JP2020162622A - Ultra-potent neutralization of cytokines by multispecific antibodies and uses thereof - Google Patents

Abstract

To provide multispecific antibodies or antigen binding fragments thereof that potently neutralize cytokines and thus may be useful in prevention and treatment of inflammatory and autoimmune diseases.SOLUTION: Disclosed is a multispecific antibody or antigen binding fragment thereof comprising at least two different domains that specifically bind to at least two different, non-overlapping sites of a cytokine. Also disclosed is a nucleic acid encoding such an antibody and antibody fragment, immortalized B cells and cultured plasma cells producing such an antibody and antibody fragment. Furthermore, disclosed is the use of the antibodies and antibody fragments of the invention in screening methods as well as in the diagnosis, prophylaxis and treatment of inflammatory and/or autoimmune diseases.SELECTED DRAWING: None

Description

Detailed description of the invention

The present invention relates to multispecific anti-cytokine (preferably anti-GM-CSF) antibodies that strongly neutralize cytokines (particularly GM-CSF) and antigen-binding fragments thereof. Multispecific anti-cytokine (preferably anti-GM-CSF) antibodies that strongly neutralize the cytokines of the invention (particularly GM-CSF) are provided against at least two different non-overlapping sites on a single cytokine molecule. It contains at least two domains that specifically bind to each other. The present invention also relates to nucleic acids encoding such antibodies and antibody fragments, as well as immortalized B cells and cultured plasma cells that produce such antibodies and antibody fragments. Furthermore, the present invention relates to screening methods and the use of the antibodies and antibody fragments of the present invention in the diagnosis, prevention and treatment of diseases (particularly inflammatory diseases and autoimmune diseases).

Cytokines are a family of immunomodulatory molecules secreted by various cells that act locally or systemically on other cells. Most cytokines have immunomodulatory activity involved in the regulation of various types of inflammatory processes and host defense mechanisms. Cytokines include chemokines, interferons, interleukins, phosphokines, tumor necrosis factor, monokines and colony stimulating factors. Cytokines are produced by a wide range of cells, including immune cells (such as macrophages, B lymphocytes, T lymphocytes and mast cells) as well as endothelial cells, fibroblasts, and various stromal cells; specific Cytokines can be produced by one or more cells.

Cytokine Granulocyte-macrophage-colony stimulator (GM-CSF) is a 127 amino acid monomeric protein having a molecular weight of 14 kDa to 35 kDa depending on the degree of glycosylation that can be changed. Non-glycosylated GM-CSF and glycosylated GM-CSF show similar activity in vitro (Cebon, J., Nicola, N., Ward, M., Gardner, I., Dempsey, P., Layton, J. ., Duhrsen, U., Burgess, AW, Nice, E., and Morstyn, G. (1990). Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity. J. Biol. Chem . 265, 4483-4491). GM-CSF exerts its bioactivity by binding to its receptor, which is expressed on the cell surface of myeloid and endothelial cells (but not on lymphocytes) (Hansen, G., Hercus). , TR, McClure, BJ, Stomski, FC, Dottore, M., Powell, J., Ramshaw, H., Woodcock, JM, Xu, Y., Guthridge, M., et al. (2008). The structure of The GM-CSF receptor complex revealsa distinct mode of cytokine receptor activation. Cell 134, 496-507). The receptor is a heterologous dimer and is composed of alpha and beta subunits. The beta subunit is also part of the interleukin-3 receptor complex and the interleukin-5 receptor complex, which associates with the GM-CSF receptor alpha subunit and GM-CSF to form a multimeric complex. It leads to the formation of (GM-CSF is bound with picomolar binding affinity).

