEQ ID NO:3.

[0254] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4, e.g. one of £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:4. In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, the nucleotide sequence of SEQ ID NO:4.

[0255] In some embodiments, following recombinase-mediated excision the animal comprises a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:5, e.g. one of £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, following recombinase-mediated excision the animal comprises the nucleotide sequence of SEQ ID NO:5. In some embodiments, an animal according to the present disclosure may comprise more than one endogenous nucleotide sequence providing for inducible inhibition of a primary immune response according to the present disclosure. In some embodiments, the animal may comprise one of 1 , 2, 3, 4, 5, 6, 7, 9 or 10 endogenous nucleotide sequences according to the present disclosure. In such embodiments, the plural endogenous nucleotide sequences may each independently conform to any embodiment of an endogenous nucleotide sequence described herein. In some embodiments wherein the animal comprises plural endogenous nucleotide sequences according to the present disclosure, the individual endogenous nucleotide sequences may provide for inducible inhibition of non-identical genes involved in mounting a primary humoral immune response (e.g. selected from: IGHM and IGHD).

[0256] In some embodiments, an animal according to the present disclosure comprises an endogenous nucleotide sequence encoding a recombinase, e.g. a Cre recombinase. In preferred embodiments, the Cre recombinase CreERT2.

[0257] In some embodiments, an animal according to the present disclosure comprises an endogenous nucleotide sequence encoding a recombinase (e.g. a Cre recombinase, e.g. CreERT2) under the control of a conditional system for controlling expression and / or activity of the recombinase. In some embodiments, the animal disclosure comprises an endogenous nucleotide sequence encoding a tamoxifen / 4-hydoxytamoxifen-controlled system for controlling activity of the recombinase.

[0258] In some embodiments, expression of the recombinase in the animal is under the control of a regulatory sequence (e.g. a promoter) driving expression in cells of hematopoietic origin. In some embodiments, expression of the recombinase in the animal is under the control of a regulatory sequence (e.g. a promoter) driving expression in B cell lineage cells. In some embodiments, expression of the recombinase in the animal is under the control of the CD79A promoter.

[0259] In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6, e.g. one of £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:6. In some embodiments, the animal comprises an endogenous nucleotide sequence comprising, or consisting of, the nucleotide sequence of SEQ ID NO:6.

[0260] An animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response may comprise such an endogenous nucleotide sequence as a consequence of having been genetically engineered to comprise such an endogenous nucleotide sequence. Accordingly, in some embodiments, the animal is a genetically-engineered animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response. A genetically engineered animal may also be referred to as a transgenic animal. Methods for genetically engineering animals to comprise a nucleotide sequence of interest are well known to the skilled person, and are described e.g. 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, both of which are hereby incorporated by reference in their entirety. Such methods include e.g. pronuclear microinjection, which is described e.g. in Pu et al., Methods Mol Biol (2019) 1874:17-41 (hereby incorporated by reference in its entirety), and e.g. SSN system-mediated genetic modification of germ cells, fertilized eggs or embryos, which is described e.g. in Lee et al., Drug Discovery Today: Disease Models (2016) 20: 13-20 (hereby incorporated by reference in its entirety).

[0261] Methods for producing genetically engineering animals include e.g. methods comprising transfecting embryonic stem cells with nucleic acid sequence(s) for genomic integration via homologous recombination, selecting cells having integrated the nucleic acid sequence(s) into their genomic DNA, introducing the genetically-modified embryonic stem cell into a blastocyst, and intrauterine implantation of the blastocyst comprising the genetically-modified embryonic stem cell, for gestation.

[0262] Nucleotide sequences of interest can be prepared using recombinant DNA techniques, and vectored into cells / embryos by viral transduction, or introduced by microinjection, electroporation, etc. Cells / embryos / animals comprising the nucleotide sequence of interest can be identified by suitable screening, e.g. by southern blot, PCR, etc.

[0263] A major obstacle to the use of monoclonal antibodies produced from immunized animals in humans is their xenogenic origin. The host mounts an immune response to the non-host antibody, resulting in elimination, and potentially also undesirable side effects. Various approaches have been undertaken to reduce or eliminate their immunogenicity, such as the production of chimeric antibodies comprising human Fc regions, and humanization in wherein the variable domains of the antibody are engineered for similarity to human antibody sequences.

[0264] More recently, transgenic techniques have been employed, in which the animal’s endogenous immunoglobulin gene loci are replaced with their human homologues. Monoclonal antibodies produced from such mice using traditional hybridoma techniques are fully human. 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 several human monoclonal antibodies that have been approved with acceptable safety and efficacy profile. Using such antibody discovery platforms a significant hurdle was overcome, by reducing the immunogenicity of the antibodies produced while retaining the practicality of mouse immunization. The mice are engineered to retain a robust B cell response, and repeat immunization with human antigens results in a robust secondary immune response and the capacity to develop a diverse repertoire of mAbs. In aspects and embodiments of the present 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.

[0265] In some embodiments, the genome of the animal encodes human immunoglobulin VH region and / or VL region sequences. In some embodiments, the genome of the animal encodes human immunoglobulin Fc region sequence. In some embodiments, the genome of the animal encodes human immunoglobulin VH region, VL region and / or Fc region sequences.

[0266] Animals comprising endogenous human immunoglobulin genes or gene segments are useful for producing antibodies comprising fully human Fv ( / .e. VH and VL regions). Transgenic mice encoding human immunoglobulin genes / gene segments are described e.g. in Lu et al., J Biomed Sci. 2020; 27: 1 and Bruggemann et al., Arch Immunol Ther Exp (Warsz). (2015) 63(2): 101 —108, and include 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) 23:1 117-1125), TransChromo™ Mouse (Ishida et al., Cloning Stem Cells. (2002) 4:91-102), and Velocimmune® (Regeneron; Murphy, PNAS (2014) 11 1 (14): 5153-5158).

[0267] In some embodiments, the animal of the present disclosure comprises an endogenous nucleotide sequence encoding human immunoglobulin V, D and / or J genes, or segments thereof.

[0268] In some embodiments, the animal of the present disclosure comprises an endogenous nucleotide sequence encoding the human immunoglobulin genes or gene segments encoded by the genome of an animal described in Lu et al., J Biomed Sci. 2020; 27: 1 , Bruggemann 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) 23:1 117-1125, Ishida et al., Cloning Stem Cells. (2002) 4:91-102 or Murphy, PNAS (2014) 111 (14): 5153-5158, US 7,135,287 B1 , US 7,105,348 B2, or US 2006 / 059575 A1 , all of which are hereby incorporated by reference in their entirety.

[0269] In some embodiments, antibodies produced by the immune system of the animal of the present disclosure comprise fully human VH region and / or VL region sequences. In some embodiments, antibodies produced by the immune system of the animal of the present disclosure comprise a fully human Fc region sequence. In some embodiments, antibodies produced by the immune system of the animal of the present disclosure comprise fully human VH region, VL region and / or Fc region sequences. In some embodiments, antibodies produced by the immune system of the animal of the present disclosure comprise a fully human amino acid sequence.

[0270] In some embodiments, an animal according to the present disclosure has been immunized with a first peptide / polypeptide comprising an amino acid sequence of interest ( / .e. (a) an amino acid sequence of a protein / protein complex of interest, or (b) an amino acid sequence which is similar to the amino acid sequence of (a)) to elicit a primary immune response directed against the amino acid sequence of interest.

[0271] In some embodiments, the animal according to the present disclosure is an individual / subject of a particular strain of mouse. In some embodiments, the animal is a C57BL / 6 mouse, a BALB / c mouse, a A / J mouse, a CD1 mouse, a ICR mouse, a 129S2 / SvPas mouse, or a FVB / N mouse. The mouse strains recited in the preceding sentence are described e.g. in The Jackson Laboratory Handbook on Genetically Standardized Mice, 6thEdition, October 2009 (Jackson Laboratory, Ed. Kevin Flurkey and Joanne M. Currer).

[0272] In some embodiments, the animal is a genetically-engineered mouse ( / .e. a transgenic mouse).

[0273] In some embodiments, the animal is a genetically-engineered mouse in which the endogenous immunoglobulin gene loci are replaced with their human homologues. In some embodiments, the animal is a Xenomouse mouse, UltiMAb mouse, TransChromo mouse or Velocimmune mouse. In some embodiments, the animal is a mouse described in Lu et al., J Biomed Sci. 2020; 27: 1 , Bruggemann 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) 23:1 117-1125, Ishida et al., Cloning Stem Cells. (2002) 4:91-102, Murphy, PNAS (2014) 111 (14): 5153-5158, US 7,135,287 B1 , US 7,105,348 B2 or US 2006 / 059575 A1 .

[0274] In some embodiments, the animal is a genetically-engineered mouse having increased longevity ( / .e. as compared to equivalent mice lacking such genetic modification). Mice comprising genetic modification resulting in an increased lifespan are described e.g. in Ladiges et al., Aging Cell. (2009) 8(4):346-52 (which is hereby incorporated by reference in its entirety); see in particular Table 1 thereof. In some embodiments, the animal is a 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 aMUPA Tg mouse, a p66shc_ / _mouse, a GHr / BP_ / ~ mouse, a Ghrhrlit / Htmouse, a Snell Dwarf mouse, a lgf1 r+ / ~ mouse, a FIRKO mouse, a Klotho Tg mouse, a Mit CAT Tg mouse, a MT Tg heart mouse, a UCP2 Tg brain mouse, a PappA_ / ~ mouse, a AC5_ / _mouse, a Surf1~'~ mouse, a PEPCKTgmuscle mouse, a Irs1_ / “ mouse, a lrs2+ / _mouse, a lrs2+ / _brain mouse, or a IGF-1 Tg heart mouse.

[0275] In some embodiments, the animal is a mouse having low incidence of spontaneous tumors, e.g. a genetically-engineered mouse having low incidence of spontaneous tumors ( / .e. as compared to equivalent mice lacking such genetic modification). In some embodiments, the animal is a mouse described in Rithidech et al., Blood Cells Mol Dis. (1999) 25(1):38-45.

[0276] In some embodiments, the animal is a mouse having defective immune cell tolerance, e.g. a genetically- engineered mouse having defective immune cell tolerance. In some embodiments, the animal is a mouse described in Khattri et al., J Immunol. (2001) 167(11):6312-6320. In some embodiments, the animal is a mouse having chronic immune activation, e.g. a genetically- engineered mouse having chronic immune activation. In some embodiments, the animal is a mouse described in Subramanian etal., Proc Natl Acad Sci U S A. (2006) 103(26):9970-9975.

[0277] In some embodiments, the animal is a mouse having a dysregulated autoimmune or hyperimmune phenotype, e.g. a genetically-engineered mouse. In some embodiments, the animal is a mouse comprising genetic variation that spontaneously gives rise to a dysregulated autoimmune or hyperimmune phenotype. In some embodiments, the animal is a mouse in which a dysregulated autoimmune or hyperimmune phenotype is / has been induced by treatment with a chemical or peptide / polypeptide. In some embodiments, the mouse is a NOD mouse, a NZB / W F1 mouse, a MRL mouse, a BXSB mouse, a Ipr mouse, a gid mouse, a motheaten mouse, a Scurfy mouse, a Baff Tg mouse, a Bc / 2 Tg mouse, a Binr1- mouse, a C1qa'- mouse, a C4'- mouse, a Cd19 Tg mouse, a Cd19Cre- Traf3fl / flmouse, a Cd?1- mouse, a Cd40l Tg mouse, a Cd45E613R KI mouse, a Ctla4 / - mouse, a Dnasel'- mouse, a FoxpS1- mouse, a G2a / _mouse, aGadd45a,mouse, aGadd45a,-p21cip1 / waf -1- mouse, a Gadd45blGadd45g-1- mouse, a 114 Tg mouse, a LatY136F KI mouse, a LckCre-Ptenfl / ~ mouse, a Lyn1- mouse, a Man2a1'- mouse, a Mark2 / - mouse, a Mfge8^ mouse, a Ox40Cre- Ptenmmouse, a p65P13K Tg mouse, a Pd1'- mouse, a Pkba g mouse, a Prkcd!mouse, a Pten+ / - mouse, a Tacr1- mouse, or a TyroS Ax Mertk1- mouse.

[0278] In some embodiments, the animal is a mouse having autoimmune encephalitis, e.g. a genetically- engineered mouse having autoimmune encephalomyelitis. In some embodiments, the animal is a mouse having autoimmune encephalitis in an SJL background, as described e.g. in Rajan et al., J Immunol (1996) 157 (2): 941-949. In some embodiments, the animal is a mouse having collagen-induced arthritis in a DBA / 1 background, as described e.g. in Courtenay etal., Nature (1980) 283(5748):666-8. In some embodiments, the animal is a mouse having Imiquimod-induced psoriasis in a BALB / c or C57BL / 6 background, as described e.g. in Van der Fits et al., J Immunol (2009) 182(9):5836-45.

[0279] In some embodiments, the animal is a hyperimmune mouse, e.g. a genetically-engineered hyperimmune mouse. Genetically-engineered hyperimmune mice produce a more robust immune response to antigenic material than equivalent mice lacking such genetic modification. 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.

[0280] The inventors have advantageously discovered that the use of a hyperimmune mouse for generating the animal(s) of the invention is useful for generating antibodies with a high affinity and titer.

[0281] Accordingly, in some embodiments, the animal is a hyperimmune mouse. “Hyperimmune mouse” (which may also be referred to as an “autoimmune mouse”) as referred to herein may refer to a mouse with a hyperimmune, dysregulated autoimmune, or highly immuno-reactive background or phenotype and / or a mouse of a hyperimmune, dysregulated autoimmune, or highly immuno-reactive strain. In some embodiments, the hyperimmune mouse is a genetically-engineered mouse.

[0282] In some embodiments, the hyperimmune mouse may have one or more of the following properties:

[0283] An elevated immune response (or a strong or overactive adaptive response) as compared to a mouse without a hyperimmune, dysregulated autoimmune or highly immune-reactive background or phenotype, e.g. as characterised by an enlarged pool of naive B cells, enhanced activation of the primary or secondary immune response following immunization with an antigen, or increased activation of B cells following immunization with an antigen.

[0284] An altered tolerance to antigens (self or foreign) as compared to a mouse without a hyperimmune, dysregulated autoimmune of highly immuno-reactive background or phenotype, e.g. as characterised by an increased retention of self-reactive B cells, unchecked stimulation and proliferation of self-reactive B cells, or loss of negative selection against somatic hypermutation-generated self-reactivity.

[0285] Normal, or close to normal, development of B and T cells.

[0286] A lifespan compatible with the length of antibody discovery campaigns.

[0287] Strong T-dependent antibody responses.

[0288] Expanded germinal centers and B cell population

[0289] Capable of production of class-switched auto-antibodies.

[0290] Capable of producing high sera antibody titers when immunized with self-epitopes e.g. epitopes or antigens with high homology (>80%) to self.

[0291] Capable of producing high sera antibody titers when immunized with poorly immunogenic antigens i.e. antigens that do not trigger a strong immune response in a mouse without a hyperimmune, dysregulated autoimmune of highly immuno-reactive background or phenotype .

[0292] Accordingly, in some embodiments, the animal (e.g. the hyperimmune mouse) may be capable of producing strong antibody titers when immunized with self-epitopes or poorly immunogenic antigens.

[0293] Generation of a hyperimmune mouse can be achieved using one or more of the following:

[0294] Mutations that affect B cell activation, proliferation and survival e.g. altered BCR and co-receptor signalling or loss of Fas-FasL dependent apoptosis (such as mice described in Miyamoto et al. Nature (2002) 416, 865-869 and Groom et al. J Clin Invest. (2002) 109(1):59-68.

[0295] Alterations to antigen presentation e.g. using negative regulation of cytokine signalling for APC recruitment and migration (such as CCL2) or using mutations in antigen processing genes (such as TAP1 or LMP2).

[0296] Mutations that affect Treg and TFH activation and function e.g. mutations that alter TCR signalling or result in loss of FOXP3-mediated differentiation (e.g. as described in Zahorsky-Reeves and Wilkinson. European Journal of Immunology (2001) 31 (1) 196-204) . spontaneously acquired genetic mutations that result in an autoimmune phenotype e.g. the NZB / W F1 mouse. In some embodiments, the hyperimmune mouse is a NOD mouse, a NZB / W F1 mouse, a MRL mouse, a BXSB mouse, a Ipr mouse, a gid mouse, a motheaten mouse, a Scurfy mouse, a Baft Tg mouse, a Bc / 2 Tg mouse, a Bim1- mouse, a Ci ar1- mouse, a C4'- mouse, a Cd19 Tg mouse, a Cd19CreTraf3fi / rimouse, a Cd22!- mouse, a Cc / 40 / Tg mouse, a Cd45E613R KI mouse, a Ctla4!- mouse, a Dnasel1- mouse, a Foxp3!- mouse, a G2a / _mouse, aGadd45a / - mouse, aGadd45a!p21cip1 Iwaf -'- mouse, a Gadd45b / - Gadd45g-!- mouse, a 114 Tg mouse, a LatY136F KI mouse, a LckCrePtenfl / ~ mouse, a Lyn1- mouse, a Man2a1!- mouse, a Mark2!- mouse, a Mfge8!- mouse, a Ox40CrePtenfmmouse, a p65P13K Tg mouse, a Pd1'- mouse, a Pkba Tg mouse, a Prkcd1- mouse, a Pten+ / - mouse, a Tact1- mouse, or a TyroS1- Axl'- Mertk'- mouse.

