WO2022162009A1 - Method for rapid identification of cross-reactive and/or rare antibodies - Google Patents

Abstract

The present invention provides a method for rapid identification of a B cell producing a cross- reactive antibody. Based thereon, the sequences of the variable regions of the cross-reactive antibody may be identified and cloned into an expression vector for expression of said cross- reactive antibody. The cross-reactive antibody may be used in the treatment of diseases. Furthermore, its epitope may be identified, e.g. to design vaccines eliciting an immune response comprising cross-reactive antibodies.

Description

METHOD FOR RAPID IDENTIFICATION OF CROSS-REACTIVE AND/OR RARE ANTIBODIES

The present invention relates to the field of screening and identification of antibodies, in particular to the identification of cross-reactive antibodies and B cell clones producing cross- reactive antibodies. Accordingly, the present invention provides a method for identification of cross-reactive antibodies and B cell clones producing cross-reactive antibodies. The present invention also provides cross-reactive antibodies and B cell clones producing cross- reactive antibodies obtained according to the method of the present invention. Such B cell clones and cross-reactive antibodies are useful in a variety of medical applications, including prevention and therapy of diseases targeted by the cross-reactive antibody as well as in vaccine design, e.g. to identify epitopes targeted by the cross-reactive antibodies for broad vaccination approaches, or for diagnostic approaches, e.g. for detection of an antigen in a (isolated) sample.

The use of therapeutic monoclonal antibodies has emerged as ground-breaking approach to specifically target a wide variety of diseases including immune disorders, cancers, and infectious diseases. The potential of therapeutic antibodies as powerful tools for treatment of numerous diseases emerged in particular since 1975, when Kohler and Milstein developed a procedure for producing monoclonal antibodies (mAbs) (Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7; 256(5517):495-7). The first mAbs were produced in mice and, when administered to patients, those murine antibodies faced serious problems as they were recognized as foreign molecules. This resulted in elimination by the human immune system and in allergic responses ranging from a mild rash to renal failure. Moreover, these murine antibodies were not able to interact properly with components of the human immune system and their biological efficacy was severely restricted (for review see Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. British Journal of Pharmacology. 2009;157(2):220-233. doi: 10.1111/j .1476- 5381 .2009.00190.x.).

To avoid those problems, strategies were developed to make the murine antibodies more "human". One approach was the development of chimeric antibodies, in which murine variable domains were fused to human constant domains, resulting in an antibody, which is approx. 70% human and which has a fully human Fc portion (Neuberger MS, Williams GT, Mitchell EB, Jouhal SS, Flanagan JG, Rabbitts TH: A hapten-specific chimaeric IgE antibody with human physiological effector function. Nature. 1985 Mar 21 -27; 314(6008):268-70). To further decrease murine part of mAbs, "humanized" antibodies were developed, in which the hypervariable loops of a fully human antibody were replaced with the hypervariable loops of the murine antibody of interest by "complementarity-determining region (CDR) grafting". Humanized antibodies contain 85 - 90% human sequences and are even less immunogenic than chimeric antibodies. Most of the approved mAbs are chimeric or humanized (for review see Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. British Journal of Pharmacology. 2009;157(2):22O-233. doi:10.1 1 1 1/j.1476-5381 .2009.00190.x.). However, humanizing is technically demanding and can result in a loss of antibody activity (i.e., in a loss of function).

Another approach for developing therapeutic antibodies relates to in vitro display technologies, such as phage display (McCafferty J, Griffiths AD, Winter G, Chiswell DJ: Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990 Dec 6; 348(6301 ):552-4). Thereby, human antibodies or antibody fragments are displayed on the surface of a simple organism, such as phage, bacteria or yeast for screening. However, such library systems do not contain full-length antibodies and the antibodies are expressed by bacteria or yeast rather than by human cells. Such expression systems cannot reflect human post-translational modifications. In particular, antibodies produced in vitro do often not resemble natural, human antibody glycosylation patterns, which are however crucial for antibody effectiveness as they influence effector functions and downstream activation of the immune system.

Furthermore , transgenic "humanized" mice may be used to produce antibodies from human genes (Lonberg N. Human monoclonal antibodies from transgenic mice. In: Chernajovsky Y, Nissim A, editors. Therapeutic Antibodies. Handbook of Experimental Pharmacology, Volume 181. Berlin Heidelberg: Springer-Verlag; 2008. pp. 69-97. Eds.). However, as this technology relies on immunization of mice with an antigen, it is limited to the production of antibodies for antigens, which can be recognized by the immune system of a mouse.

Accordingly, the "gold standard" for producing therapeutic antibodies is the use of isolated human B lymphocytes, which utilizes the "natural" way of human antibody production (Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004 Aug;10(8):871 -5. Epub 2004 Jul 1 1 ; Lanzavecchia A, Bernasconi N, Traggiai E, Ruprecht CR, Corti D, Sallusto F. Understanding and making use of human memory B cells. Immunol Rev. 2006 Jun;21 1 :303-9). Accordingly, B cells were traditionally used to obtain natural human antibodies and human antibody libraries based on the natural B cell genome (Duvall MR, Fiorini RN. Different approaches for obtaining antibodies from human B cells. Curr Drug Discov Technol. 2014 Mar;1 1 (1 ):41 -7).

While antibodies are exquisitely specific for the eliciting antigen, a number of studies have shown that certain individuals make rare antibodies that bind to multiple antigens, for instance different viruses of the same or different families. The identification of such cross- reactive antibodies with broad specificity is an important goal since these antibodies can provide broad protection and define conserved antigens and epitopes relevant for vaccine design.

However, conventional methods for identification of human monoclonal antibodies are not suited for the rapid identification of such cross-reactive antibodies and for their efficient and rapid isolation. Usually, B cell clones are selected (and antibodies isolated) based on a single, selected specificity and, optionally, the antibody may be tested later for a different specificity, which is a cumbersome and time-consuming process. For example, several studies (Wrammert J et al, Nature 2008 453:667; Tiller T et al, J Immunol Methods 2008, 329:1 12; Scheid JF et al, J Immunol Methods 2009, 343:65) have used a baiting approach to isolate antigen-specific memory B cells from which antibodies can be cloned and expressed. This method, however, does not allow to selectively identify cross-reactive antibodies.

Furthermore, WO 2010/01 1337 Al , WO 2010/107939 A2 and WO 2012/082073 A1 describe methods, wherein B cells are first isolated/purified from a sample, e.g. PBMCs, using B cell markers and cell sorting methods. Thereafter, isolated B cells are plated at low densities of very few cells per well and cultured, including stimulation/expansion, for the production of antibodies. Only thereafter, a primary screening is performed to select cultures producing the desired antibody. However, this conventional approach is very cumbersome and costintensive, because it requires a large amount of (isolated) B cell cultures, as any B cell obtained by cell sorting is cultured, irrespective of its antigen specificity.

Another approach (Traggiai E. et al Nat Med 2004, 10:871 ; WO 2004/076677 A2) is based on the isolation of human monoclonal antibodies through the efficient immortalization of memory B cells using Epstein Barr Virus (EBV) and a TLR agonist (CpG). This method requires physical isolation of memory B cells by cell sorting and high throughput cell cloning in the presence of feeder cells and is suitable for antibody screening after approximately 2 weeks. However, the method is cumbersome and cannot be used simultaneously with a large number of individuals (donors) to identify the most suitable ones and, therefore, to identify rare cross- reactive antibodies.

Corti et al. (Science 201 1 , 333:850; see also WO 2010/046775 A2) described the culture of single plasma blasts isolated seven days after a booster immunization. The method is suitable only for plasma blasts or plasma cells that do not divide and survive in culture in the presence of immortalized stromal cells. Moreover, due to single-cell culture of plasma cells, that do not divide, this method is cumbersome and time-consuming for screening of large cell numbers, e.g. from different donors. For rapid expansion of polyclonal B cells, Pinna D, et al. (Eur J of Immunology 2009, 39:1260) showed that culturing total peripheral blood mononuclear cell (PBMCs) with TLR agonists in the presence of IL-2 was able to selectively expand memory B cells and to stimulate memory B cells to secrete antibodies, and therefore allows quantification of the frequency of antigenspecific B cells. However, the described method fails short in the isolation, identification and retrieval of cross-reactive antibody sequences from antigen-specific B cells.

Accordingly, there is a need to identify a more efficient high-throughput method for the rapid screening of blood samples, e.g. of multiple donors, and for the isolation of cross-reactive monoclonal human antibodies, for example monoclonal antibodies binding to multiple alpha and beta coronaviruses.

In view of the above, it is the object of the present invention to overcome the drawbacks of current methods outlined above and to provide a method for rapid identification of cross- reactive antibodies, which enables an efficient high throughput pipeline for the identification of the most suitable PBMC donors and/or for the identification of sequences of cross-reactive antibodies, e.g. in only 7 - 8 days. It is also an object of the present invention to provide a rapid and cost-effective method for identifying cross-reactive antibodies, in particular among a large number of blood samples.

This object is achieved by means of the subject-matter set out below and in the appended claims.

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term "consist of" is a particular embodiment of the term "comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of". The term "comprising" thus encompasses "including" as well as "consisting" e.g., a composition "comprising" X may consist exclusively of X or may include something additional e.g., X + Y.

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The word "substantially" does not exclude "completely" e.g., a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention. The term "about" in relation to a numerical value x means x ± 10%, for example, x ± 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x + 15%, or x ± 20%.

The term "disease" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and therapy. The terms "subject" or "patient" are used interchangeably herein to mean all mammals including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is a human.

Doses are often expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg etc.) bodyweight", even if the term "bodyweight" is not explicitly mentioned.

The term "binding" and similar reference usually means "specifically binding", which does not encompass non-specific sticking.

In general, an "antibody" is a protein that binds specifically to an antigen. Typically, an antibody comprises a unique structure that enables it to bind specifically to its corresponding antigen, but - in general - antibodies have a similar structure and are, in particular, also known as immunoglobulins (Ig). As used herein, the term "antibody" encompasses various forms of antibodies including, without being limited to, whole antibodies, antibody fragments (such as antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties of the antibodies (e.g., regarding binding and/or neutralization) are retained. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody is a human monoclonal antibody.

As described above, the term "antibody" generally also includes antibody fragments. Fragments of the antibodies may retain the antigen-binding activity of the antibodies. Such fragments are referred to as "antigen-binding fragments". Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Fragments of the antibodies can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by recombinant means, for example by cloning and expressing a part (fragment) of the sequences of the heavy and/or light chain. Single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody are also encompassed by the term "antibody". For example, a scFv may comprise the CDRs of an antibody as described herein. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker. Antibody fragments may be contained in a variety of structures known to the person skilled in the art. Although the specification, including the claims, may, in some places, refer explicitly to antigen binding fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of antibodies, it is understood that the term "antibody" includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Cure Opin. Chem. Biol. 5 (2001 ) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Nat/. Acad. Sci. USA 90 (1993) 2551 -2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M, et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G, J. Mol. Biol. 227 (1992) 381 -388; Marks, J. D, et al, J. Mol. Biol. 222 (1991 ) 581 -597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P, et al, /. Immunol. 147 (1991 ) 86-95). Most preferably, however, human monoclonal antibodies are prepared by the method according to the present invention as described herein, which may be combined with improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871 -5. The term "human antibody" as used herein also comprises such antibodies which are modified to generate the properties as described herein.

Antibodies can be of any isotype (e.g., IgA, IgG, IgM i.e. an a, y or p heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, antibodies may be IgGI , lgG2, lgG3 or lgG4 subclass, for example IgGI . Antibodies may have a K or a A light chain. In some embodiments, the antibody is of IgGI type and has a K light chain.

Antibodies may be provided in purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

Antibodies may be immunogenic in human and/or in non-human (or heterologous) hosts e.g., in mice. For example, the antibodies may have an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.

As used herein, a "neutralizing antibody" is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone, or in combination, as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein.

The term "heavy chain" (of an antibody or antibody fragment) as used herein refers to a polypeptide which is to be associated with another polypeptide (the "light chain"). In particular, the heavy chain and the light chain are associated through a disulfide bond. The heavy chain may comprise one, two, three or four antibody heavy constant domains. Preferably, it comprises three antibody heavy constant domains: CH1 , CH2 and CH3, and a hinge region between CH1 and CH2. Said heavy chain constant domains may be derived from an antibody which is murine, chimeric, synthetic, humanized or human. Human constant domains are preferred. The heavy chain may comprise one or more variable domains, preferably a variable domain of an antibody heavy chain (VH).

The term "light chain" (of an antibody or antibody fragment) as used herein refers to a polypeptide which is to be associated with another polypeptide (the "heavy chain"). In particular, the heavy chain and the light chain of an antibody are associated through a disulfide bond. The light chain may comprise an antibody light chain constant region CL. Said light chain constant region may be derived from an antibody which is murine, chimeric, synthetic, humanized or human. Human constant regions are preferred. The second polypeptide chain may comprise one or more variable domains, preferably a variable domain of an antibody light chain (VL).