GM-CSF can be expressed by a variety of cell types, including T lymphocytes, macrophages, NK cells, mast cells, endothelial cells, fibroblasts and some malignant cells (Gasson, JC (1991). Molecular physiology of granulocyte-macrophage colony-stimulating factor. Blood 77, 1131-1145; Hamilton, JA, and Anderson, GP (2004). GM-CSF Biology. Growth Factors 22, 225-231; Sergeeva, A., Ono, Y., Rios, R., and Molldrem, JJ (2008). High titer autoantibodies to GM-CSF in patients with AML, CML and MDS are associated with active disease. Leukemia 22, 783-790). GM-CSF acts as a growth factor on hematopoietic progenitor cells to give rise to granulocytes and monocytes. GM-CSF is also essential for microglial development and alveolar macrophage function. In addition, GM-CSF also has an pro-inflammatory effect on cells of the immune system expressing the GM-CSF receptor. The most important of these functions are monocyte, macrophage and granulocyte activation (production of other cytokines and chemokines, matrix-degrading proteases, enhanced HLA expression, and adherent molecules in various inflammatory and autoimmune diseases. Or, resulting in increased expression of CC chemokine receptors) (Hamilton, JA (2002). GM-CSF in inflammation and autoimmunity. Trends Immunol 23, 403-408). GM-CSF also interacts with other inflammatory factors (such as other cytokines or LPS) (Parajuli, B., Sonobe, Y., Kawanokuchi, J., Doi, Y., Noda, M. , Takeuchi, H., Mizuno, T., and Suzumura, A. (2012). GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 inmurine microglia. J Neuroinflammation 9, 268). From these facts, GM-CSF is thus part of the immune / inflammatory cascade.

Thus, GM-CSF can be considered a target for anti-inflammatory and anti-autoimmune therapies. Chronic and acute inflammatory and / or autoimmune diseases (eg, rheumatoid arthritis (RA), polysclerosis (MS), Crohn's disease, psoriasis, asthma, atopic dermatitis or shock) are GM- Benefits can be gained on the basis of CSF neutralization and subsequent pro-inflammatory cascade. For example, in MS, elevated levels of GM-CSF correlate with the active stages of MS (McQualter, JL, Darwiche, R., Ewing, C., Onuki, M., Kay, TW, Hamilton, JA. , Reid, HH, and Bernard, CC (2001). Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med 194, 873-882; Noster, R., Riedel, R., Mashreghi, M.-F., Radbruch, H., Harms, L., Haftmann, C., Chang, H.-D., Radbruch, A., and Zielinski, CE (2014). IL-17 and GM-CSF expression Sci Transl Med 6, 241ra80-241ra80), GM-CSF-deficient mice progress the disease in an experimental autoimmune encephalomyelitis (EAE) mouse model for multiple sclerosis. Not allowed (McQualter, JL, Darwiche, R., Ewing, C., Onuki, M., Kay, TW, Hamilton, JA, Reid, HH, and Bernard, CC (2001). Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med 194, 873-882).

In asthma, elevated levels of GM-CSF are found in the respiratory tract of asthmatic patients (Broide, DH, and Firestein, GS (1991). Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization. J Clin Invest 88, 1048-1053; Sousa, AR, Poston, RN, Lane, SJ, Nakhosteen, JA, and Lee, TH (1993). Detection of GM-CSFin asthmatic bronchial epithelium and decrease by inhaled corticosteroids Am. Rev. Respir. Dis. 147, 1557-1561). In fact, GM-CSF in collaboration with IL-5 promotes eosinophil differentiation, activation and survival (Yamashita, N., Tashimo, H., Ishida, H., Kaneko, F., Nakano, J., Kato, H., Hirai, K., Horiuchi, T., and Ohta, K. (2002). Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM -CSF). Cellular Immunology 219, 92-97). The efficacy of GM-SCF neutralizing antibodies has been demonstrated in mouse models of asthma, where their administration results in increased airway response and significantly reduced inflammation (Yamashita, N., Tashimo, H., Ishida, H., Kaneko, F., Nakano, J., Kato, H., Hirai, K., Horiuchi, T., and Ohta, K. (2002). Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte -macrophage colony-stimulating factor (GM-CSF). Cellular Immunology 219, 92-97)).