[0297] In some embodiments, the hyperimmune mouse is a NZB / W F1 mouse. The NZB / W F1 mouse (also referred to herein as the “NZBWF1” or “NZBWF1 / J” mouse) is the New Zealand Black (NZB) x New Zealand White (NZW) F1 mouse as described e.g. in Dubois et al. JAMA (1966) 195(4):285-289 and in Bagavant et al., Autoimmun Rev. (2020) 19(2): 102686.

[0298] In some embodiments, the hyperimmune mouse comprises endogenous nucleotide sequence(s) providing for inducible knockout of one or both of IGHM and / or IGHD. In some embodiments, the mouse is a NZBWF1 / J mouse. In some embodiments, the mouse is a hyperimmune lghdlghmftox / TtoxCd79a+ / CreERT2mouse. In some embodiments, the mouse is a NZBWF1 / J lghdlghmfloxxflox', Cd79a+ / CreERT2mouse.

[0299] In some embodiments, the animal is a humanized mouse. The humanized mouse may have been engrafted with cells or tissue from a human. The humanized mouse may also be a transgenic mice.

[0300] In some embodiments, the humanized mouse is as described in, e.g., Chen and Murawsky. Front Immunol (2018) Volume 9 and Ma, B., Osborn, M. (2021). Transgenic Animals for the Generation of Human Antibodies. In: Ruker, F., Wozniak-Knopp, G. (eds) Introduction to Antibody Engineering. Learning Materials in Biosciences. Springer, Cham, https: / / doi.org / 10.1007 / 978-3-030-54630-4_5.

[0301] In some embodiments, the animal is a humanized hyperimmune mouse.

[0302] Producing antigen-binding molecules

[0303] Aspects of the present disclosure concern methods for eliciting the production of an antigen-binding molecules capable of binding to a protein of interest.

[0304] Aspects of the present disclosure are concerned with the generation of antigen-binding molecules, e.g. for subsequent purification. Aspects of the present disclosure are concerned with the generation of populations of cells producing antigen-binding molecules.

[0305] As referred to herein, and in the context of the various aspects and embodiments of the present disclosure, an “antigen-binding molecule” refers to a molecule capable of binding to a target antigen, and may e.g. be an antibody / immunoglobulin. In some embodiments an antibody / immunoglobulin according to the present disclosure is an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM. In preferred embodiments, the antibody / immunoglobulin is an IgG.

[0306] Antigen-binding molecules / cells producing antigen-binding molecules may be generated for downstream use, e.g. in therapeutic, research, imaging and / or diagnostic applications. The methods of the present disclosure may be employed to produce antigen-binding molecules having particular properties of interest relevant to therapeutic, research, imaging and / or diagnostic applications.

[0307] Briefly, the methods comprise introducing material into an animal which is recognized by the immune system of the animal to be foreign ( / .e. non-host), resulting in the selective production by the animal of antibodies which are capable of binding to the material. The material may comprise, or may be processed to, an antigen. The ability of the immune system to produce antibodies capable of binding specifically to antigens can be used to generate antibodies for detecting molecules of interest in various research, diagnostic, imaging, therapeutic and prophylactic applications.

[0308] Methods for producing antibodies are well known in the art, and are described, for example, in Antibodies: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference in its entirety. In particular, Chapter 6 provides detailed description of methods for immunizing animals for the production of antibodies.

[0309] In particular, aspects of the methods of the present disclosure are concerned with producing monoclonal antibodies. Such methods may comprise isolating antigen-binding molecule-producing cells from subjects. Such methods may comprise generating monoclonal hybridomas from cells isolated from subjects, wherein the hybridomas produce antibodies of a single type ( / .e. of a single specificity).

[0310] Methods of antibody production involves introducing antigen into an animal to elicit the production of antibodies which can then be recovered from the animal.

[0311] Aspects and embodiments of the methods of the present disclosure comprise:

[0312] (i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0313] (ii) treating the animal to inhibit its ability to mount a primary immune response; and

[0314] (iii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

[0315] In some embodiments, treatment of the animal to inhibit its ability to mount a primary immune response is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have undergone immunoglobulin isotype switching ( / .e. to IgG-, IgE-, or I gA-ex pressing cells). In some embodiments, treatment of the animal to inhibit its ability to mount a primary immune response is performed after a period of time sufficient for the cells activated / stimulated to proliferate by administration of the first peptide / polypeptide (or cells derived from such cells) to have differentiated into plasma B cells and / or memory B cells.

[0316] In some embodiments a method for producing an antigen-binding molecule comprises one or more of: preparing / formulating a peptide / polypeptide / nucleic acid / cell to be introduced into an animal; introducing a peptide / polypeptide / nucleic acid / cell into an animal; detecting and / or monitoring production of antigen-binding molecules by the animal; detecting and / or monitoring production of cells expressing / comprising antigen-binding molecules by the animal; collecting antigen-binding molecules produced by the animal; isolating / purifying antigen-binding molecules produced by the animal; collecting cells producing antigen-binding molecules from the animal; isolating / purifying cells producing antigen-binding molecules from the animal; generating a hybridoma producing antigen-binding molecules; culturing cells producing antigen-binding molecules; and isolating / purifying antigen-binding molecules produced by cells in culture.

[0317] In some embodiments, methods comprise isolating antigen-binding molecule-producing cells from the animal. In some embodiments, antigen-binding molecule-producing cells are harvested from the blood of the animal (e.g. from PBMCs obtained from the blood of the animal), or from an organ of the animal (e.g. the spleen). In some embodiments the isolated antigen-binding molecule-producing cells are cultured in vitro. In some embodiments the methods comprise culturing cells isolated from the subject in vitro.

[0318] In some embodiments, methods comprise isolating antigen-binding molecules capable of binding to the protein of interest. In some embodiments, the antigen-binding molecules are isolated from the animal. The antigen-binding molecules may be recovered from e.g. the blood, plasma, serum, or ascites of the animal.

[0319] In some embodiments, antigen-binding molecules may be isolated from cells obtained from the animal. In some embodiments, the cells are B cells. In some embodiments, the antigen-binding molecules may be isolated from cell culture supernatant from B cells cultured in vitro.

[0320] In some embodiments, antigen-binding molecules are obtained from a hybridoma producing antigenbinding molecules capable of binding to the protein of interest. In some embodiments, the methods comprise isolating antigen-binding molecules capable of binding to the protein of interest from a culture of a hybridoma produced according to the present disclosure. In some embodiments, the antigen-binding molecules are obtained from cell culture supernatant of a culture of a hybridoma. In some embodiments, the antigen-binding molecules are obtained from the blood, plasma, serum, or ascites of an animal immunized with a hybridoma. Methods for isolating ( / .e. purifying) antigen-binding molecules from an antigen-binding molecule containing sample (e.g. cell, cell extract, cell culture medium, blood, plasma, serum, ascites) are well known to the skilled person, and are described in detail in Antigen-binding molecules: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated by reference herein above), in particular at Chapter 10. The methods include, for example, ion exchange chromatography, protein A or protein G based purification, gel electrophoresis, dialysis, and affinity purification based on target binding.

[0321] An isolated or purified antigen-binding molecule as used herein refers to a composition comprising an antibody, of which at least 80%, 90%, 95%, 99% or 100% of the composition (by weight, or by weight of the protein component of the composition) is the antigen-binding molecule component of the composition.

[0322] In some embodiments, the methods of the present disclosure employ single B cell cloning. Single B cell cloning for the production of monoclonal antibodies is described e.g. in Carbonetti et al., J Immunol Methods (2017) 448: 66-73 and Lei et al. Front Microbiol (2019) 10:672, both of which are hereby incorporated by reference in their entirety. Such methods generally comprise culturing B cells obtained from an animal as single clones in vitro, e.g. in the presence of factors promoting proliferation of the B cells and / or production of antibodies from the B cells. B cells may be obtained from the blood of the animal (e.g. from populations of PBMCs derived from the blood of the animal), or from an organ of the animal (e.g. the spleen). B cells may be sorted into single cell cultures by FACS, or another cell sorting technique. B cells expressing antibodies having properties of interest may be sequenced in order to determine the amino acid sequence of the antibody and / or the nucleic acid sequence encoding the antibody.

[0323] Methods for hybridoma formation are well known to the skilled person, and are described, for example, in Antibodies: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated by reference herein above); in particular, at Chapter 7. Briefly, an animal is immunized in accordance with the present disclosure, stimulating an adaptive immune response, and B lymphocytes are isolated from the animal and fused with a suitable myeloma cell line to produce a hybridoma.

[0324] Antigen-binding molecule production by a subject (e.g. antigen-binding molecule titer) may be determined as described herein, prior to hybridoma production. If the titer is too low, one or more booster steps may be performed as described herein, and antigen-binding molecule production monitored (e.g. by repeated blood sampling), until a sufficiently high titer is achieved. In some embodiments, the methods comprise selecting a subject for hybridoma production on the basis of antigen-binding molecule production or titer. Antigen-binding molecule production or titer may be determined in e.g. a blood, plasma, serum or ascites sample obtained from the animal. In some embodiments, subjects and / or cells may be selected in accordance with methods for producing an antigen-binding molecule based on detection of production an antigen-binding molecule capable of binding to the protein of interest.

[0325] In some embodiments, collecting cells producing antigen-binding molecules comprises harvesting the spleen and / or lymph nodes of a subject. In some embodiments producing a hybridoma comprises fusing a cell (e.g. a B cell) capable of producing an antigen-binding molecule obtained from a subject with a myeloma cell. In some embodiments the fusing a cell (e.g. a B cell) capable of producing an antigenbinding molecule obtained from a subject with a myeloma cell comprises co-centrifugation in polyethylene glycol (PEG). In some embodiments producing a hybridoma comprises selected by culture of cells in selective media such as media containing hypoxanthine-aminopterin-thymidine (HAT).

[0326] Hybridoma colonies may be tested for production of antibody capable of binding to the protein of interest, and / or other peptides and polypeptides, as described herein, e.g. by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g. flow cytometry, ELISA, etc.). In some embodiments, antibody production or antibody titer may be determined in cell culture supernatant from hybridoma cultured in vitro.

[0327] The present disclosure also provides an antigen-binding molecule capable of binding to a protein of interest, wherein the antigen-binding molecule is obtained by, or obtainable by, a method for producing an antigen-binding molecule as described herein.

[0328] In some embodiments, the methods further comprise formulating antigen-binding molecules to a composition, e.g. a pharmaceutical composition. In some embodiments the methods comprise mixing an antigen-binding molecule with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0329] Pharmaceutical compositions may be formulated in fluid (including gel) or solid (e.g. tablet) form. Fluid formulations may be formulated for administration by injection or via catheter to a selected region of the human or animal body. The present disclosure also provides a pharmaceutical composition formed by a method according to the present disclosure.

[0330] Antigen-binding molecules produced by the methods of the present disclosure may be produced on a large scale using methods known to the skilled person.

[0331] Hybridomas can be propagated either in in vitro culture using standard methods of cell culture, or in vivo, e.g. as ascites in a host animal. In some embodiments, the methods of the present disclosure comprise propagating the hybridoma by in vitro cell culture. In some embodiments, the methods comprise propagating the hybridoma in vivo by injecting a host animal with the hybridoma.

[0332] In some embodiments, antigen-binding molecules can be made using recombinant DNA techniques known to the skilled person. For example, a polynucleotide encoding an antibody can be derived from a B cell or hybridoma cell producing an antibody, e.g., 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 can be determined. Isolated polynucleotides encoding the heavy and light chains can be cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0333] Amino acid sequence of interest

[0334] An “amino acid sequence of interest” as used herein refers to (i) an amino acid sequence of a protein / protein complex of interest, or (ii) an amino acid sequence which is similar to the amino acid sequence of the protein / protein complex of interest ( / .e. an amino acid sequence which is similar to the amino acid sequence of (i)).

[0335] As used herein, an amino acid sequence which is “similar to” a reference amino acid sequence is an amino acid sequence having some shared character with the reference amino acid sequence. In some embodiments an amino acid sequence which is “similar to” a reference amino acid sequence is an amino acid sequence comprised in a protein which is an isoform, variant or homolog of the protein comprising the reference amino acid sequence. In some embodiments, an amino acid sequence which is “similar to” a reference amino acid sequence (e.g. an amino acid sequence of a protein of interest) comprises one of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference amino acid sequence. In some embodiments, an amino acid sequence which is “similar to” a reference amino acid sequence does not differ by more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the reference amino acid sequence.

[0336] Conversely, an amino acid sequence which is “dissimilar to” a reference amino acid sequence is an amino acid sequence having comprises less than 100%, e.g. one of less than 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5% sequence identity to the reference amino acid sequence. In some embodiments, an amino acid sequence which is “dissimilar to” a reference amino acid sequence differs by more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to the reference amino acid sequence.

[0337] Comparison of a given amino acid sequence ( / .e. a query sequence) to a reference amino acid sequence can be performed by aligning the sequences and comparing the amino acids at corresponding positions. In some embodiments, the sequence comparison is performed over a region of the query sequence and reference sequences which is at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 15 to 20, 20 to 25, or 25 to 30, 30 to 60, 40 to 80, or 80 to 100 amino acids in length.

[0338] As used herein, a “protein” includes peptides and polypeptides. In some embodiments a protein of interest may be a peptide of interest, or a polypeptide of interest. A “peptide” is a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. Reference to peptides and polypeptides herein also encompasses complexes comprising such peptides / polypeptides, which may be homo- or hetero-multimeric complexes (e.g. formed by non-covalent interactions) comprising two or more (e.g. 2, 3, 4, 5, 6, 7, 8 or more) peptides / polypeptides.

[0339] A protein of interest may be any protein. A protein of interest may for example be a protein of diagnostic, prognostic, imaging or therapeutic relevance. In some embodiments a protein of interest may is a candidate therapeutic target for an antigen-binding molecule. “A protein of interest” as used herein means “one or more protein(s) of interest”. That is, an antibody capable of binding to a protein may be to more than one protein of interest. Reference to a “protein of interest” herein also includes a protein complex of interest, which may be a homo- or hetero-multimeric complex (e.g. formed by non-covalent interactions) comprising two or more (e.g. 2, 3, 4, 5, 6, 7, 8 or more) polypeptides.

[0340] In some embodiments, the protein of interest is a protein whose expression / activity, or whose upregulated expression / activity, is positively associated with a disease or disorder (e.g. a cancer, an infectious disease or an autoimmune disease). In some embodiments, the protein of interest is expressed by a pathogen / infectious agent, cell, or a cell of a tissue, which it is desirable to destroy or remove. In some embodiments, the protein of interest is expressed by a pathogen / infectious agent, cell, or a cell of a tissue to which it is desirable to direct a humoral immune response. In some embodiments the protein of interest is associated with a cancer, an infectious disease, or an autoimmune disease. Pathogens include prokaryotic (bacteria), eukaryotic (e.g. protozoan, helminth, fungus) and viral pathogens. In some embodiments, the protein of interest is expressed by a cancer cell, an infectious agent, a cell infected with an infectious agent or an autoimmune effector cell ( / .e. an effector of an autoimmune pathology). In some embodiments the protein of interest is a disease / disorder-associated (e.g. cancer-associated and / or autoimmune disease) variant of a protein.

[0341] In some embodiments the antigen-binding molecules generated in accordance with the methods of the present disclosure are capable of recognizing proteins related to the protein of interest.

[0342] A “protein related to the protein of interest” refers to a protein having some shared character with a reference protein of interest. For example, the methods of the present disclosure can be used to elicit antigen-binding molecules capable of recognizing variants of a protein of a pathogen / infectious agent. That is, the methods can be employed to elicit broadly-neutralizing antigen-binding molecules. In some embodiments a protein related to the protein of interest comprises the amino acid sequence of interest. In some embodiments a protein related to the protein of interest comprises the amino acid sequence of the protein of interest.

[0343] Related proteins include isoforms, fragments, variants or homologs (e.g. paralogs, orthologs) of the protein of interest, including proteins which are members of the same protein family. Isoforms, fragments, variants or homologs of a given protein of interest may optionally be characterized as having an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein.

[0344] As used herein, “sequence identity" refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, 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 (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0345] In some embodiments, an amino acid sequence of interest may be e.g. a consensus or majority sequence for more than one related protein in a corresponding region. In some embodiments, an amino acid sequence of interest may be a consensus sequence for the corresponding region to the amino acid sequence of a protein of interest for two or more isoforms, homologs or variants of the protein of interest.

[0346] In some embodiments, the amino acid sequence of interest is an antigenic amino acid sequence. The term “antigenic" as used herein refers to the ability to stimulate an immune response, in particular an adaptive immune response (e.g. a B cell- and / or T cell-mediated immune response). In some embodiments the amino acid sequence of interest is capable of stimulating a B cell-mediated immune response. In some embodiments the amino acid sequence of interest is a sequence of amino acids which forms, or is predicted to form, a B cell epitope. In some embodiments the amino acid sequence of interest is capable of stimulating the production of antigen-binding molecules.

