WO2022162009A1 - Procédé d'identification rapide d'anticorps à réaction croisée et/ou rares - Google Patents

Procédé d'identification rapide d'anticorps à réaction croisée et/ou rares Download PDF

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WO2022162009A1
WO2022162009A1 PCT/EP2022/051763 EP2022051763W WO2022162009A1 WO 2022162009 A1 WO2022162009 A1 WO 2022162009A1 EP 2022051763 W EP2022051763 W EP 2022051763W WO 2022162009 A1 WO2022162009 A1 WO 2022162009A1
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Prior art keywords
cross
cells
antibody
cell
reactive
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PCT/EP2022/051763
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English (en)
Inventor
Federica SALLUSTO
Antonio Lanzavecchia
Antonino Cassotta
Jun Siong LOW
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Eth Zurich
Institute For Research In Biomedicine
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Priority claimed from PCT/EP2021/051744 external-priority patent/WO2022161597A1/fr
Application filed by Eth Zurich, Institute For Research In Biomedicine filed Critical Eth Zurich
Publication of WO2022162009A1 publication Critical patent/WO2022162009A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • 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.
  • 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).
  • 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.
  • 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.
  • WO 2010/01 1337 Al describes 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.
  • 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.
  • PBMCs peripheral blood mononuclear cell
  • 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%.
  • 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".
  • a scFv may comprise the CDRs of an antibody as described herein.
  • 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.
  • human antibody 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.
  • 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.
  • Antibodies may be immunogenic in human and/or in non-human (or heterologous) hosts e.g., in mice.
  • 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.
  • the term "light chain” refers to a polypeptide which is to be associated with another polypeptide (the "heavy chain”).
  • 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).
  • 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.
  • the three CDRs are arranged non-consecutively in the variable domain.
  • 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.
  • FR framework region
  • variable domain of an antibody 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.
  • the three CDRs of a heavy chain and the three CDRs of the connected light chain form together the antigen binding site (paratope).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antigen may be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g.
  • 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.
  • antigen binding site 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".
  • epipe 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.
  • 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.
  • 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).
  • the terms “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • 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.
  • 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.
  • 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.
  • step (v) isolating and cloning B cells from the (cross-reactive) cultures selected in step (iv) to obtain monoclonal B cells;
  • 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.
  • step (v) instead of a heterogeneous PBMC culture as in step (iii).
  • 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.
  • B cell clones i.e., a monoclonal B cell population
  • desired functionalities i.e., the functionalities tested in the primary and secondary screening
  • step (vi) 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)
  • step (vi) directly follows step (v)
  • B lymphocyte and "B cell” are used herein interchangeably.
  • 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.
  • B lymphocytes can present antigens and secrete cytokines.
  • 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.
  • the B cell may be of any species.
  • the B cell is a mammalian B cell.
  • 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.
  • cross-reactive 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).
  • 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).
  • PBMCs peripheral blood mononuclear cells
  • step (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;
  • step (v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;
  • step (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.
  • 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 different targets may be different antigens or even different epitopes of the same antigen.
  • the different targets are related to each other, i.e. not unrelated, arbitrarily selected targets.
  • the different targets are preferably related antigens, such as antigens of related pathogens (e.g., related viruses, bacteria etc.).
  • 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 m
  • 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.
  • 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).
  • 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.).
  • PBMCs are provided in a plurality of cell cultures.
  • a peripheral blood mononuclear cell is any peripheral blood cell having a round nucleus.
  • PBMCs refers to a heterogeneous population of peripheral blood mononuclear cells, which includes at least antigen-producing B cells.
  • other PBMCs such as T cells, NK cells and monocytes may be present.
  • 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.
  • 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.
  • 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.
  • PBMCs of different donors e.g. obtained from different blood sample
  • PBMCs obtained from a single donor e.g., from a single blood sample
  • 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.
  • the donor of the PBMCs is a human donor (and, thus, the PBMCs are human PBMCs).
  • 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.
  • the donor 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.
  • 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)).
  • conditions for selective expansion of B cells 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.
  • 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.
  • 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.
  • TLR9 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.
  • 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.
  • the PBMCs are cultured under conditions essentially as described in Pinna D, et al. (Eur J of Immunology 2009, 39:1260).
  • 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.