As used herein, the term "variable domain" (also referred to as "variable region"; variable domain of a light chain (VL), variable domain of a heavy chain (VH)) refers to the domain of an antibody, or antibody fragment, which is the N-terminal domain in classical naturally occurring antibodies, typically the domain providing the highest variability in classical naturally occurring antibodies, and which is involved directly in the binding of the antibody to the antigen. Typically, the domains of variable human light and heavy chains have the same general structure and each domain comprises framework (FR) regions whose sequences are widely conserved (in particular four framework (FR) regions) and three "hypervariable regions" or complementarity determining regions, CDRs (in particular three "hypervariable regions'VCDRs). The framework regions typically adopt a 0-sheet conformation and the CDRs may form loops connecting the (3-sheet structure. The CDRs in each chain are usually held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. CDR and FR regions may be determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991 ). Typically, in particular in native monospecific IgG antibodies, the three CDRs (CDR1 , CDR2, and CDR3) are arranged non-consecutively in the variable domain. In other words, the CDRs on the heavy and/or light chain may be separated for example by framework regions, whereby a framework region (FR) is a region in the variable domain which is less "variable" than the CDR. For example, in an antibody a variable domain (or each variable domain, respectively) may preferably comprise four framework regions, separated by three CDRs. In particular, a variable domain of an antibody (light or heavy chain variable domain VH or VL) may comprise from N- to C-terminus the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Usually, the three CDRs of a heavy chain and the three CDRs of the connected light chain form together the antigen binding site (paratope). In other words, since antigen binding sites are typically composed of two variable domains, there are usually six CDRs for each antigen binding site (heavy chain: CDRH1 , CDRH2, and CDRH3; light chain: CDRL1 , CDRL2, and CDRL3). A single antibody, in particular a single in (native, classical IgG) antibody, may usually have two (identical) antigen binding sites and therefore contain twelve CDRs (i.e. 2 x six CDRs).

In the context of the present invention, a variable domain may be any variable domain (in particular, VH and/or VL) of a naturally occurring antibody or a variable domain may be a modified/engineered variable domain. Modified/engineered variable domains are known in the art. In some embodiments, the variable domains may be modified/engineered to delete or add one or more functions, e.g., by "germlining" somatic mutations ("removing" somatic mutations) or by humanizing.

As used herein, the term "constant domains" refers to domains of an antibody which are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Typically, a heavy chain comprises three or four constant domains, depending on the immunoglobulin class: CH1 , CH2, CH3, and, optionally, CH4 (in N-C-terminal direction). Accordingly, the constant region of a heavy chain is typically formed (in N- to C-terminal direction) by: CH1 - hinge (flexible polypeptide comprising the amino acids between the first and second constant domains of the heavy chain) - CH2 - CH3 (- CH4). A light chain typically comprises only one single constant domain, referred to as CL, which typically also forms the constant region of the light chain. In the context of the present invention, a constant domain may be any constant domain (in particular, CL, CH1 , CH2, CH3 and/or CH4) of a naturally occurring antibody or a constant domain may be a modified/engineered constant domain, e.g. a human constant domain/constant region, which may be modified. Modified/engineered constant domains are known in the art. Typically, constant domains are modified/engineered to delete or add one or more functions, e.g., in the context of the functionality of the Fc region. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, e.g. lgG1 , lgG2, lgG3, and lgG4, lgA1 and lgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, e, y, and p, respectively. The antibodies according to the invention are preferably of IgM type or IgG type. Unlike IgG, IgM does not contain a hinge region but does contain an additional constant domain and an 18 amino acid tailpiece at the carboxy terminus, which contains a cysteine and is involved in multimerisation of the molecule.

As used herein, the term "recombinant antibody" is intended to include all antibodies, which do not occur in nature. It also includes antibodies produced by recombinant means, for example antibodies produced by cloning naturally occurring VH/VL sequences into expression vectors containing constant region sequences, e.g. as described herein.

As used herein, the term "antigen" refers to any structural substance which serves as a target for the receptors of an adaptive immune response, in particular as a target for antibodies, T cell receptors, and/or B cell receptors. An "epitope", also known as "antigenic determinant", is the part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T cell receptors, and/or B cell receptors. Thus, one antigen has at least one (usually more) epitope, i.e. a single antigen has one or more epitopes. An antigen may be (i) a peptide, a polypeptide, or a protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or a toxin. Thus, an antigen may be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a small molecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof. Preferably, the antigen is selected from (i) a peptide, a polypeptide, or a protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide and (v) a glycolipid; more preferably, the antigen is a peptide, a polypeptide, or a protein.

The term "antigen binding site" as used herein refers to the part of the antibody, which comprises the area which specifically binds to (and is usually complementary to) a portion or all of an antigen. An antibody often only binds to a particular part of the antigen, which part is termed "epitope". Typically, two variable domains, in particular a heavy chain variable domain VH and a light chain variable domain VL, associate to form one an antigen binding site. In particular, the antigen binding site is formed by the three CDRs of the heavy chain variable domain and by the three CDRs of the light chain variable domain together, i.e. by six CDRs, as described above.

The term "specifically binding" and similar reference does not encompass non-specific sticking. As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded (ss) or double-stranded (ds).

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

As used herein, the term "mutation" relates to a change in the nucleic acid sequence and/or in the amino acid sequence in comparison to a reference sequence, e.g. a corresponding genomic sequence. A mutation, e.g. in comparison to a genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, chemicals or radiation, or a mutation obtained by site- directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms "mutation" or "mutating" shall be understood to also include physically making a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid, or by synthesizing a sequence variant, e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide without the need for mutating one or more nucleotides of a nucleic acid molecule. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Method for identification of a B cell producing a cross-reactive antibody

In a first aspect the present invention provides a method for identification of a B cell (capable of) producing a (cross-reactive) antibody (with variable regions conferring different functionalities), the method comprising the following steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities;

(iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which exhibit different functionalities (i.e., which are cross-reactive);

(v) isolating and cloning B cells from the (cross-reactive) cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and (vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which exhibits different functionalities (i.e., which is cross- reactive).

The present inventors surprisingly found an efficient and high-throughput screening strategy to identify B cell clones (capable of) producing rare, cross-reactive antibodies starting from total peripheral blood mononuclear cells (PBMCs) in very short time. Based thereon, the cross-reactive antibodies can be prodcued in a time- and cost-effective manner. This method enables efficient high-throughput screening of large sets of PBMCs, e.g., from blood samples of multiple donors, for rare, cross-reactive antibodies. Accordingly, the invention allows for rapid isolation of antibodies that bind to multiple antigens or exhibit multiple characteristics of interest. Moreover, the method of the present invention provides an efficient high throughput pipeline for the identification of the most suitable PBMC donors. In addition, gene sequences of cross-reactive antibodies can be identified, e.g. from a blood sample, in a very short time, e.g. in only 7-8 days. The method typically starts with unseparated total PBMCs, includes a B cell cloning step and is suited for multiple interrogations to identify B cells (capable of) producing rare antibodies having multiple desired properties.

The method for identification of a B cell (capable of) producing a cross-reactive antibody according to the present invention is illustrated in Fig. 1 together with an exemplary time line, showing that cross-reactive B cells can be identified even within only seven days (one week). In the method of the present invention, PBMCs (e.g. of a single or multiple donors) are cultured in a plurality of cell cultures under conditions to selectively expand (and activate) the B cells among the PBMCs (steps (i) and (ii)). Thereby, sufficient amounts of antibodies can be secreted from (the B cells in) the PBMC cultures to perform a primary screening using different functional assays (preferably in parallel) in order to determine whether the antibodies in the supernatant of a PBMC culture are cross-reactive ("positive" in the different tests for the desired functionalities), i.e. whether they possess multiple functionalities (steps (iii) and (iv)). The cross-reactive PBMC cultures (exhibiting the desired different functionalities tested in the primary screening) are then subdued to isolation of B cells among the PBMCs (e.g., by B cell sorting) and culture of individual B cells ("cloning"), such that monoclonal B cells (individual B cell clones) are obtained (step (v)). Next, a secondary screening is performed in a similar manner as the primary screening, but using the supernatants of the monoclonal B cells of step

(v) instead of a heterogeneous PBMC culture as in step (iii). Similarly to the primary screening, different functional assays are performed (preferably in parallel) in order to determine whether the antibodies in the supernatant of a monoclonal B cell culture are cross-reactive ("positive" in the different tests for the desired functionalities), i.e. whether they possess multiple functionalities (steps (vi) and (vii)). While in general the tests used in the primary and in the secondary screening may differ, usually (essentially) the same functionalities are tested in the primary and secondary screening. Thereby, such individual B cell clones (i.e., a monoclonal B cell population) are identified, which are (capable of) producing cross-reactive antibodies with the desired functionalities (i.e., the functionalities tested in the primary and secondary screening).

In some embodiments, no additional steps are carried out between step (i) and step (ii). In some embodiments, no additional steps are carried out between step (ii) and step (iii). Preferably, no additional steps are carried our between steps (i) and (ii) as well as between steps (ii) and (iii). In some embodiments, no additional steps are carried out between step (iii) and step (iv). In some embodiments, no additional steps are carried out between step (iv) and step (v). In some embodiments, no additional steps are carried out between step (v) and step

(vi). In some embodiments, no additional steps are carried out between step (vi) and step (viii). In some embodiments, steps (i) - (vii) of the method directly follow each other in said order. In particular, step (ii) directly follows step (i), step (iii) directly follows step (ii), step (iv) directly follows step (iii), step (v) directly follows step (iv), step (vi) directly follows step (v), and/or step

(vii) directly follows step (vi). Thereby, it can be ensured that the method is carried out in a very short time.

The terms "B lymphocyte" and "B cell" are used herein interchangeably. In general, a B cell or B lymphocyte is a type of white blood cells of the lymphocyte subtype. A major function of a B lymphocyte is to secrete antibodies. Accordingly, B lymphocytes belong to the humoral component of the adaptive immune system. In addition, B lymphocytes can present antigens and secrete cytokines. In contrast to the other two classes of lymphocytes, T cells and natural killer cells, B lymphocytes express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it usually initiates an antibody response.

In general, the B cell may be of any species. In some embodiments, the B cell is a mammalian B cell. Preferably, the B cell is a human B cell. Accordingly, in some embodiments the B cell is not a rabbit B cell or a murine B cell.

The B cell is usually an isolated B cell, i.e. a B cell, which is not part of a human or animal body. The isolated B cell may be a primary B cell. "Primary" B cells are isolated from living tissue and established for in vitro culture. In contrast to continuous (tumor or artificially immortalized) cell lines, "primary" cells are "freshly" isolated, i.e. they have undergone only very few cell divisions in vitro. Typically, primary cells have a finite life span, i.e. they are not "immortalized" like cell lines. In particular, primary cells have usually not been modified in any way (except for enzymatic and/or physical dissociation required to extract the cells from their tissue of origin).

As used herein, the term "cross-reactive" (e.g., in the context of a cross-reactive antibody, cell culture or B cell) refers in general to an antibody (or a cell culture or B cell producing such an antibody) having at least two (preferably at least three, more preferably at least four, even more preferably at least five, still more preferably more than five, e.g. 6, 7, 8, 9, 10 or more) functionalities (reactivities). In general, the multiple functionalities of an antibody can be assessed by respective functional assays known in the art, which are directed to (one of) said functionalities (also referred to herein as the "desired" functionalities).

In particular, the multiple functionalities of a cross-reactive antibody are due to the (specific) variable regions (VH/VL) of the antibody, rather than to its Fc portion (which is usually the same among different antibodies and therefore its properties are usually inherent to large populations of antibodies). Accordingly, the different "functionalities" preferably relate to different binding or neutralization characteristics of the cross-reactive antibody. For example, a cross-reactive antibody may be cross-reactive to different targets (e.g., bind to and/or neutralize different targets. Accordingly, the present invention provides a method for identification of a B cell (capable of) producing an antibody that is cross-reactive to different targets of interest, the method comprising the following steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for binding to and/or neutralization of each of the different targets of interest;

(iv) cross-comparing the results obtained in step (iii) for the different targets and selecting one or more cell cultures, which are cross-reactive to the different targets;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for binding to and/or neutralization of each of the different targets of interest; and

(vii) cross-comparing the results obtained in step (vi) for the different targets and selecting a B cell clone, which is cross-reactive to the different targets of interest.

Accordingly, it is preferred that the antibody that is cross-reactive to different targets of interest and wherein the different functionalities tested in the primary and secondary screening are binding to and/or neutralization of each of the different targets of interest. In general, the different targets may be different antigens or even different epitopes of the same antigen. Preferably, the different targets are related to each other, i.e. not unrelated, arbitrarily selected targets. For example, the different targets are preferably related antigens, such as antigens of related pathogens (e.g., related viruses, bacteria etc.).

Examples of pathogens include human immunodeficiency virus (HIV); hepatitis A virus; hepatitis B virus; hepatitis C virus; coronaviruses, such as SARS coronavirus and SARS-CoV- 2; measles virus; bunyaviridae; arenaviridae; reoviridae (including rotaviruses and orbiviruses); retroviridae (including HTLV-I, HTLV-II, HIV-1 , HrV-2); papillomaviridae (such as papillomavirus); adenoviridae; parvoviridae; herpesviridae (including herpes simplex viruses 1 and 2, cytomegaloviruses, varicella-zoster virus, herpesviruses 6A, 6B and 7); poxviridae (such as pox virus); mumps virus; rubella virus; lyssaviruses, such as rabies virus; ebolavirus; influenza virus; vaccina virus; variola virus; polio virus; rhinovirus; respiratory syncytial virus (RSV); flaviviruses, such as dengue and zika virus; Plasmodium (P.) falciparum; P. vivax; P. malaria; P. ovale; Corynebacterium diphtheriae; Clostridium tetani; Clostridium botulinum; Bordetella pertussis; Haemophilus influenzae; Neisseria meningitidis; Streptococcus pneumoniae; Streptococcus agalactiae, Streptococcus pyogenes; Staphylococcus aureus; Bacillus anthracis; Moraxella catarrhalis; Chlamydia trachomatis; Chlamydia pneumoniae; Yersinia pestis; Francisella tularensis; Salmonella species; Vibrio cholerae; toxic Escherichia coli etc.