[0347] In some embodiments, the amino acid sequence of interest is a consecutive sequence of amino acids of the protein of interest, or a similar sequence. In some embodiments, for example in embodiments where the amino acid sequence of interest folds to form a discontinuous epitope, the amino acid sequence of interest is a discontinuous sequence of amino acids of the protein of interest, or a similar sequence.

[0348] In some embodiments the amino acid sequence of interest is a continuous sequence of amino acids providing a linear epitope. In some embodiments the amino acid sequence of interest is a continuous sequence of amino acids which folds to provide a discontinuous epitope. In some embodiments the amino acid sequence of interest is a discontinuous sequence of amino acids which together form a discontinuous epitope.

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

[0350] In some embodiments the amino acid sequence of interest has a length of one 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 amino acid sequence of interest has a length of one 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 amino acid sequence of interest has a length of one of 15 to 100, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 15 to 19, 15 to 18, or 15 to 17 amino acids. In some embodiments, the amino acid sequence of interest has a length of one of 20 to 100, 20 to 50, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 amino acids. In some embodiments, the amino acid sequence of interest has a length of 5 to 30 amino acids.

[0351] Where the amino acid sequence of interest provides, or is predicted to provide, a discontinuous epitope, the amino acid sequence of interest may refer either to the continuous sequence of amino acids which folds to provide the discontinuous epitope, or the discontinuous sequence of amino acids which forms the discontinuous epitope.

[0352] Where the amino acid sequence of interest provides a discontinuous epitope, the overall length of the discontinuous epitope may be about 5 to 30 amino acids. Discontinuous epitopes may comprise e.g. 2, 3, 4, 5, 6 or 7 non-continuous sequences of amino acids. Such non-continuous sequences may each comprise e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, but preferably less than e.g. 30, 25, 20 or 15 amino acids.

[0353] The skilled person is able to identify antigenic sequences of a protein of interest by methods known in the art. For some proteins, sequences of amino acids which are known or predicted to be antigenic are provided in databases such as BciPep, AntiJen, AntigenDB, SPTR, FIMM, HPTAA, IEDB, Epitome, MHCBN, MHCPEP, MPID-T2, Protegen. Antigenic amino acid sequences of proteins can also be identified by literature searches, for example using the internet.

[0354] Sequences of amino acids which are predicted to form B cell epitopes, and that are therefore likely to be effective in raising antibodies, can be predicted from a query sequence by a variety of methods. The skilled person is able to predict whether a sequence of amino acids is antigenic by comparison to known or predicted antigenic sequences (e.g. for other proteins), and / or based on properties of the amino acid sequence. See, for example, El-Manzalawy and Honavar, Immunome Res, 2010, 6(Suppl 2): S2, which is hereby incorporated by reference in its entirety. Such methods take into account, for example, the hydrophilicity, flexibility, accessibility, turns, exposed surface, polarity and antigenic propensity of sequences of amino acids. Whether a peptide / polypeptide comprises a T cell epitope can be determined e.g. using prediction methods as described in Desai et al., 2014 Methods Mol Biol 1184:333-364.

[0355] Some of the methods take into account three-dimensional structure, and can be used to predict conformational epitopes. Software which can be used to identify / predict antigenic sequences of amino acids includes EMBOSS: antigenic, BepiPred, IEDB Analysis Resource, SVMTriP, and SCRATCH, ElliPro, COBEPro, BEPro, PEPITO and DiscoTope.

[0356] Antigenic sequences of a protein of interest can also be identified by experimentally determining whether an amino acid or sequence of amino acids is antigenic. For example, the skilled person is able to determine whether a given amino acid sequence is antigenic, for example by immunizing a subject (e.g. a mammal) with a peptide of the amino acid sequence and determining whether an adaptive immune response is elicited. One or more of the described approaches may be employed separately or in combination in the methods of the present disclosure to identify antigenic sequences of amino acids of a protein of interest.

[0357] When a sequence of amino acids is assessed for similarity to a reference amino acid sequence of a protein of interest which provides, or is predicted to provide, a discontinuous epitope, the sequence of amino acids may be aligned to the continuous sequence of amino acids which folds to form the discontinuous epitope, or the discontinuous sequence of amino acids which together form the discontinuous epitope.

[0358] First peptide / polypeptide

[0359] Methods of the present disclosure comprise administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein first peptide / polypeptide comprises an amino acid sequence of interest. In some embodiments the first peptide / polypeptide consists of, or consists essentially of, the amino acid sequence of interest.

[0360] It will be appreciated that the first peptide / polypeptide encompasses peptides / polypeptides comprising or consisting of: (i) an amino acid sequence of a protein of interest, or (ii) an amino acid sequence which is similar to the amino acid sequence of the protein of interest ( / .e. an amino acid sequence which is similar to the amino acid sequence of (i)).

[0361] In some embodiments the first peptide / polypeptide comprises the amino acid sequence of interest, and additionally comprises further amino acids. In some embodiments the first peptide / polypeptide comprises the amino acid sequence of interest and one of 1 -5, 1-10, 1-15, 1 -20, 1 -25, 1 -30, 1 -40 or 1 -50 additional amino acids, at one or both ends ( / .e. the N- or C-terminus) of the amino acid sequence of interest.

[0362] In some embodiments the additional amino acids correspond to the amino acids provided at those positions relative to the amino acid sequence of interest, in the context of the protein from which the amino acid sequence of interest is derived. By way of illustration, where an amino acid sequence of interest corresponds to amino acid positions 20 to 30 of the amino acid sequence of a protein of interest, and wherein the first peptide / polypeptide comprises the amino acid sequence of interest and an additional 5 amino acids at the N-terminus of the amino acid sequence of interest, those additional 5 amino acids may correspond to positions 15 to 19 of the amino acid sequence of the protein of interest.

[0363] The first peptide / polypeptide may comprise more than one peptide / polypeptide chain. For example, the first peptide / polypeptide may be a complex of peptides / polypeptides, e.g. comprising 2, 3, 4, 5 or 6 or more peptides / polypeptides. The peptides / polypeptides may be conjugated to one other, or administered as a composition (e.g. mixture) of two or more peptides / polypeptides that are not conjugated.

[0364] In some embodiments the first peptide / polypeptide is provided as a conjugate with a carrier protein. As used herein, a “carrier protein" refers to a protein which can be used to elicit an immune response to the peptide / polypeptide to which it is conjugated ( / .e. which it acts as a “carrier" for). Due to their size and complexity, carrier proteins induce an immune response, including to the conjugated peptide / polypeptide. Many proteins can be used as carriers and are chosen based on immunogenicity, solubility, and availability of useful functional groups through which conjugation with a peptide / polypeptide of interest can be achieved. Carrier proteins are well known in the art of immunology, and are described e.g. in “Thermo Scientific Pierce Antibody Production and Purification Technical Handbook”, Version 2, (2010); Thermo Scientific, USA (1601975 09 / 10), which is hereby incorporated by reference in its entirety.

[0365] In some embodiments the carrier protein is selected from keyhole limpet hemocyanin (KLH), Concholepas concholepas hemocyanin (CCH; also known as Blue Carrier Protein), bovine serum albumin (BSA), cationized BSA (cBSA), hepatitis B core antigen (HBc), thyroglobin and ovalbumin (OVA). In some embodiments the carrier protein is selected from KLH, BSA, HBc and OVA.

[0366] Peptide / polypeptides may be conjugated to carrier proteins via means well known to the skilled person., including e.g. amine-sulfhydryl crosslinking (e.g. using succinimidyl 6-((beta-maleimidopropionamido) hexanoate) SMPH), EDC conjugation (carboxyl and amino crosslinking), maleimide conjugation (sulfhydryl crosslinking), glutaraldehyde conjugation (amine-amine crosslinking) (see “Thermo Scientific Pierce Antibody Production and Purification Technical Handbook”, Version 2, (2010); Thermo Scientific, USA (1601975 09 / 10)).

[0367] A ’’first peptide / polypeptide” as used herein, may refer to at least one peptide / polypeptide (i.e. one or more peptides / polypeptides). Similarly, a “nucleic acid encoding a first peptide / polypeptide” may 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, a “first peptide / polypeptide” may refer to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, e.g., in a mixture or conjugated to one another. A “nucleic acid encoding a first peptide / polypeptide” may 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. A “first peptide / polypeptide” or “nucleic acid encoding a first peptide / polypeptide” may 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.

[0368] The two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially, as described herein.

[0369] Second peptide / polypeptide

[0370] Methods of the present disclosure comprise administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to an animal, wherein second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

[0371] In some embodiments, the second peptide / polypeptide is identical to the first peptide / polypeptide. In some embodiments, the second peptide / polypeptide is non-identical to the first peptide / polypeptide.

[0372] It will be appreciated that a peptide / polypeptide which is “non-identical” to a reference peptide / polypeptide comprises an amino acid sequence having less than 100% sequence identity to the amino acid sequence of the reference peptide / polypeptide. In some embodiments, the second peptide / polypeptide has an amino acid sequence having less than 100%, e.g. one of less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% sequence identity to the amino acid sequence of the first peptide / polypeptide. In some embodiments, the second peptide / polypeptide has an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of the first peptide / polypeptide.

[0373] It will be appreciated that the second peptide / polypeptide encompasses peptides / polypeptides comprising: (a) the amino acid sequence of interest which is comprised in the first peptide / polypeptide, or (b) an amino acid sequence which is similar to the amino acid sequence of interest which is comprised in the first peptide / polypeptide.

[0374] It will in turn be appreciated that (as explained hereinabove) the amino acid sequence of interest comprised in the first peptide / polypeptide can be (i) an amino acid sequence of a protein of interest, or (ii) an amino acid sequence which is similar to the amino acid sequence of the protein of interest ( / .e. an amino acid sequence which is similar to the amino acid sequence of (i)).

[0375] In some embodiments the second peptide / polypeptide comprises the amino acid sequence of interest, and additionally comprises further amino acids. In some embodiments second peptide / polypeptide comprises at least 5, 10, 15, 20, 40, 50, 80, 100, 200, or 300 additional amino acids at one or both ends ( / .e. the N- or C-terminus) of the amino acid sequence of interest. In some embodiments second peptide / polypeptide comprises one of at least 1-5, 1-10, 1-15, 1-20, 1-40, 1-50, 1-80, or 1-100, 1-200 or 1-300 additional amino acids at one or both ends ( / .e. the N- or C-terminus) of the amino acid sequence of interest. The second peptide / polypeptide may comprise more than one peptide / polypeptide chain. For example, the second peptide / polypeptide may be a complex of peptides / polypeptides, e.g. comprising 2, 3, 4, 5 or 6 or more peptides / polypeptides. The peptides / polypeptides may be conjugated to one other, or administered as a composition (e.g. mixture) of two or more peptides / polypeptides that are not conjugated.

[0376] Peptides / polypeptides and nucleic acids described herein may be administered in the form of cells comprising / expressing the peptide / polypeptide / nucleic acid, or synthetic agents comprising the peptide / polypeptide / nucleic acid.

[0377] In some embodiments, the second peptide / polypeptide may further comprise 1 or more amino acids at either or both ends of the second peptide / polypeptide. In some embodiments, the second peptide / polypeptide may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or one of 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, or 1-3 amino acids at one end, or both ends.

[0378] In some embodiments, the second peptide / polypeptide is capable of eliciting the production of antigenbinding molecules capable of binding to the protein of interest and an isoform, variant or homolog of the protein of interest.

[0379] A "second peptide / polypeptide” as used herein, may refer to at least one peptide / polypeptide (i.e. one or more peptides / polypeptides). Similarly, a “nucleic acid encoding a second peptide / polypeptide” may 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, a “second peptide / polypeptide” may refer to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptides and / or polypeptides, e.g., in a mixture or conjugated to one another. A “nucleic acid encoding a second peptide / polypeptide” may 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. A “second peptide / polypeptide” or “nucleic acid encoding a second peptide / polypeptide” may 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.

[0380] The two or more peptides / polypeptides / nucleic acids may be administered simultaneously and / or sequentially, as described herein.

[0381] Nucleic acid encoding peptides / polypeptides

[0382] As described herein, the peptides / polypeptides of the present disclosure may be expressed from nucleic acid encoding the peptides / polypeptides. The nucleic acid may be, or may be comprised in, a vector. In some embodiments, the nucleic acid may be DNA encoding a peptide / polypeptide described herein. Nucleic acid / vector may be administered to a subject in order to express a peptide / polypeptide as described herein. The nucleic acid / vector may be present in a cell and the cell may be administered to the animal. The nucleic acid / vector may be incorporated into the genome of a cell, and the cell may be administered to the animal. The nucleic acid / vector may provide for recombinant expression of a peptide / polypeptide as described herein.

[0383] A “vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure. The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcripts) may then be translated into a desired peptide(s) / polypeptide(s). Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

[0384] In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0385] Methods for immunizing a subject with nucleic acid encoding a peptide / polypeptide of interest to elicit an antibody response is described, for example, in Aurisicchio et al. (2012) J Cell Physiol 227: 3381-3388, which is hereby incorporated by reference in its entirety.

[0386] In some embodiments of the methods of the present disclosure, nucleic acid encoding a peptide / polypeptide as described herein is administered to an animal. In such embodiments, the peptide or polypeptide is expressed in the animal following immunization.

[0387] Administrations

[0388] Aspects of the present disclosure involve administration of peptides / polypeptides, or nucleic acids encoding peptides / polypeptides, to an animal. Aspects of the present disclosure also include administering agents for inducing inhibition of the ability of an animal to mount primary immune response and / or promotion of the ability of an animal to mount a secondary immune response to an animal. A step of “administering” a peptide / polypeptide / nucleic acid to an animal comprises introducing the peptide / polypeptide / nucleic acid into an animal one or more times. In some embodiments, “administering” a peptide / polypeptide / nucleic acid into an animal comprises one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate introductions of the peptide / polypeptide / nucleic acid into an animal.

[0389] By way of illustration, in some embodiments the method comprises introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal on 2 occasions, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal on 2 occasions.

[0390] In some embodiments the method comprises introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal on one of 3, 4, 5 or 6 occasions, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal on one of 1 , 2, 3 or 4 occasions.

[0391] In some embodiments the method comprises introducing a first peptide / polypeptide (or nucleic acid encoding the first peptide / polypeptide) as defined herein into an animal on at least 2 occasions, and introducing a second peptide / polypeptide (or nucleic acid encoding the second peptide / polypeptide) as defined herein into the animal on at least 1 occasion.

[0392] Similarly, a step of “administering" an agent resulting in inhibition of a primary immune response and / or promoting a secondary immune response comprises introducing the agent into the animal one or more times. In some embodiments, “administering” the agent comprises one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate introductions of the agent into the animal.

[0393] In some embodiments the method comprises introducing an agent as defined herein into the animal on one of 1 , 2, 3 or 4 occasions.

[0394] In some embodiments the time interval between individual introductions of an administration step comprising plural introductions is one of at least 24 hours, 36 hours, 48 hours, 72 hours, 4 days, 5 days, 7 days, 10 days or 12 days. In some embodiments, the time interval between individual introductions of an administration step comprising plural introductions is about 5-30 days, 7-20 days, e.g. about 10-16 days. In some embodiments, the time interval between individual introductions of an administration step comprising plural introductions is about 2-30 days, 5-20 days, e.g. about 6-8 days.

[0395] Introductions in accordance with an administration step described herein may be of identical materials, or non-identical materials, provided the materials satisfy the requirements of the material to be administered in accordance with the administration step. By way of illustration, administration of a first peptide / polypeptide as defined herein (or nucleic acid encoding the same) may comprise simultaneous or sequence introduction of the same peptide / polypeptide (or nucleic acid encoding the same), or of two or more (e.g. one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-identical peptides / polypeptides each independently meeting the requirements of a first peptide / polypeptide described herein (or nucleic acid encoding the same). Likewise, administration of a second peptide / polypeptide as defined herein (or nucleic acid encoding the same) may comprise simultaneous or sequence introduction of the same peptide / polypeptide (or nucleic acid encoding the same), or of two or more (e.g. one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-identical peptides / polypeptides each independently meeting the requirements of a second peptide / polypeptide described herein (or nucleic acid encoding the same).

[0396] Individual introductions of an administration step described herein may be sequential. That is, in some embodiments a peptide / polypeptide / nucleic acid is introduced into a subject, and after a given time interval a separate introduction is performed.

[0397] In some embodiments, a peptide / polypeptide may be administered in the form of an agent comprising or expressing the peptide / polypeptide. In some embodiments, a peptide / polypeptide may be administered in the form of an agent comprising nucleic acid encoding peptide / polypeptide. In some embodiments, a nucleic acid may be administered in the form of an agent comprising the nucleic acid.

[0398] The agent may be any agent capable of delivering the peptide / polypeptide / nucleic acid. In some embodiments the agent is a cell. In some embodiments the agent is a virus, or a virus-like particle.