  • 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, GardiquimodTM, imiquimod, TL8-506, loxoribine, single stranded (ss)RNA, CpG, LPS and combinations thereof.
  • the TLR agonist is selected from the group consisting of R848 (resiquimod), 3M001 , 3M002, CL075, CL097, CL264, CL307, GardiquimodTM, 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, GardiquimodTM, imiquimod, TL8-506, loxoribine, single stranded (ss)RNA, and combinations thereof.
  • the PBMCs may be co- cultured with a CD40L expressing cell line (e.g. K562L or 3T3 cells).
  • a CD40L expressing cell line e.g. K562L or 3T3 cells.
  • 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.
  • 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.
  • 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.
  • TLR agonist R848 is combined with IL-2.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • culturing of the PBMCs under culture conditions for selective expansion of B cells 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
  • 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.
  • 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.
  • 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).
  • 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).
  • step (iii) is performed on the PBMC cell cultures (with expanded B cells) obtained in step (ii).
  • 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.
  • 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.
  • step (iii) 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).
  • 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.
  • 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).
  • essentially the same pipetting scheme may be used as for the cell cultures of step (i) also for multi-well plates of step (iii).
  • (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 "A”
  • cross-reactive cultures are selected in step (iv).
  • 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.
  • 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).
  • the first and second tests (and any further test) may be performed in parallel as described above.
  • 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.
  • the different “functionalities” preferably relate to different binding or neutralization characteristics of the cross-reactive antibody.
  • a cross-reactive antibody may be cross-reactive to different targets (e.g., bind to and/or neutralize different targets).
  • 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.
  • 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.
  • 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.
  • potential target antigens e.g., coronavirus spike proteins of different coronaviruses
  • 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.
  • pathogen e.g., viral
  • inhibittion 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).
  • pathogen viral
  • a human target e.g., as required for pathogen/viral infection of a human cell.
  • binding of coronavirus e.g., SARS-CoV-2
  • the ability of an antibody to reduce or inhibit such binding may also be of interest.
  • 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.
  • the primary screening in step (iii) comprises a binding assay to test binding to the different targets.
  • binding to a target of interest 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.
  • step (iii) involves an ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • targets e.g. (corresponding) antigens derived from different pathogens, as described above
  • different ELISAs e.g., one per target
  • step (iii) 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.
  • the target e.g. protein/complex/particle
  • 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.
  • pathogen e.g., viral
  • the pathogens e.g. viruses
  • the pathogens are typically propagated in cells and/or cell lines.
  • 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.
  • pathogen e.g. virus
  • flow cytometry may be used.
  • B cells from the cross-reactive cultures selected in step (iv) are isolated and cloned in order to obtain monoclonal B cells.
  • 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.
  • 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
  • step (v) 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.
  • 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.
  • step (v) may be performed at the same day as steps (ii) and (iv) or at the day following step (iii) and/or step
  • B cells may be isolated by flow cytometry, magnetic cell isolation and cell separation (MACS), RosetteSep, or antibody panning.
  • MCS magnetic cell isolation and cell separation
  • One or more isolation techniques may be utilized in order to provide isolated B cells with sufficient purity, viability, and yield.
  • B cells may be isolated by magnetic cell sorting.
  • anti-CD19 microbeads may be used.
  • FACS fluorescent activated cell sorting
  • LCM lasercapture microdissection
  • microengraving and droplet microfluidics.
  • IgG secreting memory B cells may be isolated by a negative gating strategy as CD19 + IgM and IgA or as CD19 + CD27 f/ IgM and lgA ⁇ .
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Cloning of the isolated B cells in step (v) may be performed in complete medium.
  • 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).
  • feeder cells such as mesenchymal stromal cells (MSC) or other feeder cells, are commonly used, e.g. for B cell activation.
  • MSC mesenchymal stromal cells
  • 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.
  • 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.
  • cytokines e.g., cytokines as described above.
  • cytokines e.g., cytokines as described above.
  • Common cytokines used in this context include those as described above, in particular IL-2, IL-6 and IL-21.
  • 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).
  • TLR agonists e.g., TLR agonists as described above.
  • 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.
  • 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.
  • step (v) 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.
  • cloning of the isolated B cells in step (v) is performed in complete medium only without further supplements.
  • 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.
  • 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.
  • the antibodies produced by single B cell clones can be screend after only two days.
  • 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).
  • 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.