Preferably, the different targets of interest are corresponding antigens of related pathogens. Related pathogens may be pathogens of the same taxonomic group, such as the same species, genus or family. Often, (certain portions of) gene sequences are conserved among related pathogens. Such related pathogens usually exhibit similar structures (and associated similar functionalities), such that corresponding antigens can be identified. A "corresponding" antigen is an antigen, which can be found in each of the different, related pathogens, albeit usually with differences, e.g., different amino acid sequences. Non-limiting examples of such "corresponding" antigens of related pathogens include the spike (S) protein of coronaviruses; the envelope (E) protein of flaviviruses; the fusion (F) protein of RSV, MPV, influenza viruses; hemagglutinin of influenza viruses; and the glycoprotein (G) of lyssaviruses.

In general, cross-reactive antibodies can occur in nature (broadly binding or broadly neutralizing antibodies), and are, thus, usually different from (engineered) bi- or multispecific antibodies, which comprise two or more different antigen-binding sites conferring different functionalities. The cross-reactive antibody (as referred to herein) is usually monospecific, i.e. it comprises only a single type of antigen-binding site (in contrast to engineered bi- or multispecific antibodies comprising different antigen-binding sites, e.g. at different "arms" of the antibody). Accordingly, cross-reactivity is preferably mediated by a single type of antigenbinding site, e.g. by a single set of six CDRs or a single VH/VL combination (for example, binding to a highly conserved epitope in an antigen; such that the same antibody can bind to antigens of different strains, species, etc.).

In step (i), PBMCs are provided in a plurality of cell cultures. A peripheral blood mononuclear cell (PBMC) is any peripheral blood cell having a round nucleus. As used herein, the term "PBMCs" refers to a heterogeneous population of peripheral blood mononuclear cells, which includes at least antigen-producing B cells. In addition, other PBMCs, such as T cells, NK cells and monocytes may be present. In particular, the term "PBMCs" refers to total PBMCs, which may be isolated, e.g., from a blood sample. Methods for isolating PBMCs are well- known in the art. For example, total PBMCs can be extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, and gradient (density) centrifugation, which separates the blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes.

In general, the PBMCs provided in step (i) may be freshly isolated or cryopreserved PBMCs. The PBMCs may be isolated as described above, e.g., from a blood sample. As the method of the present invention is suitable for high-throughput approaches, PBMCs of different donors (e.g. obtained from different blood sample) may be used. Alternatively, also PBMCs obtained from a single donor (e.g., from a single blood sample) may be used. If PBMCs from different donors (different blood samples) are used, PBMCs of such different origin are preferably maintained in separate cultures (i.e., not mixed). Thereby, the B cell done producing the cross-reactive antibody can be tracked back to the donor and a suitable donor for cross- reactive antibodies can be identified among the different donors.

Preferably, the donor of the PBMCs is a human donor (and, thus, the PBMCs are human PBMCs). If cross-reactive antibodies against antigens of a specific (group of) pathogens are of interest, the donor may be known for previous exposure to one or more of the pathogens of interest, in particular the donor may have survived a (previous) infection with one or more of the pathogens of interest. In this case, it would be expected that the donor has produced antibodies against said pathogens, such that the likelihood to identify a cross-reactive antibody against said pathogens is higher as compared to donors, for whom previous exposure to the pathogen of interest is not known.

In step (i), the PBMCs are provided in a plurality of cell cultures. To this end, the PBMCs may be "plated", i.e. distributed in a plurality of cell culture vessels, such as wells, e.g., in one or more multiwell plates (also referred to as "microplates", "microwell" or "microtiter" plates). In general, cells may be plated at distinct densities. For high-throughput approaches higher densities are usually preferred. For example, the PBMCs may be plated at 10³ - 10⁷ cells per cell culture (well), preferably at 10⁴ - 10⁶ cells per cell culture (well), more preferably at 10⁴ - 10^s cells per cell culture (well). In some embodiments, a single cell culture of the plurality of cell cultures contains (at the beginning, i.e. when cells are plated) about 10,000 - 100,000 cells; preferably about 20,000 - 95,000 cells; more preferably about 30,000 - 90,000 cells; even more preferably about 40,000 - 80,000 cells; still more preferably about 50,000 - 70,000 cells; such as about 60,000 cells.

The PBMCs are cultured under conditions for selective expansion of B cells (step (ii)). Such conditions (also referred to as conditions to activate B cells polyclonally) are known in the art and described, for example, by Pinna D, et al. (Eur J of Immunology 2009, 39:1260), which is incorporated herein by reference. Examples of conditions for selective expansion of B cells include the use of alloreactive T helper clones, e.g. as described in Lanzavecchia (Lanzavecchia, A., One out of five peripheral blood B lymphocytes is activated to high-rate Ig production by human alloreactive T cell clones. Eur. J. Immunol. 1983. 13: 820-824) and in Lanzavecchia et al. (Lanzavecchia, A., Parodi, B. and Celada, F., Activation of human B lymphocytes: frequency of antigen-specific B cells triggered by alloreactive or by antigenspecific T cell clones. Eur. J. Immunol. 1983. 13: 733-738); the use of CD40L1 EL4 thymoma cells and IL-4 to activate total B cells, e.g., as described in Wen et al. (Wen, L., Hanvanich, M., Werner-Favre, C., Brouwers, N., Perrin, L. H. and Zubler, R. H., Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and antimalaria responder B cell frequencies. Eur. J. Immunol. 1987. 17: 887-892); the use of TLR agonists, such as the TLR9 agonist CpG (and IL-2) to drive polyclonal memory B-cell activation, e.g., as described in Bernasconi et al. (Bernasconi, N. L., Onai, N. and Lanzavecchia, A., A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 2003. 101 : 4500-4504); the combination of TLR agonists and stimulation with additional stimuli, such as Staphylococcus aureus Cowan (SAC) and Pokeweed mitogen (PWM) and a cytokine cocktail, in particular a combination of PWM, SAC and CpG (e.g., as described in Crotty, S., Aubert, R. D., Glidewell, J. and Ahmed, R., Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J. Immunol. Methods 2004. 286: 111-122) or a combination of SAC, PWM, CpG, LPS, IL-2, IL-6, IL-10 in the presence of NIH/3T3 feeder in limiting dilution assays (e.g., as described in Amanna, I. J. and Slifka, M. K., Quantitation of rare memory B cell populations by two independent and complementary approaches. J. Immunol. Methods 2006. 317: 175-185).

Preferably, the PBMCs are cultured under conditions essentially as described in Pinna D, et al. (Eur J of Immunology 2009, 39:1260). In particular, the culture conditions for selective expansion of B cells in step (ii) preferably include(s) a Toll-like receptor (TLR) agonist and/or a cytokine. In other words, the culture medium used to culture the PBMCs (for selective expansion of B cells) preferably include(s) a Toll-like receptor (TLR) agonist and/or a cytokine.

The TLR agonist is preferably an agonist of TLR7, TLR8 and/or TLR9. More preferably, the TLR agonist is an agonist of TLR7 and/or TLR8. TLR agonists, in particular agonists of TLR7, TLR8 and/or TLR9 (preferably of TLR7 and/or TLR8) are known in the art and commercially available. Non-limiting examples of TLR agonists include R848 (resiquimod), 3M001 , 3M002, CL075, CL097, CL264, CL307, Gardiquimod™, imiquimod, TL8-506, loxoribine, single stranded (ss)RNA, CpG, LPS and combinations thereof. Preferably, the TLR agonist is selected from the group consisting of R848 (resiquimod), 3M001 , 3M002, CL075, CL097, CL264, CL307, Gardiquimod™, imiquimod, TL8-506, loxoribine, single stranded (ss)RNA, CpG and combinations thereof. More preferably, the TLR agonist is an agonist of TLR7 and/or TLR8, which may be selected from the group consisting of R848 (resiquimod), 3M001 , 3M002, CL075, CL097, CL264, CL307, Gardiquimod™, imiquimod, TL8-506, loxoribine, single stranded (ss)RNA, and combinations thereof. Even more preferably, the TLR agonist is an agonist of TLR7 and TLR8, such as R848 (resiquimod), CL075, CL097, and single stranded (ss)RNA. Still more preferably, the TLR agonist is R848.

Examples of cytokines include IL-1 -like, IL-1 a, IL-lp, IL-1 RA, IL-18, CD132, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, CD131 , IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11 , G-CSF, IL-12, LIF, OSM, ILI O-like, IL-10, IL-20, IL-21 , IL-14, IL-16, IL-17, IFNa, IFNp, IFNy, CD154, LTp, TN Fa, TN Fp, 4-1 BBL, APRIL, BAFF, CD70, CD153, CD178, CD30L, CD40L, GITRL, LIGHT, OX40L, TALL- 1 , TRAIL, TWEAK, TRANCE, TGFpI, TGFp2, TGFp3, c-Kit, FLT-3, Epo, Tpo, FU-3L, SCF, M- CSF, aCD40, or any combinations thereof. In some embodiments, the PBMCs may be co- cultured with a CD40L expressing cell line (e.g. K562L or 3T3 cells). Preferably the cytokine is selected from the group consisting of IL-2, CD40L, IL-4, IL-21 , BAFF, APRIL, CD30L, TGF- pi , 4-1 BBL, IL-6, IL-7, IL-10, IL-13, c-Kit, FLT-3, IFNa, or any combination thereof. More preferably, the cytokine is selected from the group consisting of IL-2, IL-6, IL-10, CD40L, IL- 4, or any combination thereof. Even more preferably, the cytokine is IL-2.

In some embodiments, the culture medium (culture conditions) in step (ii) may include a cytokine (or a combination of cytokines), but no TLR agonist. For example, the culture medium (culture conditions) in step (ii) may include a combination of IL-2 and CD40L (optionally together with further one or more further cytokines, such as anyone of IL-4, IL-6, IL-10 or any combination thereof), but no TLR agonist.

As described above, the PBMCs are preferably cultured in a medium comprising a TLR agonist as well as a cytokine. Preferred combinations of a TLR agonist and a cytokine include the following combinations: CpG and IL-2; R848 and IL-2; 3M001 and IL-2; 3M001 , IL-4 and CD40L; 3MOO2 and IL-2; 3M002, IL-4 and CD40L; and LPS and IL-2. The following combinations of a TLR agonist and a cytokine are more preferred: CpG and IL-2; R848 and IL-2; 3M001 and IL-2; 3M001 , IL-4 and CD40L; 3M002 and IL-2; and 3M002, IL-4 and CD40L. The following combinations of a TLR agonist and a cytokine are even more preferred: R848 and IL-2; 3M001 and IL-2; 3M001 , IL-4 and CD40L; 3M002 and IL-2; and 3M002, IL- 4 and CD40L. The following combinations of a TLR agonist and a cytokine are still more preferred: R848 and IL-2; 3M001 and IL-2; and 3M002 and IL-2. Particularly preferably, TLR agonist R848 is combined with IL-2. In some embodiments, the only TLR agonist comprised in the medium (in the culture conditions of step (ii)), is the one mentioned above (i.e. no further TLR agonists are present). In some embodiments, the only cytokine (or combination of cytokines) comprised in the medium (in the culture conditions of step (ii)), is the cytokine (or combination of cytokines) mentioned above (i.e. no further cytokines are present).

As described above, TLR agonist R848 is preferably combined with IL-2. More preferably, in this combination, the only cytokines which may be present in addition to IL-2 are anyone of IL-6, IL-10, IL-4 and CD40L. Even more preferably, in the combination of R848 and IL-2, the only cytokines which may be present in addition to IL-2 are anyone of IL-6, IL-10 and IL-4. Still more preferably, in the combination of R848 and IL-2, the only cytokines which may be present in addition to IL-2 are anyone of IL-6 and IL-10. In some embodiments, the culture medium (culture conditions) used is step (ii) includes R848 as only TLR agonist and IL-2 as only cytokine.

In some embodiments, the TLR agonist, in particular R848, may be used at a concentration of 0.1 - 10 pg/ml, preferably 0.5 - 5 pg/ml, more preferably 1 - 4 pg/ml, even more preferably 2 - 3 pg/ml, and particularly preferably at a concentration of about 2.5 pg/ml.

In some embodiments, the cytokine, in particular IL-2, may be used at a concentration of 0.1

- 10000 U/ml, preferably 1 - 5000 U/ml, more preferably 100 - 4000 U/ml, even more preferably 500 - 1500 U/ml, and particularly preferably at a concentration of about 1000 U/ml.

Preferably, culturing of the PBMCs under culture conditions for selective expansion of B cells (step (ii)) is performed no longer than 9 days, preferably no longer than 8 days, more preferably no longer than 7 days, even more preferably no longer than 6 days and still more preferably no longer than 5 days; e.g. for 3 - 9 days, preferably 4 - 8 days, more preferably 5

- 7 days and particularly preferably for about 6 days. As shown in the Example section, the present inventors surprisingly found that after only five days of culturing PBMCs under conditions for selective expansion of B cells (polyclonally activating B cells), the culture supernatants are already sufficient for multiple parallel tests (screening) in order to identify cultures producing rare antibodies with multiple functionalities. Accordingly, the short culture time under culture conditions for selective expansion of B cells (step (ii)) allows the easy and rapid high throughput screening of samples from multiple donors.