[0399] In some particular embodiments, a peptide / polypeptide may be administered in the form of a cell comprising or expressing the peptide / polypeptide. In some embodiments, a peptide / polypeptide may be administered in the form of a cell comprising nucleic acid encoding peptide / polypeptide. In some embodiments, a nucleic acid may be administered in the form of a cell comprising the nucleic acid.

[0400] A cell expressing a peptide / polypeptide may endogenously express the peptide / polypeptide. That is, the peptide / polypeptide may be encoded by, and / or expressed from, nucleic acid of the cell prior to any introduction of nucleic acid encoding the peptide / polypeptide into the cell. A cell expressing a peptide / polypeptide may exogenously express the peptide / polypeptide. That is, the peptide / polypeptide may be encoded by, and / or expressed from, nucleic acid which has been introduced into the cell. In some embodiments the cell expressing a peptide / polypeptide may be or have been modified to express or overexpress the peptide / polypeptide.

[0401] In some embodiments, the peptide / polypeptide / nucleic acid / cell / agent administered is isolated or purified. An isolated or purified peptide / polypeptide / nucleic acid / cell / agent as used herein refers to a composition comprising the peptide / polypeptide / nucleic acid / cell / agent of which at least 80%, 90%, 95%, 99% or 100% of the composition (by weight) is the peptide / polypeptide / nucleic acid / cell / agent component of the composition. In some embodiments, the peptide / polypeptide / nucleic acid is administered in a cell or protein-containing extract.

[0402] A peptide / polypeptide / nucleic acid / cell / agent can be introduced into a subject by any suitable means, such as those described, for example, in Antibodies: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated by reference herein above), in particular at Chapter 6.

[0403] Materials to be introduced into an animal can be formulated as appropriate to the material, route of introduction, the animal and the desired response.

[0404] For example, peptides, polypeptides (optionally conjugated to a carrier protein such as KLH, BSA or OVA, etc.), cells and agents may be diluted in sterile saline, and may combined with an adjuvant (e.g. Complete or Incomplete Freund's Adjuvant, an aluminium salt (e.g. aluminium sulfate (alum), aluminium phosphate, aluminium hydroxide), CpG or an adjuvant described in Lee and Nguyen, Immune Netw. (2015) 15(2):51 -57, which is hereby incorporated by reference in its entirety) to form a stable emulsion. Nucleic acids (optionally provided as liposome-nucleic acid complexes), may be injected in aqueous solution in saline, introduced by pneumatic (jet) injection in aqueous solution, or coated on gold beads and introduced by gene gun.

[0405] Where materials are introduced into an animal by injection, any suitable site for injection maybe used. For example, injections may be intraperitoneal, intravascular (e.g. intravenous or intraarterial), intradermal, subcutaneous, intramuscular, intraosseous, intrathecal, epidural, intracardiac, intraarticular, intracavernous, and intravitreal. Preferred routes for injection in accordance with the methods of the present disclosure are intraperitoneal and intravascular (e.g. intravenous or intraarterial).

[0406] Appropriate amounts of peptide / polypeptide / nucleic acid / agent or number of cells for an individual introduction can be readily determined by the skilled person, e.g. by reference to Antibodies: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press (incorporated by reference herein above). Appropriate volumes and concentrations of formulations for introductions can also readily be determined by the skilled person.

[0407] In some embodiments, peptides / polypeptides / nucleic acids / cells may be formulated differently for different introductions, and / or different administration steps. For example, in some embodiments, different carrier proteins or adjuvants may be used in different introductions and / or different administrations steps. In some embodiments, one or more adjuvants may be used in one or more introductions and / or administration steps, and a different adjuvant or no adjuvant may be used in one or more other introductions and / or administration steps according to the methods of the present disclosure. By way of example, in an embodiment of a method according to the present disclosure a step of administering a first peptide / polypeptide to a subject may comprise introducing the first peptide / polypeptide formulated with e.g. Complete Freund’s Adjuvant and CpG into a subject, and a step of administering a second peptide / polypeptide to the subject may comprise introducing the second peptide / polypeptide formulated with e.g. Incomplete Freund’s Adjuvant and CpG.

[0408] A peptide / polypeptide / nucleic acid / cell / agent according to the present disclosure may be introduced into the animal in an amount appropriate to bring about the desired response.

[0409] For example, introducing a peptide / polypeptide as defined herein into an animal may comprise introducing one of 5 pg, 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 60 pg, 70 pg, 80 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 400 pg or 500 pg of the peptide / polypeptide (e.g., in total, or per administration). In some embodiments introducing a peptide / polypeptide as defined herein into an animal may comprise introducing one of 5-500 pg, 10-200 pg, 20-80 pg or ~50 pg of the peptide / polypeptide into an animal (e.g., in total, or per administration).

[0410] For example, introducing 5 pg, 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 60 pg, 70 pg, 80 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 400 pg or 500 pg of the agent (e.g., in total, or per administration). In some embodiments introducing an agent as defined herein into an animal may comprise introducing one of 5-500 pg, 10-300 pg, 50-200 pg or ~100 pg of the agent into an animal (e.g., in total, or per administration).

[0411] Introducing an agent as defined herein into an animal may comprise introducing one 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 of the agent (e.g., in total, or per administration). In some embodiments introducing an agent as defined herein into an animal may comprise introducing one of 1-25 mg / kg, 2-20 mg / kg, 5-15 mg / kg or ~10 mg / kg of the agent into an animal (e.g., in total, or per administration). Administration steps of the methods of the present disclosure may be sequential. That is, in some embodiments an administration step as described herein is performed, and after a given time interval a separate administration step is performed.

[0412] The time interval between administration steps performed sequentially is preferably a time interval appropriate to the desired response in the animal. By way of example, in an embodiment of the method of the present disclosure following administration of a first peptide / polypeptide as described herein (or nucleic acid encoding the same), a period of time sufficient for the animal to produce one or more antigen-binding molecules to the first peptide / polypeptide may be allowed to pass before administration of a second peptide / polypeptide as described herein (or nucleic acid encoding the same).

[0413] In some embodiments the time interval between administration steps performed sequentially is one of 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, administration steps performed sequentially are separated by a period of about 3 to 21 days, e.g. about 5 to 18 days, 7 to 16 days, or 12-16 days. In some embodiments, administration steps performed sequentially are separated by a period of about 14 days. In some embodiments, administration steps performed sequentially are separated by a period of about 23 days. In some embodiments, administration steps performed sequentially are separated by a period of about 30 days.

[0414] In embodiments where administration steps comprise plural, separate introductions of material into an animal, time intervals provided herein may be between the final introduction of one administration step, and the first introduction of the subsequent administration step.

[0415] In some embodiments, administration steps of the methods of the present disclosure may be performed simultaneously. That is, in some embodiments an administration step as described herein is performed at the same time as or immediately before / after another administration step as described herein.

[0416] In particular embodiments, the methods of the present disclosure involve simultaneous administration of an agent capable of inhibiting a primary immune response and / or capable of promoting a secondary immune response to the animal, along with a second peptide / polypeptide as described herein (or nucleic acid encoding the same).

[0417] The time interval between administration steps performed simultaneously is preferably less than one of 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or 6 hours.

[0418] In some embodiments, where administration steps performed simultaneously, the agents of the separate administration steps are formulated together as a single preparation for administration to the animal. In some embodiments, the agents of the separate administration steps are formulated as separate preparations for administration to the animal.

[0419] Administration steps performed sequentially or simultaneously herein may be introduced into the animal by the same route, or by different routes.

[0420] In some embodiments administration of an agent for inhibiting a primary immune response and / or promoting a secondary immune response in the animal is performed before, at the same time as (e.g., simultaneously with), and / or after administration with the second peptide / polypeptide. In some embodiments administration of an agent for inhibiting a primary immune response and / or promoting a secondary immune response in the animal is performed before and / or after administration with the second peptide / polypeptide, e.g., one or more administrations up to 5 days before administration of the second peptide / polypeptide and / or one or more administrations up to 10 days after administration of the second peptide / polypeptide.

[0421] In some embodiments an agent for inhibiting a primary immune response and / or promoting a secondary immune response in the animal is administered 3 and / or 1 day(s) before administration of the second peptide / polypeptide, and 1 , 3 and / or 6 days after administration of the second peptide / polypeptide. In some embodiments an agent for inhibiting a primary immune response and / or promoting a secondary immune response in the animal is administered 1 , 3 and / or 6 days after administration of the second peptide / polypeptide.

[0422] In aspects of the present disclosure that comprise administration of an agent capable of inhibiting a primary immune response and / or promoting a secondary immune response to the animal, it will be appreciated that administration of the agent is such that the primary immune response to the second peptide / polypeptide is inhibited, and / or such that the secondary immune response is promoted.

[0423] Likewise, it will be appreciated that administration of the first peptide / polypeptide as described herein (or nucleic acid encoding the same), administration of an agent for inducible inhibition of a primary immune response and / or promoting a secondary immune response to the animal and administration of the second peptide / polypeptide as described herein (or nucleic acid encoding the same) is such that the primary immune response to the first peptide / polypeptide is not substantially inhibited, but the primary immune response to the second peptide / polypeptide is inhibited, and / or the secondary immune response is promoted.

[0424] In some embodiments, the methods comprise one or more further administration steps as described herein.

[0425] For the purposes of the following statements, the administration steps are as follows:

[0426] (a) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal described herein, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0427] (b) administering to the animal an agent for inhibiting, e.g., inducibly inhibiting, a primary immune response and / or promoting a secondary immune response in the animal; and

[0428] (c) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest, and wherein the second peptide / polypeptide is non-identical to (e.g. larger than) the first peptide / polypeptide.

[0429] In some embodiments the methods of the present disclosure comprise one of more of the following combinations of the administration steps identified above:

[0430] (1) (a) + (c)

[0431] (2) (a) + (b) + (c)

[0432] (3) (a) + (c) + (a)

[0433] (4) (a) + (b) + (c) + (a)

[0434] (5) (a) + (b) + (c) + (b) + (a)

[0435] (6) (a) + (c) + (b) (7) (a) + (b) + (c) + (b)

[0436] In some embodiments, the administration steps are as follows:

[0437] (a) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal described herein, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0438] (b) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest, and wherein the second peptide / polypeptide is non-identical to (e.g. larger than) the first peptide / polypeptide; and

[0439] (c) administering to the animal an agent for inhibiting, e.g., inducibly inhibiting, a primary immune response and / or promoting a secondary immune response in the animal.

[0440] In some embodiments, the administration steps are as follows:

[0441] (a) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal described herein, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;

[0442] (b) administering to the animal an agent for inhibiting, e.g., inducibly inhibiting, a primary immune response and / or promoting a secondary immune response in the animal;

[0443] (c) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest, and wherein the second peptide / polypeptide is non-identical to (e.g. larger than) the first peptide / polypeptide; and

[0444] (d) administering to the animal an agent for inhibiting, e.g., inducibly inhibiting, a primary immune response and / or promoting a secondary immune response in the animal.

[0445] In some embodiments the methods further comprise a booster step. A “booster step” may comprise introduction of a peptide / polypeptide / nucleic acid as described herein into an animal in the absence of a carrier or adjuvant.

[0446] Booster steps are well known to person skilled in the art of immunology. Booster steps may be included in methods for producing antigen-binding molecules, for example to increase titer, for example prior to isolating antigen-binding molecules, or prior to harvesting cells for hybridoma generation.

[0447] Booster steps may be included in methods for generating immunity to proteins / pathogens, for example to elicit an anamnestic response.

[0448] In some embodiments, a booster step is given prior to isolation of antigen-binding molecules. In some embodiments, a booster step is given prior to harvesting of B lymphocytes for hybridoma production. In some embodiments, a booster step is performed at least about 12 hours to 5 days, about 1 -4 days, or about 2-3 days prior to harvesting B lymphocytes for hybridoma production.

[0449] In some embodiments, a booster step comprises administration to the animal of a peptide / polypeptide / nucleic acid which has already been administered to the animal, for example in a preceding administration step according to (a) or (c) above.

[0450] In some embodiments, the methods comprise performing more than one (e.g. one of 2, 3, 4, 5 or 6) booster steps. Plural booster steps may be separated by one of at least 12 hours, 24 hours, 36 hours, 48 hours or 72 hours. In some embodiments, booster steps may be separated by a period of about 12-48, e.g. ~24 hours.

[0451] A booster step may comprise introduction of the of peptide / polypeptide / nucleic acid by injection. In some embodiments, particularly embodiments relating to methods for producing antigen-binding molecules, a booster step may comprise injecting the peptide / polypeptide / nucleic acid into the abdomen of the animal.

[0452] Evaluation of antigen-binding molecule production

[0453] The methods of the present disclosure may further comprise monitoring / evaluating the immune response of an animal subjected to one or more administration steps as described herein. In some embodiments, the methods comprise detecting the presence of an immune response capable of recognizing a peptide / polypeptide / amino acid seguence of interest.

[0454] In some embodiments, the methods comprise detecting the presence of an antigen-binding molecule capable of binding to a peptide / polypeptide / amino acid seguence of interest. In some embodiments, the methods comprise detecting the presence of an immune cell / population of immune cells capable of producing an antigen-binding molecule capable of binding to a peptide / polypeptide / amino acid seguence of interest.

[0455] In some embodiments, the methods comprise detecting the presence of an antigen-binding molecule possessing one or more functional properties of interest, or an immune cell / population of immune cells capable of producing such an antigen-binding molecule. A functional property of interest may e.g. be the ability to antagonize or agonize a function of the protein / protein complex of interest (e.g. catalytic activity, binding (e.g. protein-protein interaction, such as ligand-receptor binding or multimerization), signalling, transport, storage, structural support etc.). Antigen-binding molecules can be analyzed for such functional properties e.g. using appropriate assays of such function(s) of the protein / protein complex of interest. Immune responses can be analyzed by methods well known to the skilled person. For example, at an appropriate period of time following administration in accordance with the methods described herein, a sample (e.g. a blood sample) may be obtained from the subject and analyzed for antigen-binding molecule production and / or the presence of cells displaying a property of interest (e.g. production of antigen-binding molecule capable of recognizing antigen of interest). Assays for the detection and quantification of antigen-binding molecule production are well known to the skilled person, and are described, for example, in Antibodies: A Laboratory Manual, Second Edition, 2014; Edward A. Greenfield, Cold Spring Harbor Laboratory Press, 2014 (incorporated by reference hereinabove), in particular at Chapter 15. For example, following one or more administration steps according to the present disclosure, a blood, plasma, serum or ascites sample may be collected from the subject, and analyzed e.g. by ELISA or flow cytometry. “Plasma” as used herein refers to the liquid component of blood, lacking the cellular component, and may be the fluid portion of the blood obtained after removal of the blood cells. “Serum” as used herein refers to plasma lacking clotting factors (e.g. fibrinogens), and may be the fluid portion of the blood obtained after removal of the fibrin clot and blood cells. “Ascites” as used herein refers to fluid obtained from the peritoneal cavity.

[0456] Immunoassays may be used to detect antigen-binding molecule production. Immunoassays may be used to determine whether a sample obtained from a subject contains antigen-binding molecule capable of binding to a given peptide or polypeptide.

[0457] In some embodiments the methods comprise quantifying antigen-binding molecule in a sample, e.g. by determining antibody titer. Antibody titer is a measurement of the amount of antibody a subject has produced that is capable of recognizing (i.e. binding to) a given antigen. Antibody titer is expressed as the inverse of the highest dilution of the test sample (e.g. a serum sample) which gives a positive result for the detection of the antigen in e.g. an immunoassay.

[0458] As used herein, “binding” by an antigen-binding molecule refers to specific interaction between an antigen-binding molecule and its cognate antigen. “Specific interaction” is interaction between antibody and antigen which is not non-specific. Antigen-binding molecule:antigen binding is mediated by non- covalent interactions such as Van der Waals forces, electrostatic interactions, hydrogen bonding, and hydrophobic interactions. In particular, the interaction is between the antigen-binding site of an antigenbinding molecule and its cognate epitope in an antigen. An epitope is a part of an antigen which is contacted by an antigen-binding molecule. In particular, the epitope is the part of the antigen to which the antigen-binding molecule binds. An epitope is provided by an antigenic sequence of amino acids. An epitope may be linear, consisting of a contiguous sequence of amino acids (i.e. an amino acid primary sequence). Alternatively, an epitope may be conformational, consisting of a discontinuous sequence of amino acids of an amino acid sequence of any antigen. The amino acids of the discontinuous sequence of amino acids may be located in different regions of a peptide / polypeptide, and may be positioned in close proximity when the antigen is folded, e.g. into its native structure.

[0459] The ability of a given antigen-binding molecule to bind to a given protein can be analyzed using techniques which are well known to the skilled person, which include ELISA, immunoblot (e.g. western blot), immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498- 507) and flow cytometry, amongst others. Through such analysis interaction between a protein / domain and an antigen-binding molecule can be measured and quantified. Such methods may involve expressing the protein / domain, contacting the expressed protein / domain with an antigen-binding molecule and detecting formation of a non-covalent complex of the protein / domain and the antigen-binding molecule.