In lung disease, GM-CSF also plays a role (high neutrophil count, protease induction, and occupational lung disease in which abnormal production of TNF-alpha is caused by a bioaerosol containing LPS). Is considered to be a central factor in the etiology of the disease). In fact, in a mouse model, LPS-dependent lung inflammation was alleviated using GM-CSF neutralizing antibodies (Bozinovski, S., Jones, J., Beavitt, S.-J., Cook, AD. , Hamilton, JA, and Anderson, GP (2004). Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L877-L885).

In RA, multiple groups have measured high levels of GM-CSF in synovial joint fluid (Alvaro-Gracia, JM, Zvaifler, NJ, Brown, CB, Kaushansky, K., and Firestein, GS (1991). ). Cytokines in chronic inflammatory arthritis. VI. Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha. J Immunol 146, 3365 -3371; Haworth, C., Brennan, FM, Chantry, D., Turner, M., Maini, RN, and Feldmann, M. (1991). Expression of granulocyte-macrophage colony-stimulating factor in rheumatoid arthritis: regulation by tumor necrosis factor-alpha. Eur J Immunol 21, 2575-2579; Fiehn, C., Wermann, M., Pezzutto, A., Hufner, M., and Heilig, B. (1992). [Plasma GM-CSF concentrations in rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropathy]. Z Rheumatol 51, 121-126) and treatment with recombinant GM-CSF after chemotherapy was shown to cause flares of RA (de Vries, EG, Willemse, PH, B iesma, B., Stern, A.C., Limburg, P.C., and Vellenga, E. (1991). Flare-up of rheumatoid arthritis during GM-CSF treatment after chemotherapy. The Lancet 338, 517-518). The therapeutic potential of GM-CSF neutralizing antibodies in RA has been suggested by their effectiveness in collagen-induced mouse arthritis models (Cook, AD, Braine, EL, Campbell, IK, Rich, MJ, and Hamilton, JA (2001). Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease. Arthritis Res. 3, 293-298; Cornish, AL, Campbell, IK, McKenzie, BS, Chatfield, S., and Wicks, IP (2009). G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nat Rev Rheumatol 5, 554-559).

Therefore, antibodies capable of neutralizing GM-CSF are a new effective prophylaxis for inflammatory and / or autoimmune diseases (eg, MS, RA, other autoimmune diseases and other inflammatory diseases). It can represent a method and / or a treatment. In principle, cytokine neutralization can be achieved by soluble target cytokines or antibodies that bind to cytokine receptors presented on the cell surface. MOR103 and namilumab are two human monoclonal antibodies derived from phage that neutralize GM-CSF and are being developed as therapeutic agents in RA and MS (Steidl, S., Ratsch, O., Brocks, B., Durr, M., and Thomassen-Wolf, E. (2008). In vitro affinity maturation of human GM-CSF antibodies by targetedCDR-diversification. Mol Immunol 46, 135-144; Krinner, E.-M., Raum, T ., Petsch, S., Bruckmaier, S., Schuster, I., Petersen, L., Cierpka, R., Abebe, D., Molhoj, M., Wolf, A., et al. (2007). A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF. Mol Immunol 44, 916-925; Behrens, F., Tak, PP, Ostergaard, M., Stoilov, R., Wiland, P., Huizinga, TW, Berenfus, VY, Vladeva, S., Rech, J., Rubbert-Roth, A., et al. (2014). MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib / IIa randomized, double-blind, placebo-controlled, dose-escalation trial. Ann Rheum Dis), mabrilimumab is a developing human targeting GM-CSF receptor alpha in RA patients. It is a monoclonal antibody (Burmester, GR, Feist, E., Sleeman, MA, Wang, B., White, B., and Magrini, F. (2011). Mavrilimumab, a human monoclonal antibody targeting GM-CSF receptor-α, in subjects with rheumatoid arthritis: Ann Rheum Dis 70, 1542-1549) a randomized, double-blind, placebo-controlled, phase I, first-in-human study.