In step (iii), a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities is performed. To this end, different functionalities of interest are tested with appropriate tests known in the art. The different tests are preferably performed essentially in parallel (or, at least, overlapping). Thereby, time can be saved. Step (iii), i.e. the primary screening, is preferably performed no longer than 9 days, preferably no longer than 8 days, more preferably no longer than 7 days, even more preferably no longer than 6 days and still more preferably no longer than 5 days; e.g. for 3 - 9 days, preferably 4 - 8 days, more preferably 5 - 7 days and particularly preferably for about 6 days after the start of the PBMC culture under conditions for selective expansion of B cells (start of step (ii)).

In particular, step (iii) (the primary screening) is performed on the cell cultures obtained in step (ii), i.e. after culturing the PBMCs under conditions for selective expansion of B cells (step (ii)), e.g. for 3 - 9 days, preferably 4 - 8 days, more preferably 5 - 7 days and particularly preferably for about 6 days. In particular, step (iii), i.e. the primary screening, is not performed on isolated or purified B cells (plated at low densities, e.g. with no more than 100 B cells per well). In other words, in the method of the present invention there is usually no isolation/purification of (or screening for) B cells, e.g. to isolate/purify B cells from (other) PBMCs, before the primary screening in step (iii). Instead, step (iii) is performed on the PBMC cell cultures (with expanded B cells) obtained in step (ii).

In contrast to the present invention, in conventional approaches of the prior art, B cells are usually first isolated/purified from a sample, e.g. PBMCs, for example using B cell markers and cell sorting methods. Thereafter, isolated B cells are plated at low densities of very few cells per well and cultured, including stimulation/expansion, for the production of antibodies. Only thereafter, the primary screening is typically performed in the prior art to select cultures producing the desired antibody. However, this conventional approach is very cumbersome and cost-intensive, because it requires a large amount of (isolated) B cell cultures, as any B cell obtained by cell sorting is cultured, irrespective of its antigen specificity. In contrast thereto, in the inventive method, supernatants of larger PBMC cultures with expanded B cells are screened in step (iii) - and B cells are only isolated thereafter and from those cultures only, which exhibit the desired antigen specificity. Thereby, the number of B cell cultures is considerably reduced. Furthermore, the inventive method reduces not only costs, efforts and time, but also increases the chance to identify very rare antibodies, because the entire B cell pool present in the PBMCs is expanded in step (ii) and tested in step (iii) - without B cell isolation and low density plating before testing (as in the prior art).

In step (iv), the results (outcome) of the different tests are cross-compared, i.e. for a single cell culture (of the plurality of cell cultures) the outcome of each test is compared. This is preferably done for various cultures (most preferably for as many cultures as possible); preferably essentially in parallel.

For example, the functionalities in step (iii) may be tested by using multi-well plates, e.g. with one (or more) plate per test. Each of the multi-well plates (for the different tests) may be prepared (with a portion of culture supernatant) in a similar manner (e.g. with corresponding coordinates in the different multi-well plates for each cell culture). For example, essentially the same pipetting scheme may be used as for the cell cultures of step (i) also for multi-well plates of step (iii). For instance, (a portion of) the culture supernatant of the cell culture of well "A1 " of step (i) may be transferred to well "A1 " of a new plate to test a first functionality, to well "A1" of a second new plate to test a second functionality and, optionally, to well "A1" of a third new plate to test a third functionality; (a portion of) the culture supernatant of the cell culture of well "A2" of step (i) may be transferred to well "A2" of a new plate to test a first functionality, to well "A2" of a second new plate to test a second functionality and, optionally, to well "A2" of a third new plate to test a third functionality; (a portion of) the culture supernatant of the cell culture of well "A3" of step (i) may be transferred to well "A3" of a new plate to test a first functionality, to well "A3" of a second new plate to test a second functionality and, optionally, to well "A3" of a third new plate to test a third functionality; and so on. Of course, it is understood that the exact coordinates need not to be the same for such an approach as long as essentially the same pipetting scheme is employed (e.g., for some plates "B" coordinates instead of "A" ("C" instead of "B" and so on) may be used, as long as it follows the same order). Thereby, the results can be easily cross-compared.

By cross-comparing the results of the different tests, such that such cultures can be identified, which are cross-reactive (i.e., which show each of the desired functionalities, for example a "positive" result in each of the different tests). Accordingly, cross-reactive cultures (exhibiting the different desired functionalities) are selected in step (iv).

It is understood that for the primary screening of step (iii) the plurality of cultures is usually investigated using (1 ) different tests for different functionalities; and (2) the same tests to investigate the same functionality among the plurality of cell cultures. This means that in order to screen the plurality of cultures for a first functionality the same first test (differing only in the supernatant of the different cultures to be investigated) is usually used for all cultures investigated. Likewise, to screen the plurality of cultures for a second functionality the same second test (differing only in the supernatant of the different cultures to be investigated) is usually used for all cultures investigated (and so on). As described above, the first and second tests (and any further test) may be performed in parallel as described above.

In general, the screening step may employ any immunoassay; e.g., ELISA, staining of tissues or cells (including transfected cells); neutralization assay or one or more of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigenbinding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their differentiation status, etc.

As described above, the different "functionalities" preferably relate to different binding or neutralization characteristics of the cross-reactive antibody. For example, a cross-reactive antibody may be cross-reactive to different targets (e.g., bind to and/or neutralize different targets). Accordingly, the primary screening in step (iii) may be a primary screening of the cell culture supernatants of the plurality of cell cultures for binding to and/or neutralization of each of the different targets of interest. In step (iv), the results obtained in step (iii) for the different targets may be cross-compared and those cell cultures may be selected, which are cross-reactive to the different targets. In some embodiments, parallel binding assays, in particular parallel ELISA screenings, of the plurality of cell cultures (culture supernatants) against essentially the same set of (potential target) antigens (e.g., coronavirus spike proteins of different coronaviruses) may be performed.

However, also other functionalities of an antibody may be of interest, such as to test the antibody's ability to inhibit pathogen (e.g., viral) binding to a (human) target. To this end, also "inhibition of binding" may be tested, i.e. whether an antibody is capable of reducing or inhibiting the binding of a pathogen (viral) protein (e.g. of different viral strains, species, variants or the like) to a human target (e.g., as required for pathogen/viral infection of a human cell). As a specific example, binding of coronavirus (e.g., SARS-CoV-2) spike protein to human ACE2 may be mentioned. The ability of an antibody to reduce or inhibit such binding may also be of interest.

With regard to the different functionalities, it is preferred that they relate to the antibody's reactivity to different targets, e.g., derived from different pathogens (such as different pathogen species or strains or variants, as described above). For example, with regard to coronaviruses, binding to (corresponding antigens, such as the spike protein) or neutralization of different coronaviruses may be tested; e.g., different beta-coronaviruses, such as OC43, HKU1, MERS, SARS-CoV and SARS-CoV-2, and/or different alpha-coronaviruses, such as NL63 and 229E. Similarly, binding to (corresponding antigens, such as the spike protein) or neutralization of different coronavirus variants, e.g. different SARS-CoV-2 variants alpha, beta, delta, omicron, may be tested.

However, it is also conceivable that the different functionalities relate to the same target, such as binding, neutralization and, optionally, a further related functionality, such as inhibition of (infection-related pathogen-) binding, of an antibody to a single target of interest. Accordingly, it is preferred that the primary screening in step (iii) comprises a binding assay to test binding to the different targets.

For example, binding to a target of interest (as well as inhibition of binding) may be investigated by any binding assay known in the art. Standard methods to assess binding of the antibody according to the present invention, or the antigen-binding fragment thereof, are known to those skilled in the art and include, for example, immunoassays, such as ELISA (enzyme-linked immunosorbent assay); radioimmunoassay; labelling (e.g. radio- or fluorscence-labelling) of antigens; flow cytometry; cytometric bead array; immunohistochemistry; immunocytochemistry; and affinity chromatography. Further examples of binding assays include SPR (surface plasmon resonance; e.g. as described in Hearty S, Leonard P, O'Kennedy R. Measuring antibody-antigen binding kinetics using surface plasmon resonance. Methods Mol Biol. 2012;907:41 1 -42. doi: 10.1007/978-1 - 61779-974-7_24); isothermal titration calorimetry (ITC); microscale thermoporesis (MST) thin layer chromatography (TLC); and bio-layer interference (BLI).

In some embodiments, step (iii) involves an ELISA (enzyme-linked immunosorbent assay). For example to test binding to different targets (e.g. (corresponding) antigens derived from different pathogens, as described above), different ELISAs (e.g., one per target) may be performed in step (iii), e.g. in parallel.

An exemplary standard ELISA may be performed as follows: ELISA plates may be coated with a sufficient amount (e.g., 1 pg/ml) of the target (e.g. protein/complex/particle) to which binding of the antibody is to be tested. Plates may then be incubated with the antibodies to be tested. After washing, antibody binding can be revealed. To this end, e.g., a labelled antibody recognizing the test antibody may be used, such as goat anti-human IgG coupled to alkaline phosphatase. Plates may then be washed, the required substrate (e.g., p-NPP) may be added and plates may be read, e.g. at 405 nm. Optionally, relative affinities of antibody binding may be determined, e.g. by measuring the concentration of mAb (EC₅₀) required to achieve 50% maximal binding at saturation. The EC₅o values may be calculated by interpolation of binding curves fitted with a four-parameter nonlinear regression with a variable slope.

To test the antibody's ability to inhibit pathogen (e.g., viral) binding to a human target, an ELISA may be performed as described above. Thereby, after addition (and incubation and washing) of the test antibody, the human target may be added (e.g., at saturating concentration), usually followed by another incubation and washing step. To reveal inhibition of binding, a labelled antibody recognizing the human target may be used.

For example, to study and quantitate neutralization in the laboratory the person skilled in the art knows various standard "neutralization assays". For a neutralization assay, the pathogens (to be neutralized), e.g. viruses, are typically propagated in cells and/or cell lines. For example, in a neutralization assay cultured cells may be incubated with a fixed amount of pathogen (e.g. virus) in the presence (or absence) of the antibody to be tested. As a readout, for example, flow cytometry may be used. Alternatively, also other readouts are conceivable. In step (v), B cells from the cross-reactive cultures selected in step (iv) are isolated and cloned in order to obtain monoclonal B cells. In other words, B cells are isolated from the selected cross-reactive PBMC cultures and individually cultured, such that a single B cell culture contains monoclonal B cells only. As used herein, the expression "cloning" with regard to B cells refers to culturing B cells individually. Thereby an individual B cell can be propagated, such that a "monoclonal" B cell culture is obtained. This step is required to obtain monoclonal antibodies. Cross-reactivity observed for a polyclonal cell culture in step (iv) may be due to cross-reactive antibodies or due to different mono-reactive antibodies (wherein each antibody reacts to a distinct target). However, in the present method, the latter, mono- reactive antibodies are not of interest. Therefore, monoclonal cross-reactive antibodies still need to be identified. Due to the pre-selection of cross-reactive polyclonal cultures in steps

(iii) and (iv), sorting and cloning of B cells can be performed in step (v) in a single high- throughput approach, although this step multiplies the number of cell cultures to be cultured and investigated. In general, the cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art. In general, step (v) may be performed at the same day as steps (ii) and (iv) or at the day following step (iii) and/or step

(iv).

Methods for isolating B cells are known in the art. For example, B cells may be isolated by flow cytometry, magnetic cell isolation and cell separation (MACS), RosetteSep, or antibody panning. One or more isolation techniques may be utilized in order to provide isolated B cells with sufficient purity, viability, and yield. For example, B cells may be isolated by magnetic cell sorting. To this end, for example anti-CD19 microbeads may be used. A large variety of technologies for single-cell separation, isolation, and sorting are known to the skilled person, which mainly include FACS (fluorescent activated cell sorting), LCM (lasercapture microdissection), microengraving, and droplet microfluidics. Particularly preferably, IgG secreting memory B cells (IgG secreting memory B cell blasts) may be isolated by a negative gating strategy as CD19⁺ IgM and IgA or as CD19⁺ CD27^f/ IgM and lgA~. In some embodiments, cross-reactive cultures (as selected in step (iv)) may be stained with CD19-PE- Cy7 (BD, catalog no. 341 1 13, 1 :100), lgM-AF647 Oackson Immuno, catalog no. 109-606- 129, 1 :500) and IgA AF488 (Jackson Immuno, catalog no. 109-546-01 1 , 1 .500) . Accordingly, it is preferred that lgG⁺ memory B cells may be sorted by a negative gating strategy, e.g. essentially as described in Pinto et al., 2013 (Pinto D et al. 2013 Bloods 21 (20): 41 10-4114), which is incorporated herein by reference.

Preferably, the purity of the isolated B cells is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Moreover, it is preferred that the isolated B cells are at least about 70%, 75%, 80%, 85%, 90%, 95% or more viable.

Sorted memory B cells may be seeded at no more than a single cell per culture vessel (e.g. no more than a single cell per well), preferably at no more than 0.9 cell per culture vessel (e.g., well), more preferably at no more than 0.8 cell per culture vessel (e.g., well) and even more preferably at no more than 0.75 cell per culture vessel (e.g., well), such as at 0.7 cell per well.

The number of B cells can be reduced as described below. Techniques of obtaining the number of desired cells in a culture are well known in the art. Such techniques include, but are not limited to, limiting dilution, or cell sorting and deposition. For example, cultures comprising a limited or reduced number of B cells can be achieved by single cell deposition using a cell sorter or by diluting a suspension of plasma cells with enough culture medium such that no more than a single cell is present per culture vessel (e.g., per well of a multi-well plate). Preferably, the isolated B cells are cloned by limiting dilution at no more than 0.9 cells per culture vessel (e.g., well), more preferably at no more than 0.8 cells per culture vessel (e.g., well) and even more preferably at no more than 0.75 cells per culture vessel (e.g., well), such as at about 0.7 cells per well.