[0460] The particular region of a given binding partner to which an antigen-binding molecule binds can furthermore be analyzed using methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, hydrogen-deuterium exchange analysis by mass spectrometry, cryoelectron microscopy, peptide scanning and mutagenesis mapping. 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, both of which are hereby incorporated by reference in their entirety.

[0461] Further articles of the disclosure

[0462] The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, comprising a nucleotide sequence as described herein.

[0463] In some embodiments, the nucleic acid / plurality is purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material. In some embodiments the nucleic acid / plurality comprises or consists of DNA and / or RNA.

[0464] It will be appreciated that in some embodiments, a plurality of nucleic acids according to the present disclosure may comprise one or more (e.g. one of 1 , 2, 3, 4, 5, 6, 7, 9 or 10) nucleotide sequences according to the present disclosure. In such embodiments, plural nucleotide sequences may independently conform to any embodiment of a nucleotide sequence described herein.

[0465] The present disclosure also provides a vector or a plurality of vectors comprising the nucleic acid / plurality of nucleic acids according to the present disclosure.

[0466] The nucleic acid / plurality may be contained in a vector or a plurality of vectors. A “vector” as referred to herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. A vector may be a vector for expression of the nucleic acid in the cell ( / .e. an expression vector.). Vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used.

[0467] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes).

[0468] In some embodiments, a vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression in a eukaryotic cell. In some embodiments, a vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter. The present disclosure also provides a cell comprising or expressing a nucleic acid / plurality or vector / plurality. The cell may be a eukaryotic cell, e.g. an animal cell. The cell may be a non-human animal cell, e.g. a mouse, rat, hamster, llama, guinea pig, rabbit, goat, chicken, primate (e.g. non-human primate, e.g. a monkey), sheep, donkey, cow, cat, dog, pig or horse cell. In some embodiments the cell is a mammalian cell (e.g. a non-human mammalian cell). In some embodiments the cell is from an animal of a species of the order Rodentia (e.g. a species of the genus Mus, Rattus or Cavia) or Lagomorpha (e.g. a species of the family Leporidae). In some embodiments the cell is a mouse cell.

[0469] In some embodiments, the cell is a multipotent cell, e.g. a pluripotent cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is an embryonic stem cell.

[0470] Also provided is an embryo comprising a cell according to the present disclosure. Also provided is a blastocyst comprising a cell according to the present disclosure.

[0471] Such nucleic acids, vectors, cells, embryos and blastocysts are useful in the production of animals according to the present disclosure. Accordingly, the present disclosure also provides an animal produced by intrauterine implantation of a blastocyst comprising a cell according to the present disclosure.

[0472] Preparation of peptides / polypeptides

[0473] Peptides and polypeptides for use in methods described herein may be prepared according to methods known to the skilled person.

[0474] Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides / polypeptides can by synthesized using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety. Alternatively, peptides / polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of peptides / polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety.

[0475] For recombinant production according to the present disclosure, any cell suitable for the expression of peptides / polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COS cells. In some embodiments, the cell is a CHO cell that transiently or stably expresses the polypeptides. In some cases the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilized which enhance secretion of the peptide / polypeptide into the media.

[0476] In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according using a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431 , which is hereby incorporated by reference in its entirety.

[0477] Production may involve culture or fermentation of a eukaryotic cell modified to express the peptides / polypeptides of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted peptides / polypeptides. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0478] Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0479] Following culturing the cells that express the peptides / polypeptides, the peptides / polypeptides of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the peptides / polypeptides are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted peptides / polypeptides of interest. If the peptides / polypeptides of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by Bonification, rapid freeze-thaw or osmotic lysis.

[0480] It may then be desirable to isolate the peptides / polypeptides of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins. Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be performed subsequently to precipitation.

[0481] 5

[0482] Once the peptides / polypeptides of interest have been isolated from culture it may be desired or necessary to concentrate the peptides / polypeptides. A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization. 0 Sequence Identity

[0483] Pairwise and multiple sequence alignment for the purposes of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J.

[0484] 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), 5 Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0485] Sequences

[0486] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the invention in diverse forms thereof.

[0487] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0488] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0489] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise” and “include", and variations such as “comprises", “comprising”, and “including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0490] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated. Also, where a polypeptide-encoding nucleic acid sequence is disclosed herein equivalent polypeptide-encoding sequences as a result of degeneracy of the genetic code are also expressly contemplated.

[0491] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an," and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0492] Methods disclosed herein may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term “in vitro’’ is intended to encompass experiments with materials, biological substances, cells and / or tissues in laboratory conditions or in culture whereas the term “in vivo” is intended to encompass experiments and procedures with intact multi-cellular organisms. In some embodiments, methods performed in vivo may be performed on non-human animals. “Ex vivo” refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.

[0493] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press Aspects and embodiments of the present disclosure will now be discussed. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference in their entirety. While the invention has been described in conjunction with the exemplary embodiments described below, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0494] Brief Description of the Figures

[0495] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0496] Figures 1A and 1B. Schematic representations of (1A) the genomic mouse locus targeted, the targeting vector, the targeted allele (after integration of the targeting vector insert), and the constitutive knock-in allele, for the production of the Cc / 79a-CreERT2 knock-in mouse as described in Example 8.1 ; and (1 B) the targeting vector, the nucleotide sequence of which is shown in SEQ ID NO:1 .

[0497] Figure 2. Schematic representation of the genomic mouse locus targeted, the targeting vector, the targeted allele (after integration of the targeting vector insert), conditional knockout allele and the knockout allele after Cre recombination, for the production of the Ighm-lghd CKO mouse as described in Example 8.2.

[0498] Figure 3. Immunization, dosing and sampling timeline for experiments to assess the inhibition of a primary immune response through depletion of lgM+lgD+ naive B cells using anti-lgD antibody.

[0499] Figures 4A and 4B. Graphs showing B cell proportions in the spleen, lymph nodes and PBMCs before (4A) and after (4B) anti-lgD treatment, as compared to isotype control antibody. **** p < 0.0001 , *** p < 0.001 Tukey’s multiple comparisons test.

[0500] Figure 5. Graphs showing immune responses to antigens. All mice received two initial administrations of EGFR-hFc, followed by a third administration of either PBS (left panels), EGFR-hFc (middle panels) or CD33-mFc (right panels). Data shown as mean + / - sem. N = 9 for Days 7, 14 & 26. n = 6 for Days 31 , 35 and 40. n = 3 for Day 50.

[0501] Figure 6. Graphs showing the percentage of B cells in which CreERT2-mediated recombination of the ZsGreen allele is detected in B cells isolated from the bone marrow, spleen and lymph nodes, and a graph showing the percentage of non-B cells comprising CreERT2-mediated recombination of the ZsGreen allele in tissues, of mice homozygous for a lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sortm6(CAG- zsGreeni)Hze^anc| heterozygous for Cd79aCreERT2, at the indicated number of days following treatment with 100 mg / kg bodyweight or 200 mg / kg bodyweight of tamoxifen. Figure 7. Graphs showing the percentage of B cells in which CreERT2-mediated recombination of the ZsGreen allele is detected in B cells isolated from the bone marrow, spleen and lymph nodes of mice homozygous for a lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sortm6<CAG ZsGreen1)Hze) and heterozygous for Cd79aGreERT2, at the indicated number of days following treatment with tamoxifen at 100 mg / kg bodyweight, or vehicle control (corn oil).

[0502] Figures 8A and 8B. Schematic and images illustrating CreERT2-mediated deletion of exons 1-6 of Ighm and exons 1 -3 of Ighd in vivo in the B cells of Ighmlghcf10^1Cd79a+ / GreERT2mice 60 h after treatment with tamoxifen at 200 mg / kg bodyweight. (8A) Schematic illustrating the architecture of the lghmlgh(foxlocus before (upper) and after (lower) tamoxifen-induced, CreERT2-mediated deletion of the gene region comprising exons 1-6 of Ighm and exons 1-3 of Ighd. The location of the forward and reverse primers for PCR amplification of lghmlghdflox, lghmlghdK0, and control region amplicons are indicated. (8B) Images of the products of PCR reactions performed using primers for amplification of lghmlghdflox, lghmlghdK0, and control region amplicons, using genomic DNA obtained from cells isolated from the bone marrow of Ighmlghcf10^10* Cd79a+ / CreERT2mice 60 h after treatment with tamoxifen at 200 mg / kg bodyweight (TAM) or vehicle control (corn oil; Oil), following separation by agarose gel electrophoresis. The expected positions of the lghmlghdflox, lghmlghdK0, and control region amplicons are indicated.

[0503] Figure 9. Histograms and bar charts showing the expression of IgM and IgD on Cd19+cells obtained from the spleens and lymph nodes of lghmlghdrlox / floxCd79a+ / GreERT2mice, 60 h after treatment with tamoxifen at 200 mg / kg bodyweight, or corn oil (vehicle control), as determined by flow cytometry.

[0504] Figures 10A and 10B. Schematic and graphs illustrating maintenance of CreERT2-mediated recombination of the ZsGreen allele in B cells isolated from the bone marrow, spleen and lymph nodes of mice homozygous for a lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sorim6<CAG-ZsGreen1>Hze') and heterozygous for Cd79aCreERT2, achieved by repeated administration of tamoxifen. (10A) Schematic illustrating the schedule for administration of tamoxifen and vehicle (corn oil) to the mice in Arm 1 and Arm 2. (10B) Graphs showing the percentage of B cells in which CreERT2-mediated recombination of the ZsGreen allele is detected in B cells isolated from the bone marrow, spleen and lymph nodes, at the indicated number of days following the administration of the initiating dose of tamoxifen, for mice in Arm 1 and Arm 2.

[0505] Figure 11. Schematic illustrating the schedule for administration of tamoxifen to lghmighcflox / flox; Cd79a+ / GreERT2mice for maintaining B cell-specific, CreERT2-mediated deletion of exons 1 -6 of Ighm and Ighd exons 1-3.

[0506] Figure 12. Bar charts showing the diversity of VH gene usage among B cells within samples collected at days 24 and 55 from Ighdlghmflox / flox-tCd79a+ / CreERT2mice and BALB / c mice, as determined by NGS sequencing. Figures 13A to 13D. Histograms showing binding of antibodies in the sera of (13A and 13C) Ighdlghmflox / flox-tcd79a+ / CreERT2mice and (13B and 13D) BALB / c mice, collected at the indicated timepoints, to: nontransfected CHO cells (Control), cells transiently expressing the membrane-bound version of the protein of interest (ECD w / protease cleavage site), or cells transiently expressing a membrane-bound version of the soluble version of the extracellular domain of the protein of interest lacking the protease cleavage site (ECD w / o protease cleavage site).

[0507] Figures 14A and 14B. Graphs showing binding of two representative antibodies derived from two antibody clones raised in the Ighdlghmflox / flox; Cd79a+ / CreERT2mouse to the full-length extracellular domain of the protein of interest (comprising the protease cleavage site; open circles), as determined by ELISA. Closed circles in Figure 14A show binding by an isotype control antibody.

[0508] Figures 15A to 15F. (15A) Flow cytometry data showing the proportion of lgG+, CD45R+ B cells in

[0509] BALB / c mice and Ighdlghmflox / flox; Cd79a+ / CreERT2(IgMIgDcKO) mice after tamoxifen-induced gene deletion. (15B) Graph showing quantification of lgG+ B cells in BALB / c mice and lgMlgDf / fmice prior to tamoxifen-induced gene deletion, and in BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene deletion. (15C) Graph showing quantification of lgG+, B220+B cells in BALB / c mice and lgMlgDf / fmice prior to tamoxifen-induced gene deletion, and in BALB / c mice and IgMIgDcKO mice after tamoxifen- induced gene deletion. (15D) Graph showing quantification of B220+B cells in the lymph node (LN) and spleen (SP) of BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene deletion. (15E) Graph showing quantification of lgG+, B220+B cells in BALB / c mice and lgMlgDf / fmice prior to tamoxifen- induced gene deletion, and in BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene deletion. (15F) Graph showing quantification of B220+B cells in the lymph node (LN) and spleen (SP) of BALB / c mice and IgMIgDcKO mice after tamoxifen-induced gene deletion.

[0510] Figure 16. Graph showing binding of antibodies produced in the sera in response to immunizing BALB / c or NZBWF1 mice with recombinant human DLL3-Fc tagged protein, as determined by ELISA. Sera were collected on Day 38 post-immunization.

[0511] Figure 17. Graph showing binding of antibodies produced in the sera in response to immunization with two doses of a 13-mer peptide conjugated to KLH in BALB / c or NZBWF1 mice, as determined by ELISA. Sera were collected on Day 25 post-immunization.

[0512] Figures 18A-18C. (18A) Image of the products of PCR reactions performed using primers for amplification of a 2328 bp wild-type (wt) or a 2458 bp flox (fl) amplicon detectable in the absence of CreERT2-mediated recombination of the IghmlghcP locus and a 232bp IgMIgDcKO amplicon detectable following CreERT2-mediated recombination of the lghmlghdftoxlocus, using genomic DNA obtained from cells isolated from the spleen and ear of BALB / c or lghmlghdftox / ftox', Cd79a+ / CreERT210 days after the last treatment with tamoxifen at 200 mg / kg bodyweight (tamoxifen) in a treatment regime consisting of 5 doses of tamoxifen over a course of 40 days, following separation by agarose gel electrophoresis. (18B and 18C) Flow cytometry and quantification of IgM- and IgD- expressing B cells collected from lymphoid tissues in tamoxifen-induced transgenic mice (IgMIgDcKO) compared with wild-type mice.

[0513] Figure 19. Immunization strategy for the generation of targeted epitope specific antibodies for each protein of interest. ‘Antigen 1’ refers to a peptide comprising an amino acid sequence for the targeted epitope within the protein of interest. ‘Antigen 2’ refers to the full-length protein of interest containing the targeted epitope.

[0514] Figures 20A and 20B. (20A) Graph showing binding of antibodies in sera generated in IgMIgDcKO mice and wild-type BALB / c mice, for the protein of interest 1 as defined in Example 19, as determined by ELISA. (20B) FACS analysis of binding profiles for antibodies raised against protein of interest 1 as defined in Example 19, present in the sera generated in IgMIgDcKO and wild-type mice.

[0515] Figures 21 A and 21 B. (21 A) Graph showing binding of antibodies in sera generated in IgMIgDcKO mice and wild-type BALB / c mice, for protein of interest 2 as defined in Example 19, as determined by ELISA. (21 B) FACS analysis of binding profiles for antibodies raised against protein of interest 2 as defined in Example 19, present in the sera generated in IgMIgDcKO and wild-type mice.

[0516] Figure 22. FACS analysis showing the specificity of recombinant antibodies to either the full-length extracellular domain (ECD) or the epitope-deleted (AEp) extracellular domain of protein of interest 1 as defined in Example 19.

[0517] Figures 23A and 23B. Graphs showing the dose-response curve for binding to full-length extracellular domain of protein of interest 3 as defined in Example 19, as determined by ELISA, for two representative antibody clones obtained from the Ighdlghmflox / flox-tCd79a+ / CreERT2mouse.

[0518] Examples

[0519] Example 1 : Transgenic mice providing for inducible knockout of IGHM and IGHD

[0520] Transgenic mice providing for inducible knockout of mouse homologs of IGHM and IGHD genes are produced.

[0521] Briefly, mouse embryonic stem cells are modified by CRISPR / Cas9-mediated gene editing (as described in Lee et al., Drug Discovery Today: Disease Models (2016) 20: 13-20) to comprise loxP target sequences flanking exons of IGHM and IGHD genes, and to encode CreERT under the control of a promoter providing for expression in B cell lineage cells. The modified embryonic stem cells are used to generate transgenic mice via introduction of into a blastocyst, and subsequent intrauterine implantation of the blastocyst for gestation.

[0522] The expression of mouse IgM and IgD can be inducibly knocked-out in the resulting transgenic mice by administration of tamoxifen. Example 2: Transgenic mice encoding human immunoglobulin genes providing for inducible knockout of IGHM and IGHD

[0523] Transgenic mice encoding human immunoglobulin genes providing for inducible knockout of mouse homologs of IGHM and IGHD genes are produced.

[0524] Briefly, embryonic stem cells derived from transgenic mice encoding human immunoglobulin genes are modified by CRISPR / Cas9-mediated gene editing (as described in Lee et al., Drug Discovery Today: Disease Models (2016) 20: 13-20) to comprise loxP target sequences flanking exons of IGHM and IGHD genes, and to encode CreERT under the control of a promoter providing for expression in B cell lineage cells. The modified embryonic stem cells are used to generate transgenic mice via introduction of into a blastocyst, and subsequent intrauterine implantation of the blastocyst for gestation.

[0525] The resulting transgenic mice produce antibodies having fully human variable region ( / .e. VH and VL) sequences, in which expression of mouse IgM and IgD can be inducibly knocked-out by administration of tamoxifen.

[0526] Example 3: Production of antibodies using transgenic mice providing for inducible knockout of IGHM and IGHD

[0527] The mice produced as described in Examples 1 and 2 are used for the production of antibodies capable of binding to an amino acid sequence of interest in its native presentation.