However, a single GM-CSF neutralizing antibody may be the basis for the improved activity of monoclonal antibodies against common gamma chain cytokines in vivo, still dissociating and stimulating receptors, long-lived GM-CSF. In vivo (Boyman, O., Kovar, M., Rubinstein, MP, Surh, CD, and Sprent, J. (2006). Selective stimulation of T cell subsets with antibody-cytokine immune complexes . Science 311, 1924-1927).

From the above viewpoint, an object of the present invention is to provide an antibody that neutralizes cytokines (particularly GM-CSF) more strongly and efficiently than currently available antibodies. Such antibodies can be used at lower doses, thereby reducing the risk of side effects and reducing costs. In addition, to provide antibodies that neutralize cytokines (particularly GM-CSF) that do not result in the accumulation of large pools of long-lived GM-CSF (particularly GM-CSF) that can still dissociate and stimulate receptors. Is the object of the present invention. In summary, the present invention provides improved antibodies or antigen-binding fragments thereof, as well as related nucleic acid molecules, vectors and cells, and pharmaceutical compositions that overcome the shortcomings of prior art in a cost-effective and simple manner. It is the object of the invention.

The object underlying the present invention is solved by the matters described in the claims.

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

The components of the present invention will be described below. Although these components are listed with specific embodiments, it should be understood that they can be combined in any way and in any number to make further embodiments. The variously described examples and preferred embodiments should not be construed as limiting the invention to just a plurality of expressly described embodiments. It should be understood that the specification supports and embraces a plurality of embodiments that combine the plurality of embodiments explicitly described with the disclosed and / or preferred components. Furthermore, unless otherwise specified, any order of exchange and combination of all components described herein should be considered as disclosed herein.

Throughout the specification and claims that follow, unless otherwise noted, the terms "comprise" and variations thereof (eg, "comprises" and "include (comprises)" comprising) ")") is understood to mean the inclusion of any other member, integer or step not mentioned and not the exclusion of any other member, integer or step not mentioned. The term "consisting of" is a particular embodiment of the term "contains" excluding any other member, integer or step not mentioned. In the context of the present invention, the term "contains" includes the term "consisting of". Thus, the term "comprising" includes "including" and "consiting" (eg, compositions that "contain" X are exclusive. Can consist of, or even contain, something (eg, X + Y).

Similar references used in the terms "a" and "an" and "the" as well as in the text describing the invention (particularly in the claims) are not otherwise stated in the specification or in context. It should be interpreted as covering the singular and plural unless there is a clear contradiction based on. The details of the numerical range herein are merely intended to serve as an abbreviation for each of the different values within the range. Unless otherwise stated, each of the individual values is incorporated herein as if they were individually detailed herein. Matters not included in the specification should not be understood as indicating that any unclaimed component is essential to the practice of the present invention.

The word "substantially" does not exclude "completely" (eg, a composition that does not "substantially" contain Y does not have to contain Y completely). Where necessary, the word "substantially" may be omitted from the provisions of the present invention.

The term "about" with respect to the number x means x ± 10%.

As used herein, the terms "disease" are the terms "disorder" and "condition" (as in the case of "medical condition") and (them). Are all generally synonymous (when indicating an abnormal condition (or part of it) that impairs the normal functioning of the human or animal body), are used interchangeably, and are typically by prominent signs and symptoms. Causes short lifespan or reduced quality of life for humans or animals.

As used herein, reference to a subject or patient's "treatment" is intended to include inhibition, prevention, weakening, remission and treatment. The terms "control" or "patient" are used interchangeably herein to mean all mammals (including humans). Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep and rabbits. In one embodiment, the patient is a human.

As used herein, the term "antibody" refers to various forms of an antibody, preferably monoclonal antibodies (whole antibody, antibody fragments, human antibodies, as long as the characteristic properties of the invention are maintained. , Chimera antibodies, humanized antibodies, genetically engineered antibodies, but not limited to)). Human antibodies or humanized antibodies (especially such as recombinant human antibodies) are particularly preferred.