  • 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).
  • 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.
  • the different functionalities tested in the primary screening are also tested in the secondary screening.
  • the secondary screening may comprise the same type of assays or a different type of assays as the primary screening,
  • the same functionality, e.g. binding to a target may be tested with the same or different assays in the primary and secondary screening.
  • secondary screening in step (vi) comprises the same type of assay (and the same set of targets/antigens) as primary screening in step (iii).
  • 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.
  • 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)).
  • the cross-reactive B cell identified in step (vii) is thereafter immortalized.
  • Methods for immortalizing B cells are well-known in the art.
  • 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.
  • 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.
  • VH/VL variable regions
  • BCR B cell receptor
  • the present invention also provides an isolated B cell obtained with the method according to the present invention as described above.
  • 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.
  • the B cell is a human B cell.
  • the cross-reactive antibody produced by said B cell is preferably a human antibody.
  • 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:
  • 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.
  • 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.
  • the host cell is a 293T cell.
  • the cell may be transfected with the vector (or the plurality of vectors).
  • 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.
  • RNA e.g. mRNA
  • 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.
  • 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.
  • the cell type of the host cell does not express (such) antibodies in nature.
  • 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).
  • PTM post-translational modification
  • the present invention also provides a method for producing a cross-reactive monoclonal antibody, the method comprising the following steps:
  • PBMCs peripheral blood mononuclear cells
  • step (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;
  • step (v) isolating and cloning B cells from cross-reactive cultures selected in step (iv) to obtain monoclonal B cells;
  • step (vi) performing a secondary screening of the supernatants of the B cell clones obtained in step (v) for different functionalities;
  • step (vii) cross-comparing the results obtained in step (vi) for the different functionalities and selecting a B cell clone, which is cross- reactive;
  • step (A) of the method for producing the cross- reactive monoclonal antibody applies accordingly to step (A) of the method for producing the cross- reactive monoclonal antibody.
  • 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.
  • 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.
  • protein A a 42kDa protein with high affinity for the Fc region of IgG
  • alternative IgG binding proteins e.g. protein G, protein L
  • synthetic protein A mimics e.g. protein A mimics
  • bioengineered peptides or synthetic ligands all of which may be
  • 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.
  • the antibody may be further characterized, e.g. by further functional assays in addition to those used in the primary and secondary screening step.
  • the assays performed in the primary and secondary screening step are preferably in vitro/ex vivo assays.
  • further (different) in vitro/ex vivo assays may be performed, e.g. to assess further/different functionalities of the antibody.
  • in vivo studies e.g., challenging studies may be carried out to further characterize the antibody.
  • the epitope, to which the cross-reactive antibody binds to (in the antigen) may be identified.
  • 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).
  • step (I) of the method for designing an antigenic component for a vaccine applies accordingly to step (I) of the method for designing an antigenic component for a vaccine.
  • peptide scan also referred to as “oligo-peptide scanning”
  • oligo-peptide scanning 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.
  • SPR surface plasmon resonance
  • an antigenic component for a vaccine may be designed, which comprises said epitope.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • a pharmaceutically acceptable carrier diluent, excipient and/or vehicle.
  • 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.
  • 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.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulphates
  • 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.
  • compositions may be prepared in various forms.
  • 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 SynagisTM 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 (only) active ingredient in the composition is the cross-reactive antibody.
  • the composition may be susceptible to degradation in 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.
  • 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.
  • pharmaceutical compositions may be supplied in hermetically-sealed containers.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid typically DNA or RNA
  • Suitable gene therapy and nucleic acid delivery vectors are known in the art.
  • 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.
  • a concentration of 10 ⁇ 2mg/ml NaCI is typical.
  • 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.
  • a sugar alcohol e.g., mannitol
  • a disaccharide e.g., sucrose or trehalose
  • 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.
  • compositions may also comprise one or more immunoregulatory agents.
  • 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.
  • the antibody may be used in the treatment of an infection with said pathogen.
  • pathogens are those described above.
  • 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).
  • 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.
  • 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.
  • treatment of a disease includes prophylactic as well as therapeutic treatment.
  • FIG. 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.
  • 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.
  • 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)
  • 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 )).
  • PBMCs peripheral blood mononuclear cells
  • an ELISA Enzyme-Linked immunosorbent Assay
  • 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.
  • substrate p-NPP
  • 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.