Cloning of the isolated B cells in step (v) may be performed in complete medium. Preferably, cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of feeder cells. Feeder cells are generally known as "supplementary" cells in cell cultures, which are used to provide optimal conditions for the cells to be cultured (e.g. to "feed" the cells to be cultured). In the context of B cell culture, feeder cells, such as mesenchymal stromal cells (MSC) or other feeder cells, are commonly used, e.g. for B cell activation. However, the present inventors have surprisingly found that for B cell cloning in step (v) of the method of the present invention, no feeder cells are required. While, in general, feeder cells may be used, it is preferred that step (v) is performed in the absence of feeder cells to reduce costs and complexity of the method.

It is also preferred that cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of cytokines (e.g., cytokines as described above). Similarly to feeder cells, cytokines are often used in B cell culture, e.g. to activate B cells. Common cytokines used in this context include those as described above, in particular IL-2, IL-6 and IL-21. However, the present inventors have surprisingly found that for B cell cloning in step (v) of the method of the present invention, no addition of cytokines is required. While, in general, cytokines may be added, it is preferred that step (v) is performed without the addition of cytokines to reduce costs and complexity of the method.

Furthermore, it is preferred that cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of TLR agonists (e.g., TLR agonists as described above). Similarly to feeder cells and cytokines, TLR agonists are often used in B cell culture, e.g. to activate B cells. Common TLR agonists used in this context include those as described above, in particular agonists of TLR7, TLR8 and/or TLR9, such as R848 and CpG. However, the present inventors have surprisingly found that for B cell cloning in step (v) of the method of the present invention, no addition of TLR agonists is required. While, in general, TLR agonists may be added, it is preferred that step (v) is performed without the addition of TLR agonists to reduce costs and complexity of the method.

Even more preferably, cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of feeder cells, cytokines and TLR agonists. While the skilled person usually expects at least one of feeder cells, cytokines and TLR agonists to be required for B cells to produce antibodies, the present inventors have surprisingly found that for B cell cloning in step (v) of the method of the present invention, neither feeder cells nor addition of cytokines or TLR agonists are required. While, in general, feeder cells, cytokines and/or TLR agonists may be used, it is preferred that step (v) is performed in the absence of feeder cells and without addition of cytokines and TLR agonists to reduce costs and complexity of the method.

Accordingly, it is particularly preferred that cloning of the isolated B cells in step (v) is performed in complete medium only without further supplements. As demonstrated in the appended examples, the proliferating B cells can be sorted and individually cloned in the absence of feeder cells, cytokines and TLR agonists, since they continue to proliferate in the absence of feeder cells or cytokines or TLR agonists added.

Preferably, cloning of the isolated B cells (culturing of the monoclonal B cells) in step (v) is performed for 1 - 3 days, more preferably for about two days. As demonstrated in the appended examples, even when monoclonal B cells are cultured in the absence of feeder cells or cytokines or TLR agonists added, the antibodies produced by single B cell clones can be screend after only two days.

In step (vi), a secondary screening is performed, wherein the supernatants of the B cell clones obtained in step (v) are screened for different functionalities. Thereafter, in step (vii), the results obtained in step (vi) are cross-compared for the different functionalities and a B cell clone, which is cross-reactive, is identified. Step (vi), i.e. the secondary screening, is preferably performed 1 - 3 days, more preferably about two days after B cell isolation and start of B cell cloning of step (v).

In general, the secondary screening of step (vi) is (technically) very similar to the primary screening of step (iii). The essential difference between the primary and secondary screening is that the primary screening (step (iii)) is performed on (supernatants of) polyclonal (PBMC) cultures, while the secondary screening (step (vi)) is performed on (supernatants of) monoclonal B cell cultures. Accordingly, the detailed description of the primary screening above applies accordingly to the secondary screening (step (vi)) - with the only difference that monoclonal B cells (or "B cell clones") are used instead of polyclonal cultures of PBMCs (with expanded B cells). For example, the secondary screening in step (vi) may comprise a binding assay to test binding to the different targets, such as an ELISA, as described above. Usually, the different functionalities tested in the primary screening are also tested in the secondary screening. However, the secondary screening may comprise the same type of assays or a different type of assays as the primary screening, In some embodiments, the same functionality, e.g. binding to a target, may be tested with the same or different assays in the primary and secondary screening. Preferably, secondary screening in step (vi) comprises the same type of assay (and the same set of targets/antigens) as primary screening in step (iii). For example, both, the primary and secondary screening may include ELISAs investigating binding to the different targets of interests, e.g. as described above for the primary screening.

Moreover, the subsequent cross-comparison performed in step (vii) is (technically) very similar to the cross-comparison following primary screening in step (iv). Accordingly, the detailed description of the cross-comparison after the primary screening above (step (iv) applies accordingly to the cross-comparison following the secondary screening (step (vii)).

In some embodiments, the cross-reactive B cell identified in step (vii) is thereafter immortalized. Methods for immortalizing B cells are well-known in the art. For example, Epstein-Barr Virus may be used to immortalize B cells, e.g. as described in WO 2004/076677 A2 and in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004 Aug;10(8):871 -5. Epub 2004 Jul 1 1 . Immortalized B cell clones are advantageous for further use and investigation as well as for the production of antibodies.

In other embodiments, the cross-reactive B cell identified in step (vii) is not immortalized, but may be directly subjected to optional additional steps, such as the retrieval of the sequence of the variable regions (VH/VL), e.g. of the B cell receptor (BCR) or the antibody produced by the B cell clone.

Accordingly, the present invention also provides an isolated B cell obtained with the method according to the present invention as described above. Such a B cell may be a single B cell or multiple monoclonal B cells, such as a B cell clone. The B cell is (capable of) producing a cross-reactive antibody, in particular an antibody that is cross-reactive to different targets of interest. Preferably, the B cell is a human B cell. Accordingly, the cross-reactive antibody produced by said B cell is preferably a human antibody.

Methods for identifying and generating a nucleic acid encoding a cross-reactive antibody

In a further aspect, the present invention also provides a method for identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) identification of a B cell (capable of) producing a cross-reactive antibody, in particular an antibody that is cross-reactive to different targets of interest, according to the present invention as described above; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-celL

In other words, the present invention also provides a method for identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) identification of a B cell (capable of) producing cross-reactive antibody, in particular an antibody that is cross- reactive to different targets of interest, comprising the following sub-steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities;

(iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which are cross-reactive;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and (vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross-reactive; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-cell.

The detailed description of the identification of the B cell (capable of) producing a cross- reactive antibody, in particular of steps (i) - (vii) described above, applies accordingly to step (a) of the method for identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody.

As used herein, the term "nucleic acid" refers to RNA or DNA. In the context of the present invention, DNA (i.e., a DNA sequence) is preferred.

The skilled person is well aware of methods to obtain the sequences of the heavy and light chain variable region (VH/VL) genes of a B cell. Therefore, when a B cell clone producing a cross-reactive antibody is identified (step (a)), the sequences of the heavy and light chain variable region (VH/VL) genes of said B cell may be readily obtained. In some embodiments, at first VH/VL nucleic acids (of the BCR of the B cell) may be retrieved by reverse transcription followed by nested PCR reactions (and sequencing). Methods to amplify and sequence Ig variable regions are well-known in the art (e.g., Coronella, J A et al. Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells. Nucleic acids research Vol. 28,20 (2000): E85. doi:10.1093/nar/28.20.e85; Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning [published correction appears in J Immunol Methods. 2008 May 20;334(1 -2): 142], J Immunol Methods. 2008;329(1 -2):112-124. doi:10.1016/j.jim.2007.09.017; Schanz et al. (2014). High-Throughput Sequencing of Human Immunoglobulin Variable Regions with Subtype Identification. PloS one. 9. e11 1726. 10.1371/journal.pone.01 11726; Meyer et al. (2019) A simplified workflow for monoclonal antibody sequencing. PLoS ONE 14(6): e0218717. https://doi.Org/10.1371/journal.pone.0218717) and commercially available (e.g., Human IgG and IgM Library Primer Set, PROGEN, Biotechnik GmbH, Heidelberg, Germany). For example, a reverse transcription PCR (RT-PCR) may be performed, in particular to obtain cDNA of the heavy and light chain variable region (VH/VL) genes. In this context, preferably primers specific for the constant regions of the heavy and light chains (in particular IgG), such as primers specific to the constant regions of IgG, IgK, and IgA, respectively, may be used. Sequences of the constant regions of human antibodies are well-known in the art, such that the skilled person can readily obtain respective primers (which are also commercially available). The obtained cDNA may then be amplified, for example by PCR (such as two-step PCR, e.g. using two sets of Ig-specific primers, one nested within the other) and optionally purified. Then, the (purified) cDNA (or PCR amplicons) may be sequenced, e.g. by Sanger sequencing. Accordingly, it is preferred that the sequences of the heavy and light chain variable region (VH/VL) genes are obtained by reverse transcription PCR (RT-PCR) and sequencing.

An exemplified method to obtain antibody sequences, which involves RT-PCR and sequencing is described in Meyer L, Lopez T, Espinosa R, Arias CF, Vollmers C, DuBois RM. A simplified workflow for monoclonal antibody sequencing. PLoS One. 2019;14(6):e0218717. Published 2019 Jun 24. doi:10.1371/journal. pone.0218717, which is incorporated herein by reference.

Accordingly, the present invention also provides a nucleic acid comprising the nucleic acid sequence of the heavy and/or light chain variable region (VH/VL) of the cross-reactive monoclonal antibody identified with the method according to the present invention.

Moreover, the present invention also provides a method for generating an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody, the method comprising the following steps:

(1 ) identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody according to the present invention as described above; and

(2) cloning of said sequences into an expression vector for expression of antibody heavy and light chains, respectively. In other words, the present invention provides a method for generating an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody, the method comprising the following steps:

(1 ) identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody comprising the following steps:

(a) identification of a B cell (capable of) producing cross-reactive antibody, in particular an antibody that is cross-reactive to different targets of interest, comprising the following sub-steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities;

(iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which are cross- reactive;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and

(vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross-reactive; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-cel I; and

(2) cloning of said sequences into an expression vector for expression of antibody heavy and light chains, respectively.

The detailed description of the identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody, in particular of step (a) (with its substeps) and step (b) described above, applies accordingly to step (1 ) of the method for generating an expression vector encoding the heavy and/or light chain of the cross-reactive monoclonal antibody.

A vector is usually a recombinant nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature. Accordingly, the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature). For example, the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector) and the like. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence. Thus, the vector may comprise a sequence corresponding, e.g., to (a heavy and/or light chain (variable region) of) the cross-reactive antibody. In particular, the vector is an expression vector. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. For example, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. A vector in the context of the present application may be a plasmid vector.

Expression vectors for (recombinant) expression of (human) monoclonal antibodies are known in the art commercially available. Such expression vectors may already comprise the sequences of the (human) constant regions (e.g., for IgG-type antibodies), such that only the VH/VL sequences need to be inserted by common cloning techniques well-known in the art. In some embodiments, the heavy chain and the light chain of the antibody are encoded by distinct expression vectors, such that the entire antibody is encoded by two expression vectors (one for the heavy chain and the other for the light chain). In this case, for expression of the antibody a host cell is transfected with both vectors. In some cases, the paired heavy chain and light chain can come from different sister B cell clones that exhibit the same phenotype. In other embodiments, the same vector may encode the heavy chain and the light chain of the antibody, e.g. in a multicistronic (bicistronic) manner.

While the B cell is identified according to the method of the present invention, the cloning of the antibody in an expression vector may be carried out, for example, as described in Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods. 2008, -329(1 -2): 112-124. doi:10.1016/j.jim.2007.09.01 7, which is incorporated herein by reference.

The present invention also provides a method for generating an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody, the method comprising the following steps:

(1 ) retrieval of a nucleic acid encoding (at least) VH and VL (of the BCR) of a cross- reactive B cell identified by the method for identification of a cross-reactive B-cell as described above; e.g. retrieval of DNA encoding (at least) VH and VL (of the BCR) by reverse transcription (RT-)PCR;

(2) optionally, amplification of the nucleic acid obtained in step (1 ); e.g. (nested) PCR reactions on the nucleic acid obtained in step (1 ); and

(3) cloning of the nucleic acid obtained in step (1 ) or step (2) into an expression vector for expression of antibody heavy and light chains, respectively.

As described above, to obtain VH/VL nucleic acids, preferably primers specific for the constant regions of the heavy and light chains (in particular IgG), such as primers specific to the constant regions of IgG, IgK, and IgA, respectively, may be used. Sequences of the constant regions of human antibodies are well-known in the art, such that the skilled person can readily obtain respective primers (which are also commercially available); e.g. in a reverse transcription (RT-)PCR. Thereby obtained cDNA may then be amplified, for example by PCR (such as two-step PCR, e.g. using two sets of Ig-specific primers, one nested within the other) and optionally purified. Then, the (purified) cDNA (or PCR amplicons) may be sequenced, e.g. by Sanger sequencing. Accordingly, it is preferred that the sequences of the heavy and light chain variable region (VH/VL) genes are obtained by reverse transcription PCR (RT-PCR) and sequencing. To facilitate high-throughput molecular cloning of BCR sequences into the VH/VK/VL expression vectors, the primers may contain complementary nucleotides (e.g., 10 - 50, preferably 20 - 40, more preferably 25 - 35, even more preferably about 30 complementary nucleotides) of the respective expression vectors for ligation (for example in order to be ligated by Gibson reaction using commercially available NEBuilderR HiFi DNA Assembly Master Mix (New England Biolabs)).