As used herein, the term "human antibody" is intended to include antibodies that have variable and constant regions derived from the immunoglobulin sequences of human germline. Human antibodies are well known in the latest technology (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in the absence of endogenous antibody production in all or a selection of human antibodies in transgenic animals that can be produced by immunization (eg, mice). Translocation of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies by antigen administration (eg, Jakobovits, A., et al., Proc. Natl. Acad). Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; See Bruggemann, M., et al., Year Immunol. 7 (1993) 3340) .. Human antibodies are also available in the Phage Display Library (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991). ) Can be produced in 581-597). In addition, the methods of Cole et al. And Boerner et al. Are 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., J. Immunol. 147 (1991) 86-95). As used herein, the term "human antibody" also includes such antibodies that have been modified, for example, in a variable region to produce the properties according to the invention.

As used herein, the term "recombinant human antibody" is transduced with any human antibody (eg, a human immunoglobulin gene) prepared, expressed, produced or isolated by a recombinant method. It is intended to include antibodies isolated from host cells (eg, CHO cells) or animals (eg, mice), or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in their reconstituted form. The recombinant human antibody according to the present invention has undergone in vivo somatic hypermutation. Thus, the amino acid sequences of the V _H and _VL regions of a recombinant antibody are derived from and related to the V _H and _VL sequences of the human germline and are naturally within the in vivo human germline repertoire. An array that does not exist.

As used herein, the terms "antigen-binding fragment,""fragment," and "antibody fragment" retain the specific binding activity and Fc portion of the antibody according to the invention. Used interchangeably to refer to any fragment of. Examples of antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab', F (ab') ₂ , Fv or scFv. Fragments of the antibodies of the invention can be obtained from the antibodies by methods involving digestion with enzymes (eg, pepsin or papain) and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be obtained by cloning and expression of multiple partial sequences of heavy and / or light chains. Examples of the "fragment" include a Fab fragment, a Fab'fragment, an F (ab') 2 fragment, and an Fv fragment. The present invention also includes single chain Fv fragments (scFv) obtained from heavy and light chains of the antibodies of the invention. For example, the present invention includes scFv containing multiple CDRs from the antibodies of the present invention. Also, heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies (eg, heavy and light chain variable domains are mediated by peptide linkers). The connected single chain Fv) is included. The antibody fragments of the present invention can result in monovalent or multivalent interactions and are included in the various structures described above. For example, the scFv molecule can be synthesized to produce a trivalent "triabody" or a tetravalent "tetrabody". The scFv molecule may contain a domain of the Fc region that yields a divalent minibody. In addition, the sequences of the invention can be components of a multispecific molecule in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Illustrative molecules include, but are not limited to, divalent Fab2, trivalent Fab3, divalent scFv and diabody (Holligerand Hudson, 2005, Nature Biotechnology 9: 1126-1136). The present specification, which includes the scope of claims, describes, in some places, to (s) antigen-binding fragments, (s) antibody fragments, (s) variants, and / or (s) derivatives of an antibody. Although explicitly mentioned, the term "antibody" or "antibody of the invention" refers to all categories of antibodies (ie, (s) antigen-binding fragments, (s) antibody fragments, (s) of antibodies. Includes variants and (s) derivatives). Moreover, as used herein, the term "antibody" includes both the antibody and its antigen-binding fragment.

As used herein, a "neutralizing antibody" is an antibody capable of neutralizing (ie, blocking, inhibiting, reducing, interfering with or suppressing) the ability of a pathogen to induce and / or sustain infection in a host. is there. The terms "neutralizing antibody" and "neutralizing antibody" or "neutralizing multiple antibodies" are used interchangeably herein. These antibodies can be used alone or in combination as prophylactic or therapeutic agents, as diagnostic tools, or as production tools, with appropriate formulation, in collaboration with aggressive vaccination, as described herein. ..