[0528] Peptide comprising a sequence of interest of the extracellular domain of a protein of interest is prepared synthetically using standard methods. The extracellular domain of the protein of interest is recombinantly expressed in and purified from Chinese Hamster Ovary (CHO) cells or HEK293 cells.

[0529] Peptides are conjugated to KLH and hepatitis B core antigen (HBc) carriers for immunization. Conjugation uses succinimidyl 6-((beta-maleimidopropionamido) hexanoate)) (SMPH) as a linker between the peptide and protein carriers. Successful conjugation to protein carriers is confirmed by SDS-PAGE analysis.

[0530] Mice are first immunized with 50 pg of peptide (representing a small fragment of an extracellular domain of the protein of interest [First Injection].

[0531] Subsequent to immunization with the peptide [First Injection]:

[0532] (a) one group of mice is administered with tamoxifen (by intraperitoneal injection) to induce knockout of IgM and IgD; and

[0533] (b) another group of mice is not treated to induce knockout of IgM and IgD.

[0534] Both groups of mice are subsequently immunized with 50 pg of polypeptide representing the full-length amino acid sequence of the extracellular domain of the protein of interest [Second Injection]. Subsequent to the second injection, mice are immunized a further two times with the same peptide or polypeptide that they were immunized with at the second injection [Third and Fourth Injections].

[0535] Subsequent to the third and fourth injections, the mice are given one to three booster injections, of the same peptide or polypeptide that they were immunized with at the second injection [Booster Injections].

[0536] Sites of injection include the armpits, groin, foot, back and abdomen.

[0537] Different formulations are used for the different injections, as follows:

[0538] First Injection: peptide + Freund's complete adjuvant

[0539] Second Injection: ECD + Freund's incomplete adjuvant

[0540] Third and Fourth Injections: ECD + Freund's incomplete adjuvant

[0541] Booster Injections: ECD (with or without adjuvant)

[0542] Example 4: Analysis of antibody production by ELISA

[0543] After the final Booster Injection, serum is collected from mice and analyzed for antibody binding to

[0544] (1) the peptide, and

[0545] (2) the extracellular domain of the protein of interest (ECD).

[0546] Analysis is performed by Enzyme Linked Immunosorbent assay (ELISA).

[0547] Briefly, ELISA plates are coated overnight with the peptide or ECD (1 pg / ml in PBS) at 4°C After coating, ELISA plates are washed with sashing buffer (0.05% Tween 20 in 1x PBS) and then blocked with 1% BSA in 1x PBS for 1 hour at room temperature, and subsequently washed thrice with washing buffer.

[0548] Serum is collected from mice, serial dilutions are applied to wells of ELISA plates, and plates are incubated at room temperature for 1 hour. After washing with washing buffer, a Horseradish Peroxidase (HRP)-conjugated antibody is added to wells and plates are incubated at room temperature for 1 hour

[0549] ELISA plates are then developed using TMB substrate solution at room temperature for 10 mins. Development is terminated by addition of 2M sulfuric acid (H2SO4), and plates are read at O.D. 450 nm, within 30 min of termination.

[0550] Example 5: Hybridoma production

[0551] Hybridomas are produced, and antibody production by hybridomas is analyzed as follows.

[0552] Fusion (Dav 1): Mice are dissected under a sterile environment to obtain spleen and lymph nodes, and the single cell suspensions of cells of these tissues were prepared.

[0553] Cells are fused with myeloma cells either by polyethylene glycol (PEG) fusion or by electrofusion, For PEG fusion, ClonaCell-HY Hybridoma Cloning Kit is used and cells are fused in accordance with the manufacturer’s instructions (Stemcell Technologies, Canada). Fused cells are cultured in ClonaCell-HY Medium C (Stemcell Technologies, Canada) overnight at 37°C in a 5% CO2 incubator. The next day, fused cells are centrifuged and resuspended in 10 ml of ClonaCell-HY Medium C and then gently mixed with 90 ml of semisolid methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies, Canada) containing HAT components and plated into 96 well plates. Cells are allowed to grow at 37 °C in a 5% CO2 incubator. After 7-10 days, single hybridoma clones are identified and antibody producing hybridomas are selected by screening the supernatants by Enzyme-linked immunosorbent assay (ELISA).

[0554] Alternatively, for Electrofusion, a NEPA21 Super Electroporator is used and cells are fused according to manufacturer’s protocol (Nepagene). Fused cells are let to recover in ClonaCell-HY Medium C (Stemcell Technologies, Canada) overnight at 37°C in a 5% CO2 incubator. The next day, fused cells are centrifuged and resuspended in 1 ml of ClonaCell-HY Medium C and then gently mixed with 90 ml of semisolid methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies, Canada) containing HAT components and 500ug of FITC-labelled anti-mouse antibody (Jackson Immunoresearch). The cells are then plated into 8 to 16 x 6-well plates. Colonies are allowed to grow at 37 °C in a 5% CO2 incubator for 7 days. Colonies are scanned for FITC fluorescence and picked using Clonepix (Fortebio) device and transferred into a 96 well plate containing to AOF Media. Picked colonies were allowed to grow for 5 days after which supernatants were screened by Enzyme-linked immunosorbent assay (ELISA).

[0555] Second round ELISA: At Day 13 a second round ELISA is performed on cell culture supernatant of wells of the 96 well plate.

[0556] Expansion / storaqe: At Day 14, cells from wells which give a positive result in the second round ELISA are transferred to wells of a 24 well plate for culture and expansion. When cultures are almost confluent, cells are harvested and frozen for storage.

[0557] Example 6: Peptide- and ECD-bindinq antibodies and hybridomas

[0558] Mice immunized according to the protocol wherein the mice are administered tamoxifen for inducible knockout of IgM and IgD prior to the Second Injection ( / .e. (a) group mice - See Example 3) produce a much higher titer of antibody capable of binding to the extracellular domain of the protein of interest as compared to mice in which IgM and IgD are not knocked-out prior to the Second Injection ( / .e. (b) group mice).

[0559] No difference is observed between (a) and (b) group mice in terms of titer of antibody capable of binding to the peptide.

[0560] The proportion of mice having sera containing antibodies capable of binding to both the peptide and the extracellular domain of the protein of interest is greater in the (a) group as compared to the (b) group. The proportion of mice producing hybridomas which produce antibody displaying binding to both the peptide and the extracellular domain of the protein of interest is greater in the (a) group as compared to the (b) group.

[0561] Example 7: Hybridomas producing antibody binding peptide and ECD

[0562] Cells from wells giving positive signal in ELISA are diluted to ~1 cell per well of a multiwell plate, and cultured in vitro for 1-2 weeks.

[0563] The cell culture supernatant from the wells is then analyzed by ELISA as described in Example 4 for binding to:

[0564] (1) Recombinant Fc-tagged protein of interest;

[0565] (2) Optionally, Fc alone; and

[0566] (3) The peptide with which the antibody was raised.

[0567] Cells from wells producing antibody capable of binding (1) and (3) only are diluted to ~1 cell per well of a multiwell plate, and cultured in vitro for 1-2 weeks.

[0568] The cell culture supernatant from the wells was then analyzed by ELISA as above. Cells from these wells are considered to be monoclonal.

[0569] Mice immunized according to the protocol wherein the mice are administered tamoxifen for inducible knockout of IgM and IgD prior to the Second Injection ( / .e. (a) group mice - See Example 3) generate hybridomas producing monoclonal antibodies capable of binding to both of the peptide and the extracellular domain comprising the peptide with a success rate which is greater than the success rate mice in which IgM and IgD are not knocked-out prior to the Second Injection ( / .e. (b) group mice).

[0570] Cells from the wells are transferred to T25 flasks for culture and expansion. After a period of 3-4 weeks, cells are frozen for storage in liquid nitrogen, or used for ascites production.

[0571] Example 8: Exemplary transgenic mice

[0572] Example 8. 1 Constitutive Cd79a-CreERT2 knock-in mouse

[0573] A mouse comprising an endogenous nucleotide sequence providing for constitutive knock-in of CreERT2 at Cd79a is produced.

[0574] A targeting vector was designed based on the mouse Cd79a transcript NCBI Ref: NM_007655.4. The targeting vector insert is shown schematically in Figure 1 A, and comprises (from 5’ to 3’):

[0575] A ~3 kb short homology arm, comprising exon 1 of Cd79a having a mutated translation initiation codon;

[0576] A puromycin resistance gene PuroR, flanked by FRT sites (providing for its flippase-mediated excision);

[0577] The first 7 nucleotides of Cd79a exon 2, followed by the Kozak sequence and ORF of CreERT2; The Cd79a 3’ UTR and human growth hormone polyadenylation signal (hGHpA; in order to prevent transcriptional read-through);

[0578] A ~6 kb long homology arm, including exons 3 to 5 of Cd79a and Arhgefl; and Nucleic acid encoding thymidine kinase.

[0579] The targeting vector is represented schematically in Figure 1 B, and the nucleotide sequence of the targeting vector is shown in SEQ ID NO:1 .

[0580] The targeting vector is transfected into cells of a Balb / c embryonic stem cell line. Embryonic stem cell clones comprising a successfully integrated sequence are identified by positive selection for puromycin resistance, and negative selection for thymidine kinase activity. The selected cells are subsequently treated to effect flippase-mediated excision of PuroR, yielding the mature knock-in allele.

[0581] Embryonic stem cells comprising the knock-in allele are used to produce transgenic mice by microinjection into blastocysts from Balb / c mice (as described in Sumiyama et al., PLoS One (2018) 13(9):e0203056), which are then implanted into female Balb / c mice for gestation.

[0582] The Cc / 79a-CreERT2 mice constitutively express CreERT2 in B cell lineage cells, under the control of the Cd79a promoter.

[0583] Example 8.2 Conditional Ighm and Icihd knockout mouse

[0584] A mouse comprising an endogenous nucleotide sequence providing for inducible knockout of Ighm and Ighd is produced.

[0585] A targeting vector was designed based on the mouse Ighm transcript Ensembl Ref: ENSMUST00000177715, and the mouse Ighd transcript Ensembl Ref: ENSMUST00000194162. The targeting vector insert is shown schematically in Figure 2, and comprises (from 5’ to 3’):

[0586] A ~4 kb short homology arm; loxP sites flanking a region comprising: neomycin resistance gene NeoR, flanked by FRT sites (providing for its flippase-mediated excision); exons 1 to 6 of Ighm; exons 1 to 3 of Ighd; and puromycin resistance gene PuroR, flanked by F3 sites (providing for its flippase-mediated excision);

[0587] A ~6 kb long homology arm; and Nucleic acid encoding thymidine kinase.

[0588] The nucleotide sequence of the targeting region of the vector is shown in SEQ ID NO:2.

[0589] The targeting vector is transfected into cells of a Balb / c embryonic stem cell line. Embryonic stem cell clones comprising a successfully integrated sequence are identified by positive selection for neomycin and puromycin resistance, and negative selection for thymidine kinase activity. The selected cells are subsequently treated to effect flippase-mediated excision of NeoR and PuroR, yielding the mature conditional knockout allele.

[0590] Cre recombinase-mediated excision of exons 1 to 6 of Ighm (including the proximal promoter) is predicted to result in loss of function, and removal of Ighd exons 1 to 3 (and the proximal promoter) is predicted to prevent transcription of Ighd.

[0591] Embryonic stem cells comprising the conditional knockout allele are used to produce transgenic mice by microinjection into blastocysts from Balb / c mice (as described in Sumiyama et al., PLoS One (2018) 13(9):e0203056), which are then implanted into female Balb / c mice for gestation.

[0592] The Ighm-lghd CKO mice provide for Cre-conditional knockout of expression of Ighm and Ighd.

[0593] Ighm-lghd CKO mice are crossed with Cc / 79a-CreERT2 knock-in mice described in Example 8.1 , yielding mice that are homozygous for the lghmlgh(foxallele, and heterozygous for the Cd79aCreERT2allele, and providing for knockout of expression of Ighm and Ighd in B cells in response to treatment with tamoxifen. The resulting transgenic mice had the following genotype: lghmlghcftox / TtoxCd79a+ / CreERT2

[0594] Example 9: Inhibition of a primary immune response through depletion of lqM+lqD+ naive B cells using anti-lqD antibody

[0595] Experiments were performed to test the ability of anti-lgD antibody treatment to restrict the primary B cell response to a second antigen.

[0596] The anti-lgD antibody binds to surface IgD-expressing B cell populations. Treatment with anti-lgD antibody can lead to depletion of immature naive B cells which express both IgM and IgD. Removal of lgM+lgD+B cell populations prior to immunization was found to reduce the ability of the animal to mount a primary response to new antigens.

[0597] The immunization, dosing and sampling timelines are shown in Figure 3.

[0598] 8-9 week old mice were immunized with the first antigen, EGFR-hFc (50pg in complete Freund's adjuvant) on Day 1 to induce a primary response to EGFR. Mice were injected again with EGFR-hFc (50pg in incomplete Freund's adjuvant) on Day 7 to augment the primary response to EGFR. On day 30, mice were immunized for a third time, either with the same antigen (EGFR-hFc, 50pg in incomplete Freund's adjuvant) or a new antigen (CD33-mFc, 50pg in incomplete Freund's adjuvant), or injected with equal volume of PBS in incomplete Freund's adjuvant.

[0599] Immunized mice were injected intra-peritoneally with 5 doses of either lOmg / kg of anti-lgD antibody (antimouse IgDa; clone AMS-9.1) or isotype antibody (IgGaK). The 5 doses were given 1 and 3 days prior to the third immunization, and 1 , 3, and 6 days subsequent to the third immunization (Days 27, 29, 31 , 33 and 36 in the full experimental timeline). B cell proportions present in the spleen, lymph nodes and peripheral blood mononuclear cells (PBMCs) were determined in mice 1 day prior to anti-lgD antibody treatment on Day 26, and 4 and 14 days after the last dose of anti-lgD antibody on Days 40 and Day 50. Cells were isolated, labelled with CD45R- VioBlue, IgM-APC and IgD-FITC conjugated antibodies, and FACS analyzed for the proportions of lgM+, lgD+, lgM+lgD+ and IgM-lgD- B cell populations.

[0600] Sera was collected from the mice on days 7, 14, 26, 31 (to assess primary response to EGFR), and on days 36, 40 and 50 (to assess primary response to CD33 and secondary response to EGFR). Sera was assessed for antibody binding to EGFR-His and CD33-His proteins, followed by detection with anti-mouse IgG-HRP secondary antibody and development of colorimetric substrate 3,3',5,5'-tetramethylbenzidine in ELISA assays. Samples were first diluted 1 :100, then serially diluted 3-fold to yield 11 dilutions for the ELISA assays. Responses to antigens were plotted as the dilution factor at 50% of maximum binding.

[0601] Results

[0602] Figure 4 shows the proportions of B cells in the spleen, lymph nodes and PBMCs before (4A; Day 26) and after (4B; Days 40 and 50) anti-lgD treatment.

[0603] Anti-lgD treatment resulted in depletion of lgM+lgD+ B cells in the spleen, lymph nodes and PBMCs. Depletion of 40-60% of lgM+lgD+ cells were observed on Day 40, 4 days after the last anti-lgD treatment. A concomitant increase in lgM+ population was observed. At Day 50, lgM+lgD+ cell proportion had recovered to > 80% of control, although reduced lgM+lgD+ and increased lgM+ cell populations were still significant on both Days 40 and 50 (p <0.0001, Tukey’s multiple comparisons test).

[0604] Figure 5 shows the immune responses observed after two immunizations with EGFR-hFc to generate a primary immune response and a third immunization with either PBS (left panels), EGFR-hFc (middle panels) or CD33-mFc (right panels), according to the timeline in Figure 3.

[0605] A secondary immune response was only observed after a third administration of EGFR, seen by Day 36 (top middle panel). Anti-lgD antibody treated mice exhibited reduced EGFR response at 78% of isotype control treated mice on Day 36 and 40, and at 88% of control on Day 50.

[0606] Anti-lgD antibody treatment reduced the primary response to CD33 on Days 40 and 50, at 26% and 56% of control respectively (bottom right panel).

[0607] Thus, treatment with anti-lgD antibody can result in depletion of lgM+lgD+ B cells and reduction in the primary response to new antigens with a smaller impact on secondary responses. Example 10: Characterization of CreERT2 activity in transgenic mice carrying the Cd79aCreERT2constitutive knock-in allele

[0608] Mice homozygous for a lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sortm6<CAG ZsGreen1)Hze) and heterozygous for Cd79aGreERT2were used to assess induction of B cell-specific cre / lox recombination in vivo. In such mice, tamoxifen-induced expression of CreERT2 is expected to result in B cell-specific expression of ZsGreen.

[0609] Briefly, 6-8 week-old mice were injected intra-peritoneally with single dose of tamoxifen at 100 mg / kg or 200 mg / kg body weight. Mice were euthanized at 1-, 2-, 4- and 5- days post-treatment for analyzes. Cells isolated from bone marrow, spleen, lymph nodes and blood were stained with Zombie NIR reagent and anti-mouse Cd45r antibody, and assessed by flow cytometry to determine the proportions of Cd45r+B cells and Cd45r non-B cells that were ZsGreen-fluorescent.