Accordingly, the present invention also provides an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody generated with the method as described above.

The expression vector may be used to transfect a host cell in order to express the cross-reactive monoclonal antibody. Accordingly, the present invention also provides a method for producing a recombinant cell expressing a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) generating an expression vector encoding the heavy and/or light chain of a cross- reactive monoclonal antibody according to the present invention as described above;

(P) transfecting a host cell with said expression vector; and

(y) optionally, culturing said host cell.

In other words, the present invention also provides a method for producing a recombinant cell expressing a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) generating an expression vector encoding the heavy and/or light chain of a cross- reactive monoclonal antibody comprising the following steps:

(1 ) identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody comprising the following steps:

(a) identification of a B cell (capable of) producing cross-reactive antibody, in particular an antibody that is cross-reactive to different targets of interest, comprising the following sub-steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures; (ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities;

(iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which are cross-reactive;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and

(vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross-reactive; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-cell; and

(2) cloning of said sequences into an expression vector for expression of antibody heavy and light chains, respectively;

(P) transfecting a host cell with said expression vector; and

(y) optionally, culturing said host cell.

The detailed description of the method for generating an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody, in particular of step (1 ) (with its substeps) and step (2) described above, applies accordingly to step (a) of the method for producing a recombinant cell expressing the cross-reactive monoclonal antibody.

Examples of suitable cells include, but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells. Other examples of such cells include, but are not limited to, prokaryotic cells, in particular bacterial cells, e.g. £ coli. In some embodiments, the cells are mammalian cells, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NSO cells, human liver cells, myeloma cells or hybridoma cells. In some embodiments, the host cell is a 293T cell.

The cell may be transfected with the vector (or the plurality of vectors). The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, e.g. into eukaryotic or prokaryotic cells. In the context of the present invention, the term "transfection" encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. In some embodiments, the introduction is non-viral.

Moreover, the cells of the present invention may be transfected stably or transiently with the vector, e.g. for expressing the cross-reactive antibody. In some embodiments, the cells are stably transfected with the vector (or the two vectors) encoding the cross-reactive antibody. In other embodiments, the cells are transiently transfected with the vector (or the two vectors) encoding the cross-reactive antibody. A stably transfected cell may be used to establish a cell line. A cell line is typically continuous (i.e., it can proliferate indefinitely), in particular due to tumor or artificial immortalization, e.g. Epstein-Barr virus (EBV)-immortalization.

The host cell (and, optionally, its progeny; e.g., a cell line) may be cultured, in particular for expression of the encoded antibody. Suitable culture conditions for culturing host cells are well-known in the art, e.g. using a commercially available culture medium. The detailed conditions may be selected depending on the cell type of the host cell, as known by the person skilled in the art.

In a further aspect, the present invention also provides a recombinant cell (expressing the cross-reactive monoclonal antibody) obtained with the method according to the present invention as described above. The recombinant cell usually heterologously expresses the cross-reactive antibody or an antigen-binding fragment thereof. In some embodiments, the cell type of the host cell does not express (such) antibodies in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the antibody that is not present in their native state. Such a PTM may result in a functional difference (e.g., reduced immunogenicity).

Production of cross-reactive antibodies and uses thereof

In a further aspect, the present invention also provides a method for producing a cross- reactive monoclonal antibody, the method comprising the following steps:

(A) producing a recombinant cell expressing the cross-reactive monoclonal antibody according to the present invention as described above; and

(B) isolating the cross-reactive monoclonal antibody.

In other words, the present invention also provides a method for producing a cross-reactive monoclonal antibody, the method comprising the following steps:

(A) producing a recombinant cell expressing the cross-reactive monoclonal antibody comprising the following steps:

(a) generating an expression vector encoding the heavy and/or light chain of a cross- reactive monoclonal antibody comprising the following steps:

(1 ) identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody comprising the following steps:

(a) identification of a B cell (capable of) producing cross-reactive antibody, in particular an antibody that is cross-reactive to different targets of interest, comprising the following sub-steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities; (iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which are cross-reactive;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and

(vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross- reactive; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-cell; and

(2) cloning of said sequences into an expression vector for expression of antibody heavy and light chains, respectively;

(P) transfecting a host cell with said expression vector; and

(y) optionally, culturing said host cell; and

(B) isolating the cross-reactive monoclonal antibody.

The detailed description of the method for producing a recombinant cell expressing a cross- reactive monoclonal antibody, in particular of step (a) (with its substeps), step (0) and step (y) described above, applies accordingly to step (A) of the method for producing the cross- reactive monoclonal antibody. In general, the identification of the VH/VL sequences, the cloning of nucleic acids in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.

In general, a host cell transfected with the expression vector (or a combination of expression vectors) encoding the cross-reactive antibody usually expresses said antibody, which can then be isolated from the supernatant of the host cell culture. Various methods are known in the art for isolation of an antibody from cell culture supernatant. Non-limiting examples include the use of protein A (a 42kDa protein with high affinity for the Fc region of IgG), of alternative IgG binding proteins (e.g. protein G, protein L), of synthetic protein A mimics, or of bioengineered peptides or synthetic ligands, all of which may be coupled to a support (e.g., for chromatography), in order to capture the desired antibody. Thereafter, the antibody may be separated and, optionally, further purification steps may be performed. The antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art. For isolation and/or purification of the cross-reactive antibody, tags introduced into the antibody (e.g. by means of the recombinant expression vector) may be used.

While the B cell is identified according to the method of the present invention, the subsequent steps of identification of antibody sequences and cloning and expression of the antibody may be carried out, for example, as described in Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods. 2008;329(1 -2):112-124. doi:10.1016/j.jim.2007.09.017, which is incorporated herein by reference.

After its (recombinant) expression, the antibody may be further characterized, e.g. by further functional assays in addition to those used in the primary and secondary screening step. For example, the assays performed in the primary and secondary screening step are preferably in vitro/ex vivo assays. For further characterization, further (different) in vitro/ex vivo assays may be performed, e.g. to assess further/different functionalities of the antibody. Moreover, in vivo studies (e.g., challenging studies) may be carried out to further characterize the antibody.

Moreover, for further characterization, the epitope, to which the cross-reactive antibody binds to (in the antigen) may be identified. As the method of the present invention is directed to the identification of cross-reactive antibodies, in particular of rare antibodies cross-reactive to multiple pathogens, such antibodies can be very useful for the identification of (highly) conserved epitopes, which are important to design broadly protecting vaccines (containing such epitopes).

Accordingly, the present invention also provides a method for designing an antigenic component for a vaccine comprising the following steps:

(I) identifying a cross-reactive antibody using the method according to the present invention as described above;

(II) identifying the epitope to which the cross-reactive antibody binds to;

(III) designing an antigenic component for a vaccine comprising the epitope to which the cross-reactive antibody binds to; and

(IV) optionally, producing said vaccine.

The detailed description of the above methods for identification of a B cell (capable of) producing a cross-reactive antibody and identification (and, optionally, production) of said cross-reactive antibody applies accordingly to step (I) of the method for designing an antigenic component for a vaccine.

Various methods for identification of an epitope (epitope mapping) in step (II) are known in the art. Non-limiting examples include a peptide scan (also referred to as "oligo-peptide scanning"), wherein usually the binding of the antibody to a number of short, overlapping peptides (e.g. of about 5 - 25 amino acids in length, preferably of about 10 - 20 amino acids in length, such as about 15 amino acids in length) covering the entire sequence (or a portion thereof) of the larger antigen is investigated. This method is particularly useful to identify linear (continuous) epitopes. Other non-limiting examples of methods for epitope mapping known in the art include SPR (surface plasmon resonance; e.g. as described in any one of Karlsson, R. (2013). Surface Plasmon Resonance in Binding Site, Kinetic, and Concentration Analyses. In Wild, D. (Ed.) The Immunoassay Handbook (Fourth Edition) (pp. 209-221 ). Doi: 10.1016/B978-0-08-097037-0.00015-4; Thomsen, L, Gurevich, L. (2018). A surface plasmon resonance assay for characterisation and epitope mapping of anti-GLP-1 antibodies. J Mol Recognit. 31 (8). Doi: 10.1002/jmr.271 14; Bhandari, D., Chen, F., Hamal, S., Bridgman, R. (2019). Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor. Antibodies, 8(1 ). Doi: 10.3390/antib8010022); ELISA; X-ray co-crystallography; cryogenic electron microscopy (cryo-EM); site-directed mutagenesis mapping; high-throughput shotgun mutagenesis epitope mapping; hydrogen-deuterium exchange (HDX); and cross-linking-coupled mass spectrometry.

After identification of the epitope, an antigenic component for a vaccine may be designed, which comprises said epitope. In general, the antigenic component is required in a vaccine to elicit a specific immune response (e.g., to elicit or enhance production of specific antibodies in a subject). In addition to said antigenic component, a vaccine may also comprise other components to elicit or enhance the immune response, which are usually not antigen-specific, such as adjuvants, as known in the art. The antigenic component is preferably a recombinant molecule, which differs from the (naturally occurring) antigen. In particular to trigger an immune response with cross-reactive antibodies, the antigenic component of the vaccine may be restricted to a fragment of the (naturally occurring) antigen, the fragment containing the identified epitope of the cross-reactive antibody (which may be a conserved sequence among different pathogens), but preferably as few as possible nonconserved sequences outside said epitope (to avoid triggering antibodies to non-conserved antigenic sequence stretches). In some embodiments, the antigenic component may consist of the epitope of the cross-reactive antibody (e.g., the epitope fragment of the naturally occurring antigen). In some embodiments, the antigenic component may be a recombinant peptide, polypeptide or protein containing the epitope of the cross-reactive antibody as well as other sequences (e.g. of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length), which do not occur in the (naturally occurring) antigen. In some embodiments, the antigenic component may be a molecule comprising (i) the epitope or a (recombinant) peptide, polypeptide or protein containing the epitope; and (ii) a distinct molecule (e.g. for support, immunogenic or targeting/transport purposes).

Using said antigenic component comprising the epitope, to which the cross-reactive antibody binds to, a vaccine may be produced, e.g. using an adjuvant and/or pharmaceutically acceptable carriers, diluents or vehicles known in the art. In a further aspect, the present invention also provides a cross-reactive monoclonal antibody obtained with the method according to the present invention as described above.

As outlined above, the cross-reactive antibody is preferably a human antibody, i.e. an antibody which comprises human VH/VL sequences as well as human constant region sequences. It is understood that a human antibody may carry one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations (mutated amino acids), in comparison to a corresponding human reference antibody occurring in nature. Such mutations may be introduced, for example, by site-directed mutagenesis known in the art. For example, one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations may be introduced into the Fc region of the antibody, e.g. to modify its half-life, complement and/or Fc receptor binding functionalities. In some instances, one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations may be introduced in the VH and/or VL sequences, e.g. to modify the antibody's binding to the antigen.

The cross-reactive antibody may be provided in a pharmaceutical composition. Accordingly, the present invention also provides a pharmaceutical composition comprising said cross- reactive antibody.

The pharmaceutical composition may optionally also contain a pharmaceutically acceptable carrier, diluent, excipient and/or vehicle. Although the carrier, diluent, vehicle or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as weting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.

A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present invention.

Pharmaceutical compositions may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis™ and Herceptin®, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

In some embodiments, the (only) active ingredient in the composition is the cross-reactive antibody. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract. A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions usually have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, for example about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen free. The composition may be isotonic with respect to humans. In some embodiments pharmaceutical compositions may be supplied in hermetically-sealed containers.

Pharmaceutical compositions typically include an "effective" amount of the cross-reactive antibody, i.e. an amount that is sufficient to treat, ameliorate, attenuate, reduce or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction or attenuation in pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend upon their size, weight, and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of a clinician.

In some embodiments, the composition may include cross-reactive antibodies, wherein the cross-reactive antibodies may make up at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) of the total protein in the composition. In the composition, the antibodies may be in purified form.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: (i) preparing the cross-reactive antibody as described above; and (ii) admixing the purified antibody with one or more pharmaceutically acceptable excipients, diluents or carriers.

In some embodiments, a method of preparing a pharmaceutical composition comprises the step of: admixing a cross-reactive antibody with one or more pharmaceutical ly-acceptable carriers, wherein the antibody is a monoclonal antibody that was obtained from a B cell identified with the method of the invention. As an alternative to delivering antibodies or B cells for therapeutic purposes, it is possible to deliver nucleic acid (typically DNA or RNA) that encodes the monoclonal antibody of interest derived from the B cell to a subject, such that the nucleic acid can be expressed in the subject in situto provide a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

Pharmaceutical compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80. Detergents are general ly present at low levels e.g., less than 0.01 %. Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity. For example, a concentration of 10±2mg/ml NaCI is typical.

Further, pharmaceutical compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material. The pH of a composition for lyophilization may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilization.

The compositions may also comprise one or more immunoregulatory agents. In some embodiments, one or more of the immunoregulatory agents include(s) an adjuvant.