As used herein, the term "variable region" (variable region of the light chain ( _VL ), variable region of the heavy chain _VH ) is lightly involved in binding the antibody directly to the antigen. Means each of a chain and a pair of heavy chains. In native antibodies, multiple domains of variable human light and heavy chains have the same general structure, with each domain having three "hypervariable regions" (ie, complementarity determining regions, It contains four framework (FR) regions whose sequences are widely conserved, connected by CDRs). The framework region has a β-sheet three-dimensional structure, and the CDR can form a loop connecting the β-sheet structures. The CDRs in each strand are held in their three-dimensional structure by the framework region and, together with the CDRs from the other strands, form an antigen binding site. The CDR3 region of the antibody heavy chain and the antibody light chain plays a particularly important role in the binding specificity / affinity of the antibody according to the present invention, and brings about a further object of the present invention.

As used herein, the term "hypervariable region" refers to multiple amino acid residues of an antibody that are responsible for antibody binding. The hypervariable regions contain multiple amino acid residues from the "complementarity determining regions" (ie, "CDRs"). The "framework" (ie, "FR") regions are those regions of the variable domain other than the hypervariable regions as defined herein. Therefore, the light and heavy chains of a natural antibody contain multiple domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus. The multiple CDRs on each chain are separated by such multiple framework amino acids. In particular, the heavy chain CDR3 is the region that contributes most to antigen binding. The CDR and FR regions are determined according to the standard description of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

As used herein, the term "stationary region" refers to a domain of an antibody that is not directly involved in the binding of the antibody to the antigen but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies or immunoglobulins are divided into multiple classes: IgA, IgD, IgE, IgG, IgM, some of which are subclasses (eg IgG1, IgG2). , IgG3 and IgG4, IgA1 and IgA2). The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, ε, γ and μ, respectively. The antibody according to the present invention is preferably of the IgG type.

As used herein, the term "nucleic acid or nucleic acid molecule" is intended to include DNA and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

As used herein, "cells," "cell lines," and "cell cultures" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cells" include primary target cells and cultures obtained from them regardless of the number of passages. It is understood that all progeny do not have to be exactly the same in the DNA contents due to intentional or accidental mutations. Various progeny with the same function or bioactivity as when screened for the originally transformed cells are included. Where other meanings are intended is clear from the text.

Dose is usually expressed in association with body weight. Therefore, the dose expressed as [g, mg or other unit] / kg (or g, mg, etc.) per "kg (or g, mg, etc.) body weight, even if the term" body weight "is not explicitly stated. Usually refers to [g, mg or other unit].

The terms "specifically bind" and similar references do not include non-specific fixation.

As used herein, the term "vaccine" is generally understood to be a prophylactic or therapeutic material that presents at least one antigen (preferably one source of immunity). The antigen or immune source can be derived from any material suitable for vaccination. For example, the antigen or immune source can be derived from a pathogen (eg, bacterial or viral particles, etc.) or tumor or cancer tissue. An antigen or source of immunity stimulates the body's adaptive immune system to () provide an adaptive immune response. In particular, an "antigen" or "immune source" can elicit an antigen-specific immune response that can be recognized by the immune system (preferably the adaptive immune system) (eg, antibodies and / or antigens as part of the adaptive immune response). Generally refers to a substance (by the formation of specific T cells). In general, the antigen can be a peptide or protein that can be presented to T cells by MHC, or can include such peptide or protein.

As used herein, "sequence variant" refers to any change in the reference sequence (where the reference sequence is the sequence listed in the "Table of Sequences and SEQ ID NOs" (sequence). That is, it is any one of SEQ ID NOs: 1 to 190)). Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In particular, in "sequence variants", functionality (of the reference sequence) is preserved. (Ie sequence variants are functional). As used herein, a "sequence variant" is at least 70% identical to a reference sequence, preferably at least 80% identical to a reference sequence (more preferably at least 90% identical to a reference sequence, and more preferably at least at least. It generally has 90% identical and particularly preferably at least 99% identical) sequences.