[0610] The results are shown in Figure 6. Cre-mediated recombination giving rise to ZsGreen fluorescence was detected specifically in Cd45r+ B cells in all lymphoid tissues. ZsGreen fluorescence was detected in 60- 90% of total Cd45r+ B cells in the bone marrow, spleen, lymph nodes and blood, while less than 5% of Cd45r- non-B cells in these tissues exhibited ZsGreen fluorescence. Effective B cell-specific cre-mediated recombination was achieved within 2 days of tamoxifen treatment in a dose-dependent manner. These data indicate that transgenic mice carrying a single Cd79aCreERT2allele can achieve effective, tamoxifen- induced, B cell-specific, cre / lox-mediated genetic knockout, in a dose-dependent manner.

[0611] In further experiments, the kinetics of cre / lox-mediated genetic knockout in the mice was evaluated.

[0612] Briefly, 6-8 week-old mice were injected intra-peritoneally with a single dose of tamoxifen at 100 mg / kg body weight or corn oil (vehicle control). Mice were euthanized at 1-, 2-, 6-, 11- and 17- days posttreatment for analyzes. Cells isolated from bone marrow, spleen, lymph nodes and blood were stained with Zombie NIR reagent and anti-mouse Cd45r antibody, and assessed by flow cytometry to determine the proportions of Cd45r+B cells that were ZsGreen-fluorescent.

[0613] The results are shown in Figure 7. Cre-mediated recombination giving rise to ZsGreen fluorescence was efficiently induced by tamoxifen in Cd45r+B cells in the spleen, bone marrow, lymph nodes and blood. The highest proportions of recombined, ZsGreen-fluorescing Cd45r+B cells were detected in all tissue types at the Day 2 timepoint. Slight declines (-10%) in the total number of recombined Cd45r+B cells were observed in the spleen, lymph nodes and blood by Day 17 post-treatment. The proportion of recombined Cd45r+B cells in the bone marrow decreased rapidly from 2 days post tamoxifen injection. By Day 17, B cells in the bone marrow consisted predominantly of ZsGreen-negative, non-recombined cells. These data indicate that transgenic mice carrying a single Cd79aGreERT2allele can achieve effective, tamoxifen-induced, B cell-specific, cre / lox-mediated genetic knockout. While all lymphoid tissues are highly responsive to tamoxifen-induced CreERT2 activity, high proportions of recombined B cells persist in the peripheral lymphoid tissues for more than 10 days post tamoxifen treatment, but the proportion of recombined cells among cells of the bone marrow decrease more rapidly. Example 11 : Characterization of CreERT2-induced knockout of Ighm and Ighd in lqhmlqhdflox / flox; Cd79a+ / CreERT2transgenic mice

[0614] Tamoxifen-induced deletion of Ighm and Ighd in lghmlghcfbx / TtoxCd79a+ / CreERT2mice (described in Example 8) was evaluated. Briefly, 3 week-old mice were injected intra-peritoneally either with a single dose of tamoxifen at 200 mg / kg bodyweight or corn oil (vehicle control). Mice were euthanized 60 h posttreatment for analyzes. Genomic DNA was isolated from bone marrow cells and used in genotyping PCRs. Three sets of primers were used in order to detect deletion of the gene region comprising exons 1- 6 of Ighm and exons 1-3 of Ighd (represented schematically in Figure 8A). A first primer set provides for the amplification of a 554bp amplicon detectable in the absence of CreERT2-mediated recombination of the lghmlghdfloxlocus (lghmlghdfloxamplicon). A second primer set provides for the amplification of a 232bp amplicon detectable following CreERT2-mediated recombination of the lghmlgh(foxlocus (lghmlghdK0amplicon). A third primer set provides for the amplification of a 187bp control amplicon, detectable both in the presence and absence of the CreERT2-mediated recombination of the IghmlghcP locus (control region).

[0615] The results are shown in Figure 8B. The control amplicon was intact and detected in both tamoxifen- and vehicle- treated mice. The lghmlghdK0amplicon was detected only in cells isolated from tamoxifen- treated mice. The lghmlghdfloxamplicon was detected only in cells isolated from corn oil-treated mice. These data indicate that deletion of the floxed region comprising exons 1-6 of Ighm and exons 1-3 of Ighd was achieved by tamoxifen treatment in lghmlghcfhx“oxCd79a+ / CreERT2mice.

[0616] In further experiments, tamoxifen-induced knockout of Ighm and Ighd in lghmlghdnox“oxCd79a+ / CreERT2mice was evaluated at the cellular level, by analysis of IgM and IgD expression on B cells in peripheral lymphoid tissues.

[0617] Briefly, 3 week-old lghmlghcftox / TtoxCd79a+ / CreERT2mice were injected intra-peritoneally with a single dose of tamoxifen at 200 mg / kg body weight or corn oil (vehicle control). Mice were euthanized 60 h posttamoxifen treatment for analyzes. Cells isolated from the spleen and lymph nodes were stained with Zombie NIR reagent, anti-mouse Cd19 antibody, anti-mouse IgM antibody and anti-mouse IgD antibody, and the expression of Cd19, IgM and IgD on the cells was evaluated by flow cytometry.

[0618] The results are shown in Figure 9. The proportion of IgM IgD Cdl 9+B cells was >5-fold higher in B cells isolated from the spleen and lymph nodes of tamoxifen-treated mice, compared to those isolated from the spleen and lymph nodes of vehicle-treated mice. These data indicate that tamoxifen treatment results in substantial disruption of expression of Ighm and Ighm in Ighmlghd^"0'', Cd79a+ / CreERT2mice, thereby inhibiting expression of IgM and IgD on B cells.

[0619] Example 12: Maintenance of high levels of CreERT2-induced knockout

[0620] The ability of repeated administration of tamoxifen to maintain high levels CreERT2-mediated knockout of floxed loci was investigated. Briefly, 6-8 week-old mice homozygous for a lox-Stop-lox ZsGreen allele (Gt(ROSA)26Sorim6(CAG- zsGreeni)Hze^wereinjected intra-peritoneally with tamoxifen at 200 mg / kg body weight. The animals were then split into 2 groups (represented schematically in Figure 10A). In Arm 1 , mice were injected with 4 additional doses of tamoxifen at 100 mg / kg body weight, at 5 to 9-day intervals. In Arm 2, mice were injected with corn oil (vehicle control) according to the same schedule. Mice were euthanized for analyzes at Days 3, 10, 17 and 32 after the initial tamoxifen administration. Cells isolated from bone marrow, spleen and lymph nodes were stained with Zombie NIR reagent and anti-mouse Cd45r antibody, and assessed by flow cytometry to determine the proportions of Cd45r+B cells that were ZsGreen- fluorescent.

[0621] The results are shown in Figure 10B. Mice that received only a single, initiating dose of tamoxifen displayed a ~ 90% decrease in recombined B cells in the bone marrow and ~ 25% decrease in recombined B cells in the spleen and lymph nodes over the course of experiment. Mice in Arm 1 (which received repeated injections of tamoxifen at intervals of 5-9 days) maintained high proportions of recombined B cells in all lymphoid tissues throughout the course of experiment. These data indicate that high proportions of B cells with CreERT2-mediated knockout of floxed loci can be maintained over time by repeated administration of tamoxifen at regular intervals.

[0622] In further experiments, the ability of repeated administration of tamoxifen to maintain high levels of Ighm and Ighd knockout in B cells of Ighdlghm Cd79a+ / CreERT2mice (described in Example 8) is investigated (see Figure 11).

[0623] Briefly, 3 week-old Ighdlghmfbx / flCiX- Cd79a+ / CreERT2mice are injected intra-peritoneally with tamoxifen at 200 mg / kg body weight. The mice are injected with additional doses of tamoxifen at 100 mg / kg body weight, at intervals from 5 to 10 days. Mice are euthanized for analyzes at Days 3, 10, 17 and 32 after the initial tamoxifen administration. Cells isolated from the spleen and lymph nodes are stained with Zombie NIR reagent, anti-mouse Cd19 antibody, anti-mouse IgM and anti-mouse IgD antibody, and the expression of Cd 19, IgM and IgD on the cells was evaluated by flow cytometry.

[0624] High proportions of the Cd19+B cells isolated from the spleen, lymph nodes and bone marrow of tamoxifen-treated mice are Ighm lghd-, across all time points, indicating that high levels of CreERT2- mediated knockout of Ighm and Ighd expression in B cells can be maintained over time by repeated administration of tamoxifen at regular intervals.

[0625] Example 13: Application of transgenic mice providing for inducible knockout of Ighm and Ighd to obtain antibodies directed to a particular region of a protein of interest

[0626] The inventors employed the transgenic mice providing for inducible knockout of Ighm and Ighd to obtain antibodies that bind to the membrane-proximal protease cleavage site within the extracellular domain of a protein of interest. 8-9 week old lghdlghmflox / flox; Cd79a+ / CreERT2mice (described in Example 8) or BALB / c mice were immunized with a peptide comprising the protease cleavage site (‘Antigen 1’; 50pg in complete Freund's adjuvant) on Days 0 and 7 to induce a primary response. On days 24 and 25, mice were injected intraperitoneally with tamoxifen at 300 mg / kg body weight to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the full extracellular domain of the protein (comprising the protease cleavage site, ‘Antigen 2’; 50 g in incomplete Freund's adjuvant). Mice were injected with 3 additional doses of tamoxifen at 200 mg / kg body weight on Days 34, 42 and 52, to maintain Ighm and Ighd knockout in B cells.

[0627] Peripheral lymphoid samples were collected from the mice at day 28 (‘first timepoint’, prior to induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein) and day 63 (‘second timepoint', after induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein), for analysis of V-gene usage of lgG+ B cells that bound to the extracellular domain of the protein of interest, and for analysis of binding of antibodies produced in the mice to (i) the soluble, cleaved form of the extracellular domain ( / .e. lacking the protease cleavage site), and (ii) the non-cleaved form of the extracellular domain ( / .e. lacking the protease cleavage site).

[0628] Briefly, antibody gene sequences from B cells within the samples collected at days 28 and 63 were obtained by NGS sequencing, and clustered into clonotypes using Cell Ranger (which computationally groups B cells belonging to a common lineage based on their antibody gene sequences). Antibody sequences were then aligned to an in-house antibody database to determine V(D)J gene usage. Sequences were processed to remove all clones with non-functional or multiple functional heavy and / or light chain sequences. The resulting antibody amino acid sequences were then aligned to their closest germline sequence to quantify the number of somatic hypermutations (SHMs), based on the number of distinct residues from the identified germline sequences. For simplicity, the analysis assumed no backmutations to germline residues.

[0629] Figure 12 shows that the diversity of VH gene usage was found to be preserved in the Ighdlghm Cd79a+ / CreERT2mice ( / .e. relative to usage in their wildtype counterparts), both pre- and post-induction of Ighd and Ighm knockout.

[0630] The following tables illustrate that Ighd and Ighm knockout did not prevent somatic hypermutation. Indeed, the number of somatic hypermutations in VH and VL genes of representative antibody clones expressing antibodies that bind specifically the protease cleavage site of the protein of interest, produced from the Ighdlghm Cd79a+ / CreERT2mice was found to be increased at the second timepoint ( / .e. after Ighd and Ighm knockout).

[0631] ‘Representative clones

[0632] This data shows that somatic hypermutation during the B cell affinity maturation process is maintained in the IgMIgDcKO mouse.

[0633] Antibodies in sera collected at 1stand 2ndtimepoints were evaluated for their ability to bind to the soluble extracellular domain of the protein of interest (lacking the protease cleavage site) or the full-length extracellular domain of the protein of interest (comprising the protease cleavage site).

[0634] Briefly, CHO cells were transiently transfected with (i) a plasmid encoding the membrane-bound version of the protein of interest (comprising the protease cleavage site), or (ii) a plasmid comprising the soluble version of the extracellular domain of the protein of interest (lacking the protease cleavage site) and the native transmembrane domain (for membrane localization and surface expression). Non-transfected CHO cells served as negative controls. After 18 hr, cells were harvested, and 3 x 104cells / well of each cell type were incubated with 50pL of 3-fold serial dilutions (starting from a 1 :200 dilution in PBS) of sera obtained from the mice on the indicated days, and incubated at 4°C for 1 hr. Cells were then washed twice with 150pL of FACS buffer (1xPBS + 1 % FBS) and resuspended in 50pL of Alexa Fluor 647 AffiniPure Goat Anti-Mouse IgG (Jackson Immuno, 1 in 1000 dilution) antibody for 20 minutes at 4°C. Cells were washed twice with FACS buffer and resuspended in a final volume of 40pL DAPI solution except the unstained well in 40pL of FACS buffer without DAPI. Samples were then run on iQue3 flow cytometry and data were analyzed using FlowJo v10.8.1 software.

[0635] The results are shown in Figures 13A to 13D. Sera from the lghdlghmflox / flox; Cd79a+ / CreERT2mice following Ighd and Ighm knockout and immunization with Antigen 2 did not contain antibodies capable of binding to the soluble extracellular domain of the protein of interest (lacking the protease cleavage site), whereas sera from BALB / c mice obtained post-immunization with Antigen 2 contained antibodies that bound to this protein.

[0636] In further experiments, the antibody gene sequences from the 20 most represented clonotypes obtained from the lghdlghmflox / flox; Cd79a+ / CreERT2and BALB / c mice were cloned into plasmids for expression as chimeric mouse / human antibodies (comprising murine VH and VL regions, and human lgG1 CH1 , hinge, CH2 and CH3 regions (heavy chain), and human K CL (light chain)) expressed in ExpiCHO (ThermoFisher) cells and Protein A-purified for subsequent characterization.

[0637] The antibodies were evaluated for their ability to bind to the protease cleavage site by ELISA. Briefly, wells of a polypropylene plate were coated with 1 pg / ml Neutravidin (Invitrogen), incubated overnight at 4°C, washed and blocked with 1x phosphate-buffered saline (PBS) with 1% BSA for 2 hours at room temperature. The plate was then washed two times with 1x PBS with 0.05% Tween 20 before the addition of 1 pg / ml of (i) biotinylated, His-tagged soluble extracellular domain of the protein of interest (lacking the protease cleavage site) or (ii) biotinylated, His-tagged full-length extracellular domain of the protein of interest (comprising the protease cleavage site). Plates were washed three times with 1x PBS with 0.05% Tween 20 and dried in between each step. Nine points of a four-fold dilution series (serially diluted with 1x PBS + 1% BSA, starting from 10 pg / ml) of the antibodies were then added and incubated for 1 hr at room temperature. After washing three times with 1x PBS with 0.05% Tween 20 and dried in between each step, HRP-conjugated goat anti-human secondary antibody (Invitrogen) was prepared at 1 :7000 dilution with 1x PBS with 1% BSA and added to plates. Plates was incubated in the dark, at room temperature, for 1 hr. Plates were developed with colorimetric detection substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA). The reaction was stopped with Invitrogen ELISA Stop Solution (Cat. No. #SS04), and OD was measured at 450 nM using a BioTek PowerWave HT.

[0638] Figures 14A and 14B show the dose-response curve for binding to full-length extracellular domain of the protein of interest (comprising the protease cleavage site) as determined by ELISA, for two representative antibody clones obtained from the IghdlghmflCiX / flCiX- Cd79a+ / CreERT2mouse. Both are shown to bind to the target antigen with high affinity (EC50 = 2.01 x 10-11M for the antibody shown in Figure 14A and EC50 = 3.81 x 1011M for the antibody shown in Figure 14B).

[0639] Taken together, these data demonstrate that transgenic mice providing for inducible knockout of Ighm and Ighd are useful to obtain a diverse, high-affinity antibody repertoire focused to regions of interest of a given protein of interest.

[0640] Example 14: IqG class-switched response initiated prior to gene knockout is maintained after tamoxifen-induced conditional gene deletion

[0641] The inventors employed the transgenic mice providing for inducible knockout of Ighm and Ighd to investigate whether the IgG class-switched response is maintained following tamoxifen-induced conditional gene deletion.

[0642] 8-9 week old Ighdlghmflox / flox; Cd79a+ / CreERT2mice (described in Example 8) or BALB / c mice were immunized with a peptide comprising the protease cleavage site (‘Antigen 1’; 50pg in complete Freund's adjuvant) on Days 0 and 7 to induce a primary response. On days 24 and 25, mice were injected intraperitoneally with tamoxifen at 300 mg / kg body weight to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the full extracellular domain of the protein (comprising the protease cleavage site, ‘Antigen 2’; 50pg in incomplete Freund's adjuvant). Mice were injected with 3 additional doses of tamoxifen at 200 mg / kg body weight on Days 34, 42 and 52, to maintain Ighm and Ighd knockout in B cells.

[0643] Peripheral lymphoid samples were collected from the mice at day 28 (‘first timepoint’, prior to induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein) and day 63 (‘second timepoint’, after induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein). Cells isolated from lymph nodes were stained with Zombie NIR reagent, antimouse CD45R, IgG 1 , lgG2a, and lgG2b, and assessed for the proportion of lgG+, CD45R+B cells by flow cytometry.