The cross-reactive antibody or the pharmaceutical composition comprising said antibody may be used as a medicament. Accordingly, the present invention also provides a method for treating a subject in need thereof comprising administration of (an effective amount of) the cross-reactive antibody or the pharmaceutical composition comprising said antibody to the subject. Depending on the different functionalities of the antibody, the cross-reactive antibody or the pharmaceutical composition comprising said antibody may be used for the treatment of various diseases, such as infectious diseases, autoimmune disorders or cancers. Accordingly, the present invention also provides the use of the cross-reactive antibody or the pharmaceutical composition comprising said antibody for the manufacture of a medicament for the treatment of an infectious diseases, an autoimmune disorder or a cancer. For example, if the antigen targeted by the antibody is derived from a pathogen, the antibody may be used in the treatment of an infection with said pathogen. Exemplified pathogens are those described above. For example, if the antigen targeted by the antibody is a cancer or tumor antigen (such as a tumor-associated or tumor-specific antigen), the antibody may be used in the treatment of a cancer or tumor (which is preferably known or shown to express said antigen). For example, if the antigen targeted by the antibody is a self-antigen involved in an autoimmune disorder, the antibody may be used in the treatment of said autoimmune disorder. In summary, as well-known to the skilled person, the disease to be treated is usually selected according to the antibody's functionality, in particular the disease to be treated is usually related to the antigen targeted by the antibody. As used herein, "treatment" of a disease includes prophylactic as well as therapeutic treatment.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

Figure 1 shows schematically the method of the present invention for identification of a B cell (capable of) producing a cross-reactive antibody and an exemplary timeline with the experimental day. In step (i) (providing PBMCs in a plurality of cell cultures) PBMCs are provided in a plurality of cell cultures, e.g. using a plate with distinct wells for distinct cultures. PBMCs are cultured under conditions for selective expansion of B cells, e.g. for 5 - 7 days (step (ii): selective expansion of B cells among the PBMCs). Thereafter (e.g., on experimental day 5, 6 or 7), supernatants of the cultures are used in primary screening, i.e. in parallel tests for distinct functionalities (such as binding to or neutralization of distinct antigens) (step (iii): primary screening with multiple functional assays performed in parallel). Cross-comparison of the results of the different assays reveals cross-reactive cell cultures (i.e., cell cultures producing antibodies exhibiting the desired multiple functionalities, which were investigated in the different parallel tests) (step (iv): cross-comparison to identify cross-reactive cultures). In these cross-reactive cultures only, B cells are isolated and cultured separately to obtain monoclonal B cells (separate B cell clones) (step (v): isolating B cells from cross-reactive cultures and culturing B cells to obtain monoclonal B cells from heterogeneous pool of B cells), e.g. for about two days. Thereafter (e.g., on experimental day 7, 8 or 9), supernatants of the monoclonal B cell cultures are used in secondary screening, i.e. in parallel tests for distinct functionalities (such as binding to or neutralization of distinct antigens) (step (vi): secondary screening with multiple functional assays in parallel). The tests performed in the secondary screening may be essentially the same as in the primary screening. Cross-comparison of the results of the different assays reveals cross-reactive monoclonal B cells (i.e., monoclonal B cells producing antibodies exhibiting the desired multiple functionalities, which were investigated in the different parallel tests) (step (vii): cross-comparison to identify cross-reactive B cell clones). Thereby, specific B cell clones (capable of) producing a cross-reactive antibody are identified.

Figure 2 shows for Example 1 the results of primary screening of PBMCs against six different antigens (whole spike protein of human coronaviruses OC43, HKU1 , NL63 and 229E, as well as tetanus toxoid, influenza HA antigen of H1 N1 and PBS as negative control; as indicated in the figure).

Figure 3 shows for Example 1 that by cross comparing the OD of each individual well to different exemplary antigens, cultures with antibodies with multiple reactivities could be identified. Results are shown in for the spike protein of human coronavirus OC43 vs. (i) spike protein of human coronavirus HKU1 , (ii) spike protein of human coronavirus NL63, (iii) spike protein of human coronavirus 229E and (iv) tetanus toxin.

Figure 4 shows for Example 2 the number of sorted memory B cells positive for the different antigens as indicated, which were cultured in distinct conditions (complete medium alone; complete medium with IL-2/6/21 ; complete medium with CpG; complete medium with R848; complete medium with mesenchymal stromal cells (MSC); complete medium with CD40L-expressing MSC).

Figure 5 shows for Example 6 the ELISA binding profiles of recombinant antibodies towards a panel of distinct antigens as indicated. Binding data of various concentrations of the antibodies purified from EXPI293 cells transfected with VH and VL of CLM20_B8 (A) and CLM20_C9 (B) to the spike proteins of the different coronaviruses as indicated, and EC50 values calculated based on these curves are indicated in the table in ng/ml unit.

Figure 6 shows for Example 7 the results of the epitope mapping study, wherein the CLM20_B8 antibody of Example 3 was tested against 1 18 15-mer peptides (overlapping of 10 peptides) spanning the entire S2 protein, as illustrated in the schematic drawing of the spike protein. The coronavirus spike protein is schematically shown with signal sequence (SS), N-terminal domain (NTD), receptor-binding domain (RBD), subdomains 1 and 2 (SD1 and SD2), S2' protease cleavage site (S2'), fusion peptide (FP), heptad repeat 1 (HR1 ), central helix (CH), connector domain (CD), heptad repeat 2 (HR2), transmembrane domain (TM), and cytoplasmic tail (CT). The epitope of CLM20J38 corresponds to the FP (sequence KPSKRSFIEDLLFNK (SEQ ID NO: 1 )).

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1 : Identification of antigen-specific, cross-reactive B-cells

Total peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and plated at 60,000 cells per well in 96-well plate, in the presence of TLR agonist R848 and IL- 2. The complete media composition was as follow: RPMI-1640 supplemented with 10% fetal calf serum (Hyclone), 1 X Glutamax (Gibco), 1 X MEM Non-Essential Amino Acids Solution (Gibco), 1 mM Sodium Pyruvate, 100ug/ml Kanamycin, 50U/mL Pen-Strep, 30ug/ml Transferrin, 50uM 2-Mercaptoethanol, 1000U/mL IL-2 and 2.5ug/ml R848.

Seven days post culturing, supernatants of each well were screened against six different antigens, namely the whole spike protein of common human coronaviruses (namely OC43, HKU1 , NL63 and 229E), as well as tetanus toxoid, influenza hemagglutinin (HA) antigen of H1 N1 and PBS as negative control.

To this end, an ELISA (Enzyme-Linked immunosorbent Assay) was performed. Briefly, plates were coated with the different antigens (antigen(s)-of-interest) and later washed and blocked with Casein Blocker (Thermo Scientific). Subsequently, antibodies-containing supernatants were added to allow binding of antigen-specific antibodies (if any). The plates were washed, and alkaline-phosphatase-conjugated goat anti human IgG were added to bind to any IgG that remains bound to the antigen. Plates underwent a final wash, and substrate (p-NPP) was added and plates were read at 405 nm.

Results are shown in Fig. 2. The number of wells positive for each of the tested antigen is indicated at the top of the diagram. This primary screening allows quantification and comparison of the relative frequency of antigen-specific memory B cells to the respective antigens.

To identify cross-reactive cultures (wells) (i.e., cultures with antibodies with multiple reactivities (i.e., against distinct antigens)), the reactivity of the cultures to the different antigens (as obtained by ELISA) was then cross-compared for each individual well. Exemplary results are shown in Fig. 3 for spike protein of human coronavirus OC43 vs. (i) spike protein of human coronavirus HKU1 , (ii) spike protein of human coronavirus NL63, (iii) spike protein of human coronavirus 229E and (iv) tetanus toxin.

Example 2: Identification of culture conditions for rapid B cell culture and cloning

Next, the inventors sought to identify a way to isolate and clone the memory B cells from these multi-reactive cultures to validate their cross-reactivity. Once a culture of interest has been identified, lgG⁺ memory B cells were sorted by negative gating strategy as described in Pinto et al., 2013 (Pinto D et al. 2013 Blood VIA (20): 41 10-41 14), which is incorporated herein by reference.

Sorted memory B cells were then seeded at 0.7 cell per well in several conditions to identify the condition that was the most efficient and cost-effective to keep these memory B cell clones alive and sufficiently healthy to produce enough antibodies for secondary screening. To this end, sorted cells were seeded in 6 different conditions (culture media and supplements):

1 . Complete media only (composition as described in Example 1 above)

2. Complete media supplemented with 10ng/ml of IL-6 and 5ng/ml of IL-21

• Rationale: IL-6 and IL-21 have been reported to promote antibody production (T Hirano, T Taga, N Nakano, K Yasukawa, S Kashiwamura, K Shimizu, K Nakajima, K H Pyun, and T Kishimoto (1983) Purification to homogeneity and characterization of human B-cell differentiation factor (BCDF or BSFp-2). doi: 10.1073/pnas.82.16.5490; Ettinger R, Sims CP, Fairhurst AM, Robbins R, da Silva YS, Spolski R, Leonard WJ, Peter E Lipsky (2005) IL-21 Induces Differentiation of Human Naive and Memory B Cells into Antibody-Secreting Plasma Cells. DOI: 10.4049/jimmunol.175.12.7867).

3. Complete media supplemented with 2.5ug/ml of CpG

• Rationale: Memory B cells constitutively express high levels of TLR9, the receptor for CpG. Activation of TLR9 was reported to lead to proliferation of memory B cells and secretion of antibodies (NL Bernasconi, N Onai, A Lanzavecchia (2003) A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. DOI: 10.1 182/blood-2002-1 1 -3569).

4. Complete media supplement with 2.5ug/ml of R848

• Rationale: Memory B cells proliferation and antibody production is enhanced by R848 (D Pinna, D Corti, D Jarrossay, F Sallusto, A Lanzavecchia (2009) Clonal dissection of the human memory B cell repertoire following infection and vaccination, doi: 10.1002/eji.200839129).

5. Complete media with monolayer of mesenchymal stromal cells (MSCs)

• Rationale: MSCs promote plasma cell survival (Minges Wols HA, Underhill GH, Kansas GS, Witte PL. (2002) The Role of Bone Marrow-Derived Stromal Cells in the Maintenance of Plasma Cell Longevity, doi: 10.4049/jimmunol.169.8.4213).

6. Complete media with monolayer of CD40L-overexpressing MSCs

• Rationale: CD40L-expressing MSCs have been reported to enhance activation and proliferation of B cells by mimicking T cell help (XM Luo, E Maarschalk, RM O'Connell, P Wang, L Yang, D Baltimore (2009) Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes. DOI: 10.1 182/blood- 2008-09-177139).

Results are shown in Fig. 4. These data show that surprisingly any one of the conditions tested

- including complete medium alone (i.e., without any feeder cells, cytokines or TLR agonists)

- was found to support survival of the memory B cells. Culturing in the presence of mesenchymal stromal cells (MSCs) enhanced recovery of clones, however, merely cloning with complete medium alone was unexpectedly sufficient to (1 .) sustain survival of memory B cells and (2.) support antibody production. Example 3: Deconvolution of multi-reactive B cell clones

Next, it was investigated if an exemplary culture of the multi-reactive cultures of Example 1 contained a single cross-reactive B cell clonotype or multiple B cell clonotypes producing different antibodies targeting different antigens (e.g., spike protein of human coronaviruses OC43, HKU1 , NL63, 229E and SARS-CoV2, tetanus toxin). To this end, the inventors picked a parent culture (Culture E7) which, at primary screening exhibited triple-reactivity to the spike proteins of coronaviruses OC43, HKU1 and 229E, shown in Table 1. IgM and IgA memory B cells were sorted as described in Example 1 , and cloned at 0.7 cell per well in complete medium only as described in Example 2. Two days post cloning, the supernatants of each well were evaluated through secondary ELISA screening (essentially as described in Example 1 ). Results are shown in Table 2 below.

Table 1 : OD values of primary ELISAs screening performed with PBMC culture supernatants and the indicated antigens

Original Well OC43 HKU1 NL63 229E CoV2 Tetanus PBS Xreactivity

E7 3.124 2.484 0.098 3.149 0.092 0.185 0.092 3

Table 2: OD values of secondary ELISA screening performed with supernatants of single B cell clones and the indicated antigens

OC43 HKU1 229E

2.501 2.421 0.12

2.215 1.899 0.1

2.852 2.706 0.114

1.506 1.501 0.092

2.752 2.76 0.103

3.043 3.035 0.103

0.109 0.1 1 2.922

0.102 0.098 3.069

0.1 15 0.109 3.103

0.108 0.106 3.068 These data show that the triple reactivity identified in the first ELISA for this exemplary multi- reactive culture E7, is due to two distinct B cell clonotypes: one which produces cross reactive antibodies to spike proteins of coronaviruses OC43 and HKU1 and the other producing antibodies only specific to NL63 spike protein. These results highlight the importance of the secondary screening of B cell clones, as the inventors were able to show that the parent well which exhibited triple-reactivity during primary screening contained two separate B cel! clonotypes that yielded the reactivity pattern as described above.

Example 4: Identification of broadly reactive B cell clones

The method described in Examples 1 and 3 was used to identify and isolate broadly cross- reactive antibodies against coronaviruses from donors with previous SARS-CoV-2 infection. Briefly, total PBMCs were isolated from the donors, plated at 10,000 cells per well in 96-well plate, and cultured in the presence of TLR agonist R848 and IL-2 for five days. From the primary screening performed on day 5, the inventors identified three parent cultures which exhibited quintuple-reactivity to the spike protein of endemic beta-coronaviruses (OC43 and HKU1 ) and alpha-coronaviruses (NL63 and 229E) as well as pandemic strains of coronaviruses SARS-CoV and SARS-CoV-2, but not to irrelevant antigens such as HA or Tetanus toxoid. Table 3 below shows the results of the primary screening, namely, the OD values of the ELISAs performed with culture supernatants of the three parent cultures and the indicated antigens.