Sequence identity is often calculated relative to the total length of the reference sequence (ie, the sequences detailed in this application). As referred to herein, the percentage of identity is the default parameter specified by, for example, the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/. Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1] can be used for BLAST.

As used herein, a "nucleic acid sequence variant" is one or more nucleotides deleted or substituted in the reference sequence, or one or more nucleotides inserted into the sequence of the reference nucleotide sequence. , Has a modified sequence. Nucleotides are referred to herein by standard one-letter notation (A, C, G or T). Due to the degeneracy of the genetic code, "nucleotide sequence variants may or may not result in changes in their respective reference amino acid sequences (ie," amino acid sequence variants "). Preferred sequence variants are nucleotide sequence variants that do not give rise to amino acid sequence variants (silent mutations), but other non-silent mutations (particularly at least 70% identical to the reference sequence (preferably at least 80% identical to the reference sequence, more preferably). Mutant nucleotide sequences that give rise to amino acid sequences that are at least 90% identical, more preferably at least 95% identical and particularly preferably 99% identical) are also within range.

As used herein, the term "mutation" or "mutating" is used, for example, in a nucleic acid sequence (eg, a codon in a nucleic acid molecule that encodes one amino acid to give rise to a different amino acid. To modify (eg, site-specific mutagenesis) or to synthesize a sequence variant (eg, to know the nucleotide sequence of a nucleic acid molecule encoding a polypeptide, and to encode a variant of that polypeptide. By designing the synthesis of a nucleic acid molecule containing a nucleotide sequence without the need to modify one or more of the nucleotides of the nucleic acid molecule)), it is understood to include the physical generation of mutations.

An "amino acid sequence variant" has a modified sequence in which one or more amino acids in the reference sequence have been deleted or substituted, or one or more amino acids have been inserted into the sequence of the reference amino acid sequence. ing. As a result of the modification, the amino acid sequence variants are at least 70% identical to the reference sequence (preferably at least 80% identical to the reference sequence, more preferably at least 80% identical, more preferably at least 90% identical, particularly preferably 99. % Same) amino acid sequence. Variant sequences that are at least 10% identical have no more than 10 modifications (ie, any combination of deletions, insertions or substitutions) per 100 amino acid reference sequence.

In a peptide / protein statement, a "linear sequence" or "sequence" is a plurality of amino acids in the amino-terminal to carboxyl-terminal direction in which adjacent sequences in the sequence are contiguous in the peptide / protein primary structure. Is an array of.

Although it is possible to have a non-conservative amino acid substitution, the substitution is a conservative amino acid substitution in which the substituted amino acid has structural or chemical properties similar to the corresponding amino acid in the reference sequence. Is preferable. As an example, conservative amino acid substitutions are substitutions of one aliphatic or hydrophobic amino acid (eg, alanine, valine, leucine and isoleucine) with another aliphatic or hydrophobic amino acid; Substitution of hydroxyl-containing amino acids (eg, serine and threonine) with other species of hydroxyl-containing amino acids; substitution of one acidic amino acid (eg, glutamate or aspartic acid) with another species of acidic amino acid; one fragrance Substitution of group residues (eg, phenylalanine and tyrosine) with aromatic residues of other species; substitution of one basic amino acid (eg, lysine, arginine and histidine) with other species of basic amino acid; And includes substitution of one small amino acid (eg, alanine, serine, threonine, methionine and glycine) with another small amino acid.

Amino acid sequence insertions range from one residue to a polypeptide containing 100 or more residues, amino-terminal fusion and / or carboxy-terminal fusion, and intra-sequence insertion of one or more amino acid residues. Including. Examples of terminal insertions include fusion of the amino acid sequence to the reporter molecule or enzyme to the N-terminus or C-terminus.