  • Example 2 Identification of culture conditions for rapid B cell culture and cloning
  • 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):
  • 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).
  • Example 1 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).
  • different antigens e.g., spike protein of human coronaviruses OC43, HKU1 , NL63, 229E and SARS-CoV2, tetanus toxin.
  • 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
  • Table 2 OD values of secondary ELISA screening performed with supernatants of single B cell clones and the indicated antigens

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Abstract

La présente invention concerne un procédé d'identification rapide d'une cellule B produisant un anticorps à réaction croisée. Ainsi, les séquences des régions variables de l'anticorps à réaction croisée peuvent être identifiées et clonées en un vecteur d'expression pour l'expression dudit anticorps à réaction croisée. L'anticorps à réaction croisée peut être utilisé dans le traitement de maladies. De plus, son épitope peut être identifié, par exemple pour concevoir des vaccins provoquant une réponse immunitaire comprenant des anticorps à réaction croisée.
PCT/EP2022/051763 2021-01-26 2022-01-26 Procédé d'identification rapide d'anticorps à réaction croisée et/ou rares WO2022162009A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004076677A2 (fr) 2003-02-26 2004-09-10 Institute For Research In Biomedicine Production d'anticorps monoclonaux par transformation de lymphocytes b par le virus d'epstein barr
WO2010011337A1 (fr) 2008-07-25 2010-01-28 Theraclone Sciences Procédés et compositions pour la découverte d’anticorps spécifiques d’une cible à l’aide de puces à anticorps (ara)
WO2010046775A2 (fr) 2008-10-22 2010-04-29 Institute For Research In Biomedicine Procédés pour produire des anticorps à partir de plasmocytes
WO2010107939A2 (fr) 2009-03-17 2010-09-23 Theraclone Sciences, Inc. Anticorps neutralisants anti-virus de l'immunodéficience humaine (vih)
WO2012082073A1 (fr) 2010-12-14 2012-06-21 National University Of Singapore Anticorps monoclonal humain ayant une spécificité pour la protéine e du virus de la dengue de sérotype 1 et utilisations de celui-ci

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004076677A2 (fr) 2003-02-26 2004-09-10 Institute For Research In Biomedicine Production d'anticorps monoclonaux par transformation de lymphocytes b par le virus d'epstein barr
WO2010011337A1 (fr) 2008-07-25 2010-01-28 Theraclone Sciences Procédés et compositions pour la découverte d’anticorps spécifiques d’une cible à l’aide de puces à anticorps (ara)
WO2010046775A2 (fr) 2008-10-22 2010-04-29 Institute For Research In Biomedicine Procédés pour produire des anticorps à partir de plasmocytes
WO2010107939A2 (fr) 2009-03-17 2010-09-23 Theraclone Sciences, Inc. Anticorps neutralisants anti-virus de l'immunodéficience humaine (vih)
WO2012082073A1 (fr) 2010-12-14 2012-06-21 National University Of Singapore Anticorps monoclonal humain ayant une spécificité pour la protéine e du virus de la dengue de sérotype 1 et utilisations de celui-ci

Non-Patent Citations (50)

* Cited by examiner, † Cited by third party
Title
AMANNA, I. J.SLIFKA, M. K.: "Quantitation of rare memory B cell populations by two independent and complementary approaches.", J. IMMUNOL. METHODS, vol. 317, 2006, pages 175 - 185, XP028017625, DOI: 10.1016/j.jim.2006.09.005
BERNASCONI, N. L.ONAI, N.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, vol. 101, 2003, pages 4500 - 4504