[0644] The results are shown in Figures 15A and 15B. The proportions of lgG+ B cells in lymph nodes were not significantly different between normal wildtype BALB / c and lgMlgDf / fmice prior to tamoxifen-induced gene deletion. The proportions of lgG+ B cells were similar in both normal BALB / c mice and IgMIgD conditional knockout (IgMIgDcKO) mice after tamoxifen-induced gene deletion (Figure 15B and 15C).

[0645] Further investigation demonstrated that the total number of CD45R+B cells in peripheral lymphoid tissues is decreased in IgMIgDcKO mice after tamoxifen-induced gene deletion, compared with wild-type (Figure 15D; LN = lymph node; SP = spleen).

[0646] The above experiments were repeated with a second protein of interest. 8-9 week old Ighdlghm Cd79a+ / CreERT2mice (described in Example 8) or BALB / c mice were immunized with a peptide comprising a specific target domain in the protein (‘Antigen 3’; 50pg in complete Freund's adjuvant) on Days 0 and 7 to induce a primary response. On days 24 and 25, mice were injected intra-peritoneally with tamoxifen at 300 mg / kg body weight to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the full extracellular domain of the protein (comprising the target domain, ‘Antigen 4’;

[0647] 50pg in incomplete Freund's adjuvant). Mice were injected with 3 additional doses of tamoxifen at 200 mg / kg body weight on Days 34, 42 and 52, to maintain Ighm and Ighd knockout in B cells.

[0648] Peripheral lymphoid samples were collected from the mice at day 28 (‘first timepoint’, prior to induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein) and day 63 (‘second timepoint’, after induction of Ighd and Ighm knockout and immunization with the full extracellular domain of the protein). Cells isolated from lymph nodes were stained with Zombie NIR reagent, antimouse CD45R, IgG 1 , lgG2a, and lgG2b, and assessed for the proportion of lgG+, CD45R+B cells by flow cytometry.

[0649] The results are shown in Figures 15E and 15F. The proportions of lgG+B cells in lymph nodes were not significantly different between normal wildtype BALB / c and lgMlgDf / fmice prior to tamoxifen-induced gene deletion. The proportions of lgG+B cells were similar in both normal BALB / c mice and IgMIgD conditional knockout (IgMIgDcKO) mice after tamoxifen-induced gene deletion (Figure 15E). The total number of CD45R+B cells in the spleen is decreased in IgMIgDcKO mice after tamoxifen-induced gene deletion, compared with wild-type, but not in the lymph node (Figure 15F; LN = lymph node; SP = spleen).

[0650] The following table illustrates that epitope-specific clonotypes developed in response to antigen immunization by the first timepoint are not lost upon Ighd and Ighm knockout. The same epitope-specific clonotypes are present in the mice both pre- and post-induction of Ighd and Ighm knockout. This demonstrates that epitope-specific clonotypes are maintained in IgMIgDcKO mice.

[0651] Taken together, these results demonstrate that the IgG class-switched response to immunization initiated prior to gene knockout is maintained after tamoxifen-induced conditional gene deletion in IgMIgDcKO mice.

[0652] Example 15: NZBWF1 mice respond better to immunization to antigens with high homology to self

[0653] The inventors used a highly immuno-reactive mouse strain (NZBWF1) to analyse responses to antigens with high homology to self.

[0654] 6-12 weeks old BALB / c or NZBWF1 mice were immunized with recombinant human DLL3-His tagged protein. Sera from animals were analysed for antibody titer against human DLL3-Fc tagged protein on Day 38 post-immunization by indirect Enzyme Linked Immunosorbent Assay (ELISA).

[0655] The results are shown in Figure 16. Human DLL3 has 85.8 % homology to mouse DLL3 protein. NZBWF1 mice exhibited stronger titers against the antigen when compared to the BALB / c mice.

[0656] These results show that NZBWF1 mice respond to immunization with protein antigens, and develop strong antibody titers to antigens with high homology (>80%) to self.

[0657] Example 16: NZBWF1 mice produce a stronger antibody response to poorly immunogenic antigens

[0658] The inventors used the highly immuno-reactive mouse strain (NZBWF1) to analyse responses to poorly immunogenic antigens.

[0659] 6-10 weeks old BALB / c or NZBWF1 mice were immunized with two doses of a 13-mer peptide conjugated to KLH, seven days apart. Sera from animals were analysed for antibody titer against the peptide conjugated to biotin on Day 21 post-immunization by indirect Enzyme Linked Immunosorbent Assay (ELISA).

[0660] The results are shown in Figure 17. NZBWF1 mice exhibited stronger titers against peptide antigen compared to BALB / c mice. These results show that NZBWF1 mice respond to immunization with small peptides and produce antibody titers to poorly immunogenic antigens. Example 17: Additional characterisation of transgenic mice providing for inducible knockout of IGHM and IGHD

[0661] Tamoxifen-induced deletion of Ighm and Ighd 'n lghmlghcfbx / TtoxCd79a+ / CreERT2mice (described in Example 8.2) was further evaluated in mice that had undergone immunization and tamoxifen treatment as described in Example 13. Briefly 8-9 week old lghdlghmflox / flox; Cd79a+ / CreERT2mice (described in Example 8) or BALB / c mice were immunized with a peptide comprising the protease cleavage site (‘Antigen 1’;

[0662] 50pg in complete Freund's adjuvant) on Days 0 and 7 to induce a primary response. On days 24 and 25, mice were injected intra-peritoneally with tamoxifen at 300 mg / kg body weight to induce Ighm and Ighd knockout in B cells. On days 30 and 44, mice were immunized with the full extracellular domain of the protein (comprising the protease cleavage site, ‘Antigen 2’; 50pg in incomplete Freund's adjuvant). Mice were injected with 3 additional doses of tamoxifen at 200 mg / kg body weight on Days 34, 42 and 52, to maintain Ighm and Ighd knockout in B cells.

[0663] Mice were euthanized 10 days after the last tamoxifen treatment for analysis. Genomic DNA was isolated from cells in the spleen and ear and used in genotyping PCRs. A first primer set provided for the amplification of a 2328 bp wild-type or a 2458 bp lghmlghdflox / floxamplicon detectable in the absence of CreERT2-mediated recombination of the lghmlgh(foxlocus. A second primer set provided for the amplification of a 232bp amplicon, indicating deletion of the floxed Ighm-lghd gene locus, detectable following CreERT2-mediated recombination of the lghmlgh(foxlocus.

[0664] Genotyping revealed B-cell-specific, tamoxifen-induced conditional deletion of Ighm and Ighd (Figure 18A). This is demonstrated by the presence of the band at 232 bp in B cells isolated from the spleen of tamoxifen-induced transgenic mice (IgMIgD cKO (fl / fl + tamoxifen)). The 232 bp band is not present in B cells isolated from the spleens of mice not treated with tamoxifen, or B cells isolated from the spleens of wild-type mice treated with tamoxifen. In addition, the 232 bp band was not detected in ear cells taken from either wild-type or IgMIgD cKO mice, which demonstrates the specificity of the inducible knockout to B cells.

[0665] Flow cytometry of B cells collected from lymphoid tissues demonstrated a 50-60% deletion of lgM+lgD+B cells in the lymphoid tissues in tamoxifen-induced transgenic mice (IgMIgD cKO) compared with wild-type mice (Figures 18B and 18C).

[0666] Taken together, these results demonstrate that the tamoxifen-induced conditional deletion of Ighm and Ighd is specific to B cells.

[0667] Example 18: Rate of targeted epitope antibody discovery using transgenic mice providing for inducible knockout of IGHM and IGHD is improved

[0668] Three proteins of interest were used to analyse the generation of targeted epitope specific antibodies. The results are summarized below. The immunization strategy for the generation of targeted epitope specific antibodies (for each protein of interest (“POI”) is shown in Figure 19.

[0669] The following tables show the % of targeted epitope-specific sera generated in IgMIgDcKO mice compared with wild-type.

[0670] Protein of interest 1

[0671] Protein of interest 2

[0672] The antibodies were evaluated for their ability to bind to the protease cleavage site by ELISA. Briefly, wells of a polypropylene plate were coated with 1 pg / ml Neutravidin (Invitrogen), incubated overnight at 4°C, washed and blocked with 1x phosphate-buffered saline (PBS) with 1% BSA for 2 hours at room temperature. The plate was then washed two times with 1x PBS with 0.05% Tween 20 before the addition of 1 pg / ml of (i) biotinylated, His-tagged soluble extracellular domain of the protein of interest (lacking the protease cleavage site) or (ii) biotinylated, His-tagged full-length extracellular domain of the protein of interest (comprising the protease cleavage site). Plates were washed three times with 1x PBS with 0.05% Tween 20 and dried in between each step. Nine points of a four-fold dilution series (serially diluted with 1x PBS + 1% BSA, starting from 10 pg / ml) of the antibodies were then added and incubated for 1 hr at room temperature. After washing three times with 1x PBS with 0.05% Tween 20 and dried in between each step, HRP-conjugated goat anti-human secondary antibody (Invitrogen) was prepared at 1 :7000 dilution with 1x PBS with 1% BSA and added to plates. Plates was incubated in the dark, at room temperature, for 1 hr. Plates were developed with colorimetric detection substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA). The reaction was stopped with Invitrogen ELISA Stop Solution (Cat. No. #SS04), and OD was measured at 450 nM using a BioTek PowerWave HT.

[0673] FACS analysis was also performed to assess the sera binding profiles. Briefly, CHO cells were transiently transfected with (i) a plasmid encoding the membrane-bound version of the protein of interest (comprising the protease cleavage site), or (ii) a plasmid comprising the soluble version of the extracellular domain of the protein of interest (lacking the protease cleavage site) and the native transmembrane domain (for membrane localization and surface expression). Non-transfected CHO cells served as negative controls. After 18 hr, cells were harvested, and 3 x 104cells / well of each cell type were incubated with 50pL of 3- fold serial dilutions (starting from a 1 :200 dilution in PBS) of sera obtained from the mice on the indicated days, and incubated at 4°C for 1 hr. Cells were then washed twice with 150|JL of FACS buffer (1xPBS + 1% FBS) and resuspended in 50pL of Alexa Fluor 647 AffiniPure Goat Anti-Mouse IgG (Jackson Immuno, 1 in 1000 dilution) antibody for 20 minutes at 4°C. Cells were washed twice with FACS buffer and resuspended in a final volume of 40pL DAPI solution except the unstained well in 40pL of FACS buffer without DAPI. Samples were then run on iQue3 flow cytometry and data were analyzed using FlowJo v10.8.1 software.

[0674] The results for protein of interest 1 (POI1) are shown in Figures 20A and 20B and the results for protein of interest 2 (POI2) are shown in Figures 21 A and 21 B.

[0675] The following tables show the rate of obtaining epitope specific antibody clones from clonal B cell screens performed for POI1 and a third protein of interest (POI3).

[0676] Protein of interest 1

[0677] Protein of interest 3

[0678] Figure 22 shows the specificity of recombinant antibodies as assayed by FACS analysis for POI1 . Representative recombinant antibodies derived from the IgMIgDcKO mice were evaluated for their ability to bind to cells expressing the full-length protein of interest (comprising the protease cleavage site), or a version of the protein of interest that lack the protease cleavage site by FACS analysis. The results show that these antibodies are specific for the targeted protease cleavage site and do not bind to other domains in the full-length protein.

[0679] Figures 23A and 23B show the dose-response curve for binding to full-length extracellular domain of POI3 (comprising the protease cleavage site) as determined by ELISA, for two representative antibody clones obtained from the Ighdlghmfl0X / Tl0X', Cd79a+ / CreERT2mouse. Both are shown to bind to the target antigen with high affinity (EC50 = 7.40 x 1011M for the antibody shown in Figure 23A and EC50 = 3.96 x 10-11M for the antibody shown in Figure 23B).

[0680] Example 20: Generation of a hyperimmune Ighdlghmflox / flox; Cd79a+ / CreERT2mouse

[0681] Hyperimmune IghdlghmflCiX / flCiX- Cd79a+ / CreERT2mice were generated by mating mice carrying the Ighdlghm fiox / noxanc| cd79a+ / CreERT2alleles (in a BALB / c background) (for example the mice generated in Example 8.2) with the autoimmune parental strains NZB or NZW, and subsequently mating the NZB and NZW mice to generate NZBWF1 progeny that carry the two alleles.

[0682] The expression of mouse IgM and IgD can be inducibly knocked-out in the resulting transgenic mice by administration of tamoxifen.

Claims

Claims:

1. An animal comprising an endogenous nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

2. The animal according to claim 1 , wherein the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in mounting a primary humoral immune response.

3. The animal according to claim 1 or claim 2, wherein the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in immunoglobulin isotype switching and / or B cell maturation.

4. The animal according to any one of claims 1 to 3, wherein the animal comprises an endogenous nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or both genes selected from IGHM and IGHD.

5. The animal according to any one of claims 1 to 4, wherein the animal comprises an endogenous nucleotide sequence providing for recombinase-mediated disruption of expression of one or more genes involved in mounting a primary humoral immune response.

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

7. The animal according to claim 6, wherein the animal comprises an endogenous nucleotide sequence providing for inducible expression or activity of the recombinase.

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

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

10. The animal according to any one of claims 1 to 9, wherein the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.11 . The animal according to any one of claims 1 to 10, wherein the animal is a mouse, a rat or a rabbit.

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

13. The animal according to any one of claims 1 to 12, wherein the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4.

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

15. The animal according to claim 14, wherein expression of the Cre recombinase is under the control of a promoter driving expression in B cell lineage cells.

16. The animal according to any one of claims 1 to 15, wherein the animal comprises an endogenous nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6.

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

18. The method according to claim 17, wherein the method comprises:(i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;(ii) treating the animal to inhibit its ability to mount a primary immune response; and(iii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

19. The method according to claim 17, wherein the method comprises:(i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;(ii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest; and(iii) treating the animal to inhibit its ability to mount a primary immune response.

20. The method according to claim 18 or claim 19, wherein treating the animal to inhibit its ability to mount a primary immune response comprises administering an agent for inducing expression or activity of the recombinase.

21. A method for producing an antigen-binding molecule, wherein the method comprises:(i) administering a first peptide / polypeptide, or nucleic acid encoding the first peptide / polypeptide, to an animal, wherein the first peptide / polypeptide comprises an amino acid sequence of interest;(ii) treating the animal to inhibit its ability to mount a primary immune response; and(iii) administering a second peptide / polypeptide, or nucleic acid encoding the second peptide / polypeptide, to the animal, wherein the second peptide / polypeptide comprises the amino acid sequence of interest or an amino acid sequence which is similar to the amino acid sequence of interest.

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

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

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

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

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

27. The method according to any one of claims 23 to 26, wherein the agent is selected from, or comprises, an antibody, antigen-binding molecule, polypeptide, decoy receptor, aptamer, sequestering agent or small molecule.

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

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

30. The method according to any one of claims 21 to 29, wherein the animal comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.31 . The method according to any one of claims 21 to 30, wherein the animal is a mouse, a rat or a rabbit.

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

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

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

35. A nucleic acid, or a plurality of nucleic acids, comprising a nucleotide sequence providing for inducible inhibition of a primary humoral immune response.

36. The nucleic acid, or a plurality of nucleic acids according to claim 35, comprising a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in mounting a primary humoral immune response.

37. The nucleic acid, or a plurality of nucleic acids according to claim 35 or claim 36, comprising a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or more genes involved in immunoglobulin isotype switching and / or B cell maturation.

38. The nucleic acid, or a plurality of nucleic acids according to any one of claims 35 to 37, comprising a nucleotide sequence providing for inducible inhibition of the expression of, or inhibition of the activity of the product of, one or both genes selected from IGHM and IGHD.

39. The nucleic acid, or a plurality of nucleic acids according to any one of claims 35 to 38, comprising a nucleotide sequence providing for recombinase-mediated disruption of expression of one or more genes involved in mounting a primary humoral immune response.

40. The nucleic acid, or a plurality of nucleic acids according to any one of claims 35 to 39, comprising a nucleotide sequence encoding all or part of the nucleotide sequence of a gene involved in mounting a primary humoral immune response, flanked by target sequences for a recombinase.41 . The nucleic acid, or a plurality of nucleic acids according to any one of claims 35 to 40, further comprising a nucleotide sequence encoding a conditional system for controlling expression or activity of the recombinase.

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

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

44. The nucleic acid or plurality of nucleic acids according to any one of claims 35 to 43, comprising a nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:4.

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

46. The nucleic acid or plurality of nucleic acids according to claim 45, wherein expression of the Cre recombinase is under the control of a promoter driving expression in B cell lineage cells.

47. The nucleic acid or plurality of nucleic acids according to any one of claims 35 to 46, comprising a nucleotide sequence comprising, or consisting of, a nucleotide sequence having 60% or greater nucleotide sequence identity to SEQ ID NO:6.

48. A vector, or a plurality of vectors, comprising the nucleic acid or plurality according to any one of claims 35 to 47.

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

50. The cell according to claim 49, wherein the cell comprises an endogenous nucleotide sequence encoding one or more human immunoglobulin genes or gene segments.51 . The cell according to claim 49 or claim 50, wherein the cell is a mammalian cell.

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

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

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

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

Images