Table 3. OD values of ELISAs performed with PBMC culture supernatants and the indicated antigens

Xrea

SARS- SARS- Tetac-

Culture PBS OC43 HKU1 NL63 229E CoV CoV-2 HA nus tivity

CLM20_A7 0.131 3.334 3.338 3.322 3.337 3.336 1.629 0.139 0.187 6

CLM20 B8 0.117 3.198 3.177 3.188 3.185 3.145 2.007 0.146 0.149 6

CLM20_C9 0.134 2.885 2.808 2.694 2.57 2.806 3.004 0.173 0.165 6

To affirm that these parental cultures contain indeed broadly-reactive B cells that target all human coronaviruses, the inventors sorted memory B cells from all three cultures, as described in Example 3, and cloned at 0.7 cell per well in complete medium in the absence of feeder cells or other supplements, as described in Example 2. Two days post cloning, the supernatants of each well were evaluated through secondary ELISA screening, as described in Example 3. Results are shown in Table 4 below.

These data show that the selected three independent cultures indeed contained B cell clones that were broadly-reactive to all common coronaviruses. In addition, this strategy also allowed the inventors to obtain multiple daughter clones that exhibited the same reactivity, thereby facilitating subsequent retrieval of antibody gene sequences. Example 5: Retrieval of antibody gene sequences

Next, six clones of interest derived from three selected parent cultures of Example 4, cultures CLM20_A7, CLM20_B8, CLM20_C9 (clones of wells C7, E7, J8, M8, C21 and H15 of Table 4 above), were selected and subjected to RT-PCR to generate cDNA, using three primers specific to the constant regions of IgG, Ig , and Ig , respectively. Native IgG, Ig , and Ig sequences were subsequently amplified by two-step PGR using Q5 High-Fidelity DNA polymerase (NEB) and two sets of Ig-specific primers, one nested within the other. The PGR amplicons were later purified for Sanger sequencing.

Thereby, six paired VH/VL genes were retrieved and sequenced. The VHA/L sequences (VDJ sequences) of daughter clones from different wells derived from within the same parent cultures (i.e., clones C7 and E7 of culture CLM20_A7; clones J8 and M8 of CLM20_B8; and clones C21 and H15 of CLM20_C9 see table above) were identical. Therefore, hereafter the antibodies are referred to by their culture ID only.

Interestingly, antibodies CLM20_B8 and CLM20_A7 had very similar VHA/L sequences (with 7 nucleotide mutations resulting in 2 amino acid differences), suggesting that they were derived from the same progenitor in the donor, which had undergone somatic hypermutations; whereas two selected clones of CLM20_C9 (clones C21 and H15; see table 4 above) were of completely different lineage than CLM20_B8 and CLM20_A7. In general, all selected cultures (antibodies produced by those cultures) exhibit the same pan-reactive binding patterns to all coronavirus spike protein, as shown in Example 4.

Example 6: Validation of cross-reactivity of antibodies obtained from selected clones

To confirm cross-reactivity of antibodies obtained from the selected clones, the VDJ sequences of the antibodies CLM20_A7, CLM20_B8, CLM20_C9 (of examples 4 and 5), as well as five additional antibodies (CLM99_G12, CLM99_D10, CLM99_E3, CLM13_G9 and CLM20_Bis_E3) obtained from other donors essentially in a similar manner as described in Examples 4 and 5 (primary and secondary data not shown), were cloned into expression vectors for expression of a light chain (lambda) and of a human IgGI heavy chain, as described in Tiller T., Meffre E., Yurasov S., Tsuiji M, Nussenzweig M.C., and Wardemann H. (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, doi: 10.1016/j.jim.2007.09.017.

The IgG antibodies were produced by transient transfection of EXPI293 cells and the supernatants were tested against the same panel of antigens to validate their cross-reactivity. Briefly, EXPI293 cells were co-transfected with expression vectors carrying the VDJ sequences of the IgH and IgL using polyethylenimine (PEI), a stable cationic polymer as transfection reagent. After overnight incubation, the EXPI293 supernatant was tested in ELISA to validate the reactivity of secreted antibodies to various antigens. Results are shown in Table 5 below:

Table 5: Binding data for recombinantly produced antibodies, including antibodies CLM20_A7, CLM20_B8, CLM20_C9 (of examples 4 and 5) and CLM99_G12, CLM99_D10, CLM99_E3, CLM13_G9 and CLM20_Bis_E3 obtained in the same manner as described in Examples 4 and 5 _ _ _ _ _ _ _

SARS- SARS-

Antibody OC43 HKU1 NL63 229E CoV CoV-2 MERS HA

CLM20J38 4.457 4.461 4.519 4.495 4.408 4.451 4.444 0.131

CLM20_A7 4.391 4.361 4.44 4.34 4.394 4.308 4.447 0.133

CLM20_C9 4.359 4.306 4.39 4.305 4.359 4.318 4.372 0.128

CLM99_G12 4.246 4.215 0.1 15 0.107 4.103 3.428 4.198 0.1 13

CLM99_D10 4.253 4.199 0.125 0.103 4.23 4.231 4.213 0.102

CLM99JE3 4.179 4.144 0.092 0.087 4.1 4.1 13 4.1 18 0.079

CLM13_G9 0.084 0.086 0.084 0.082 4.175 4.142 0.082 0.08

CLM20_Bis_B3 3.616 4.151 0.104 0.095 4.153 2.281 0.223 0.095

Indeed, ectopic expression of VH/VL sequences of CLM20_B8, CLM20_A7 and CLM20_C9 results in production of pan-reactive antibodies to beta-coronavirus (OC43, HKU1, SARS- CoV1 , SARS-CoV2, MERS) and alpha-coronavirus (NL63, 229E), while no reactivity against unrelated antigen influenza HA was detected. Ectopic expression of VH/VL sequences of CLM99_G12, CLM99_D10, CLM99_E3, CLM99JG9, CLM13_G9 and CLM20_Bis_E3 results in production of broadly-reactive antibodies to beta-coronavirus (OC43, HKU1 , SARS-CoV1 , SARS-C0V2, MERS), while no reactivity against unrelated antigen influenza HA was detected. Next, antibodies CLM20J38 and CLM20_C9 were purified and different concentrations of the purified antibodies were tested in an ELISA for binding to spike proteins of the different coronaviruses OC43, HKU1 , NL63, 229E, SARS-CoV1 , SARS-CoV2 and MERS. Results are shown in Fig. 5. Accordingly, Fig. 5 shows the binding data of titrating concentrations of purified CLM20_B8 and CLM20„C9 antibodies against spike proteins of the different coronaviruses as indicated, as well as the EC50 values calculated based on these curves.

In summary, these data confirm that the antibodies identified with the method according to the present invention are broadly reactive human coronavirus antibodies displaying high affinity to the spike protein of various distinct human coronaviruses.

Example 7: Epitope identification

Purified antibodies were tested against a panel of 1 18 15-mer peptides (overlapping of 10) spanning the entire S2 protein (Spi ke676-Spi kel 273). Briefly, each well of the plate was coated with 8ug/ml of each of the 1 18 15-mer peptides. ELISA was performed as described above using 0.6ug/ml of purified antibody as primary antibody in each individual well.

Results are shown in Fig. 6. These data show that the antibodies were found to be specific for the peptide "KPSKRSFIEDLLFNK" (SEQ ID NO: 1 ; which comprises the epitope to which the antibodies bind to). As illustrated in Fig. 6, said peptide maps to the fusion peptide (FP) Spi ke81 1 -Spike825 (S2'). Accordingly, the data show that the epitope of the spike protein, to which the cross-reactive antibody binds to, is located in the fusion peptide of the spike protein.

Claims

CLAIMS A method for identification of a B cell capable of producing a cross-reactive antibody, the method comprising the following steps:

(i) providing peripheral blood mononuclear cells (PBMCs) in a plurality of cell cultures;

(ii) culturing the PBMCs under culture conditions for selective expansion of B cells;

(iii) performing a primary screening of the cell culture supernatants of the plurality of cell cultures for different functionalities;

(iv) cross-comparing the results obtained in step (iii) for the different functionalities and selecting one or more cell cultures, which are cross-reactive;

(v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;

(vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities; and

(vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross-reactive. The method according to claim 2, wherein the antibody that is cross-reactive to different targets of interest and wherein the different functionalities tested in the primary and secondary screening are binding to and/or neutralization of each of the different targets of interest. The method according to claim 2, wherein the different targets of interest are corresponding antigens of related pathogens. The method according to any one of the previous claims, wherein steps (i) - (vii) directly follow each other in said order. The method according to any one of the previous claims, wherein the PBMCs provided in step (i) are obtained from a blood sample of a human donor, of a single donor or of several donors. The method according to any one of the previous claims, wherein the PBMCs provided in step (i) are freshly isolated or cryopreserved PBMCs. The method according to any one of the previous claims, wherein the PBMCs provided in step (i) are plated at 10⁴ - 10⁶ cells per cell culture. The method according to any one of the previous claims, wherein the culture conditions for selective expansion of B cells in step (ii) include a TLR agonist and/or a cytokine. The method according to claim 8, wherein the TLR agonist is an agonist of TLR7, TLR8 and/or TLR9; preferably an agonist of TLR7 and/or TLR8. The method according to claim 8 or 9, wherein the cytokine is IL-2. The method according to any one of the previous claims, wherein the culturing of PBMCs under culture conditions for selective expansion of B cells in step (ii) is performed no longer than 9 days, preferably no longer than 8 days, more preferably no longer than 7 days, even more preferably no longer than 6 days and still more preferably no longer than 5 days; e.g. for 5 - 7 days. The method according to any one of the previous claims, wherein the primary screening (step (iii)) is performed directly on the cell cultures obtained in step (ii). The method according to any one of the previous claims, wherein no isolation or purification of B cells (from the PBMCs) is performed before the primary screening (step (iii)). The method according to any one of the previous claims, wherein primary screening in step (iii) comprises a binding assay to test binding to the different targets, such as an ELISA. The method according to any one of the previous claims, wherein cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of feeder cells. The method according to any one of the previous claims, wherein cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of cytokines. The method according to any one of the previous claims, wherein cloning of the isolated B cells in step (v) is performed under culture conditions in the absence of TLR agonists. The method according to any one of the previous claims, wherein cloning of the isolated B cells in step (v) is performed in complete medium only without further supplements. The method according to any one of the previous claims, wherein cloning of the isolated B cells in step (v) is performed for 1 - 3 days, preferably for about two days. The method according to any one of the previous claims, wherein secondary screening in step (vi) comprises a binding assay to test binding to the different targets, such as an ELISA. The method according to any one of the previous claims, wherein secondary screening in step (vi) comprises the same type of assay as primary screening in step (iii). The method according to any one of the previous claims, wherein the cross-reactive B cell identified in step (vii) is thereafter immortalized. The method according to any one of the previous claims, wherein the cross-reactive B cell identified in step (vii) is a human B cell. An isolated B cell obtained with the method according to any one of the previous claims. A method for identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) identification of a B cell capable of producing a cross-reactive antibody (in particular an antibody that is cross-reactive to different targets of interest) according to the method of any one of claims 1 to 23; and

(b) obtaining the sequences of the heavy and light chain variable region (VH/VL) genes of said cross-reactive B-cell. The method according to claim 25, wherein the sequences of the heavy and light chain variable region (VH/VL) genes are obtained by reverse transcription PCR (RT-PCR) and sequencing. A nucleic acid comprising the nucleic acid sequence of the heavy and/or light chain variable region (VH/VL) of the cross-reactive monoclonal antibody identified with a method according to claim 25 or 26. A method for generating an expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody, the method comprising the following steps:

(1 ) identification of nucleic acid sequences of the heavy and light chain variable region (VH/VL) of a cross-reactive monoclonal antibody according to the method of claim 25 or 26; and

(2) cloning of said sequences into an expression vector for expression of antibody heavy and light chains, respectively. An expression vector encoding the heavy and/or light chain of a cross-reactive monoclonal antibody generated with the method according to claim 28. A method for producing a recombinant cell expressing a cross-reactive monoclonal antibody, the method comprising the following steps:

(a) generating an expression vector encoding the heavy and/or light chain of a cross- reactive monoclonal antibody according to the method of claim 28;

(P) transfecting a host cell with said expression vector; and

(y) optionally, culturing said host cell. A recombinant cell expressing the cross-reactive monoclonal antibody obtained with the method according to claim 30. A method for producing a cross-reactive monoclonal antibody, the method comprising the following steps:

(A) producing a recombinant cell expressing the cross-reactive monoclonal antibody according to the method of claim 30; and

(B) isolating the cross-reactive monoclonal antibody. The method according to claim 32, wherein the antibody is a human antibody. The method according to claim 32 or 33, wherein the antibody is further characterized after its expression and isolation. A cross-reactive monoclonal antibody obtained with the method according to any one of claims 32 to 34. A pharmaceutical composition comprising the antibody according to claim 35. The antibody according to claim 35 or the pharmaceutical composition of claim 36 for use as a medicament. Use of the antibody according to claim 35 or the pharmaceutical composition of claim 36 for the manufacture of a medicament for the treatment of an infectious diseases, an autoimmune disorder or a cancer.

39. A method for treating a subject in need thereof comprising administration of the antibody according to claim 35 or the pharmaceutical composition of claim 36 to the subject.