BHANDARI, D.CHEN, F.HAMAL, S.BRIDGMAN, R.: "Kinetic Analysis and Epitope Mapping of Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface Plasmon Resonance Biosensor", ANTIBODIES, vol. 8, no. 1, 2019
BOERNER, P. ET AL., I /MMUNOL, vol. 147, 1991, pages 86 - 95
BRUGGEMANN, M. ET AL., YEAR IMMUNOL., vol. 7, 1993, pages 3340
CHAMES PVAN REGENMORTEL MWEISS EBATY D: "Therapeutic antibodies: successes, limitations and hopes for the future", BRITISH JOURNAL OF PHARMACOLOGY, vol. 157, no. 2, 2009, pages 220 - 233, XP002714017, DOI: 10.1111/j.1476-5381.2009.00190.x
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN R. LISS, pages: 77
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, no. 20, 2000, pages E85, XP002533174, DOI: 10.1093/nar/28.20.e85
CORTI ET AL., SCIENCE, vol. 333, 2011, pages 850
CROTTY, S.AUBERT, R. D.GLIDEWELL, J.AHMED, R.: "Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system", J. IMMUNOL. METHODS, vol. 286, 2004, pages 111 - 122, XP004503451, DOI: 10.1016/j.jim.2003.12.015
D PINNAD CORTID JARROSSAYF SALLUSTOA LANZAVECCHIA, CLONAL DISSECTION OF THE HUMAN MEMORY B CELL REPERTOIRE FOLLOWING INFECTION AND VACCINATION, 2009
DEBORA PINNA ET AL: "Clonal dissection of the human memory B-cell repertoire following infection and vaccination", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 39, no. 5, 29 April 2009 (2009-04-29), pages 1260 - 1270, XP055250859, ISSN: 0014-2980, DOI: 10.1002/eji.200839129 *
DUVALL MRFIORINI RN: "Different approaches for obtaining antibodies from human B cells", CURR DRUG DISCOV TECHNOL, vol. 11, no. 1, March 2014 (2014-03-01), pages 41 - 7, XP009190830
ETTINGER RSIMS GPFAIRHURST AMROBBINS RDA SILVA YSSPOLSKI RLEONARD WJPETER E LIPSKY, IL-21 INDUCES DIFFERENTIATION OF HUMAN NAIVE AND MEMORY B CELLS INTO ANTIBODY-SECRETING PLASMA CELLS, 2005
GENNARO: "Remington: The Science and Practice of Pharmacy", 2000
HEARTY SLEONARD PO'KENNEDY R: "Measuring antibody-antigen binding kinetics using surface plasmon resonance", METHODS MOL BIOL, vol. 907, 2012, pages 411 - 42
HOOGENBOOM, H. R.WINTER, G., J. MOL. BIOL., vol. 227, 1992, pages 381 - 388
JAKOBOVITS, A. ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS, A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 2551 - 2555
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
KARLSSON, R: "The Immunoassay Handbook", 2013, article "Surface Plasmon Resonance in Binding Site, Kinetic, and Concentration Analyses", pages: 209 - 221
KOHLER GMILSTEIN C: "ontinuous cultures of fused cells secreting antibody of predefined specificity", NATURE, vol. 256, no. 5517, 7 August 1975 (1975-08-07), pages 495 - 7
LANZAVECCHIA ABERNASCONI NTRAGGIAI ERUPRECHT CRCORTI DSALLUSTO F: "Understanding and making use of human memory B cells", IMMUNOL REV, vol. 211, June 2006 (2006-06-01), pages 303 - 9
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., vol. 13, 1983, pages 820 - 824
LANZAVECCHIA, A.PARODI, B.CELADA, F.: "Activation of human B lymphocytes: frequency of antigen-specific B cells triggered by alloreactive or by antigen-specific T cell clones", EUR. J. IMMUNOL., vol. 13, 1983, pages 733 - 738
LONBERG N: "Therapeutic Antibodies. Handbook of Experimental Pharmacology", vol. 181, 2008, SPRINGER-VERLAG, article "Human monoclonal antibodies from transgenic mice", pages: 69 - 97
MARKS, J. D. ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MCCAFFERTY JGRIFFITHS ADWINTER GCHISWELL DJ: "Phage antibodies: filamentous phage displaying antibody variable domains", NATURE, vol. 348, no. 6301, 6 December 1990 (1990-12-06), pages 552 - 4, XP002196759, DOI: 10.1038/348552a0
MEYER ET AL.: "A simplified workflow for monoclonal antibody sequencing", PLOS ONE, vol. 14, no. 6, 2019, pages e0218717, XP055751203, Retrieved from the Internet <URL:https://doi.org/10.1371/journal.pone.0218717> DOI: 10.1371/journal.pone.0218717
MEYER LLOPEZ TESPINOSA RARIAS CFVOLLMERS CDUBOIS RM: "A simplified workflow for monoclonal antibody sequencing", PLOS ONE, vol. 14, no. 6, 24 June 2019 (2019-06-24), pages e0218717, XP055751203, DOI: 10.1371/journal.pone.0218717
MINGES WOLS HAUNDERHILL GHKANSAS GSWITTE PL, THE ROLE OF BONE MARROW-DERIVED STROMAL CELLS IN THE MAINTENANCE OF PLASMA CELL LONGEVITY, 2002
NEUBERGER MSWILLIAMS GTMITCHELL EBJOUHAL SSFLANAGAN JGRABBITTS TH: "A hapten-specific chimaeric IgE antibody with human physiological effector function", NATURE, vol. 314, no. 6008, 21 March 1985 (1985-03-21), pages 268 - 70, XP001084105, DOI: 10.1038/314268a0
NL BERNASCONIN ONAIA LANZAVECCHIA, A ROLE FOR TOLL-LIKE RECEPTORS IN ACQUIRED IMMUNITY: UP-REGULATION OF TLR9 BY, 2003
PINNA D ET AL., EUR J OF IMMUNOLOGY, vol. 39, 2009, pages 1260
PINTO D ET AL., BLOOCL, vol. 21, no. 20, 2013, pages 4110 - 4114
PINTO D ET AL., BLOOD, vol. 121, no. 20, 2013, pages 4110 - 4114
SCHANZ ET AL.: "High-Throughput Sequencing of Human Immunoglobulin Variable Regions with Subtype Identification", PLOS ONE, vol. 9, 2014, pages e111726
SCHEID JF ET AL., J IMMUNOL METHODS, vol. 343, 2009, pages 65
T HIRANOT TAGAN NAKANOK YASUKAWAS KASHIWAMURAK SHIMIZUK NAKAJIMAK H PYUNT KISHIMOTO, PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF HUMAN B-CELL DIFFERENTIATION FACTOR (BCDF OR BSFP-2, 1983
THOMSEN, L.GUREVICH, L.: "A surface plasmon resonance assay for characterisation and epitope mapping of anti-GLP-1 antibodies", J MOL RECOGNIT, vol. 31, no. 8, 2018
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, vol. 334, no. 1-2, 20 May 2008 (2008-05-20), pages 142, XP022614842, DOI: 10.1016/j.jim.2008.02.010
TILLER T ET AL.: "Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning", J IMMUNOL METHODS, vol. 329, no. 1-2, 2008, pages 112 - 124, XP022389335
TILLER T.MEFFRE E.YURASOV S.TSUIJI MNUSSENZWEIG M.C.WARDEMANN H., EFFICIENT GENERATION OF MONOCLONAL ANTIBODIES FROM SINGLE HUMAN B CELLS BY SINGLE CELL RT-PCR AND EXPRESSION VECTOR CLONING, 2008
TRAGGIAI EBECKER SSUBBARAO KKOLESNIKOVA LUEMATSU YGISMONDO MRMURPHY BRRAPPUOLI RLANZAVECCHIA A: "An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus", NAT MED, vol. 10, no. 8, 11 July 2004 (2004-07-11), pages 871 - 5, XP037065944, DOI: 10.1038/nm1080
TRAGGIAI EBECKER SSUBBARAO KKOLESNIKOVA LUEMATSU YGISMONDO MRMURPHY BRRAPPUOLI RLANZAVECCHIA A: "An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus", NAT MED, vol. 10, no. 8, 2004, pages 871 - 5, XP037065944, DOI: 10.1038/nm1080
VAN DIJK, M. A.VAN DE WINKEL, J. G., CURR. OPIN. CHEM. BIOL., vol. 5, 2001, pages 368 - 374
WANG CHUNYAN ET AL: "Abstract", BIORXIV, 20 October 2020 (2020-10-20), XP055847620, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.10.20.346916v1.full.pdf> [retrieved on 20211005], DOI: 10.1101/2020.10.20.346916 *
WEN, L.HANVANICH, M.WERNER-FAVRE, C.BROUWERS, N.PERRIN, L. H.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., vol. 17, 1987, pages 887 - 892
WRAMMERT J ET AL., NATURE, vol. 453, 2008, pages 667
XM LUOE MAARSCHALKRM O'CONNELLP WANGL YANGD BALTIMORE, ENGINEERING HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS TO PRODUCE A BROADLY NEUTRALIZING ANTI-HIV ANTIBODY AFTER IN VITRO MATURATION TO HUMAN B LYMPHOCYTES, 2009

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