Importantly, sequence variants are such that modifications in the sequence variants bind to the same non-overlapping epitope / site of the functionality of their respective reference sequences (eg, cytokines (particularly GM-CSF)) and / or cytokines (particularly). It is a functional sequence that sufficiently neutralizes GM-CSF) and does not lose the functionality of the antibody or antigen-binding fragment thereof. Guidance for determining each of the nucleotide and amino acid residues that can be substituted, inserted or deleted without losing such functionality can be found by using computer programs well known in the art.

As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide or protein refers to the origin of the polypeptide. Preferably, the nucleic acid or amino acid sequence obtained from a particular sequence is an amino acid sequence that is substantially identical to the sequence from which it is obtained or a portion thereof, thereby making "substantially identical". , Includes sequence variants as defined above. Preferably, the nucleic acid sequence or amino acid sequence obtained from the particular peptide or protein is obtained from the corresponding domain in the particular peptide or protein. Thus, "corresponding" specifically refers to the same functionality. For example, a CDR (ie CDRH1) amino acid sequence or nucleic acid sequence in a multispecific antibody that is "obtained from a (specific) monospecific antibody" is the (ie) CDR (ie, corresponding) CDR of this monospecific antibody. CDRH1) It is often obtained from an amino acid sequence or nucleic acid sequence (not the other (non-corresponding) portion of the monospecific antibody). The "corresponding" portions of peptides, proteins and nucleic acids are thus readily identifiable to those of skill in the art (eg, CDR1 corresponds to CDR1, CDR2 corresponds to CDR2, and so on). Similarly, sequences "derived from" other sequences are often readily identifiable to those skilled in the art as having their origin in the sequence.

Preferably, the nucleic acid sequence or amino acid sequence obtained from another nucleic acid, peptide, polypeptide or protein can be identical to the starting nucleic acid, peptide, polypeptide or protein (where it is obtained). However, nucleic acid sequences or amino acid sequences obtained from other nucleic acids, peptides, polypeptides or proteins also have one or more mutations to the starting nucleic acid, peptide, polypeptide or protein (where it is obtained). (In particular, a nucleic acid sequence or amino acid sequence obtained from another nucleic acid, peptide, polypeptide or protein is of the starting nucleic acid, peptide, polypeptide or protein (it is obtained). It can be a functional sequence variant as described above). For example, in a peptide / protein, one or more amino acid residues can be replaced with other amino acid residues, or insertions or deletions of one or more amino acid residues can occur.

A number of references are cited throughout this specification. Each of the above or below references cited herein (all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is incorporated herein by reference in its entirety. What is not described herein should not be construed as an admission that the invention does not have the right to date back to such disclosure by the prior invention.

It should be understood that the invention is not limited to the particular methodologies, protocols and reagents described herein as they may vary. It is understood that the methodology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited solely by the appended claims. Should. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The present invention, among other findings, is that a cocktail of three antibodies that bind to different, non-overlapping sites on the same cytokine (particularly GM-CSF) is based on mass from the currently available antibodies MOR103 or namilumab. Based on the discovery that it is powerful. In particular, when a cocktail of three antibodies is used, an immune complex in which a large pool of long-lived GM-CSF does not accumulate and multiple antibodies are rapidly degraded in vivo in an Fc-dependent manner. To form. This prompted us to design a multispecific antibody resulting from the combination of antibodies described above. Surprisingly, such multispecific antibodies have significantly improved neutralizing activity compared to currently available GM-CSF antibodies as well as antibody cocktails (here). , Each antibody is specific only for a single site of cytokine). It has been suggested that in the multispecific construct according to the invention, cytokines (particularly GM-CSF) are irreversibly sequestered and are not utilized for interaction with the receptor. These multispecific antibodies can be used at very low doses to treat a variety of cytokine (particularly GM-CSF) -dependent diseases such as autoimmune and inflammatory diseases.

(Multispecific antibody or antigen-binding fragment thereof)
In the first aspect, the present invention
It contains (a) at least two different epitope binding sites; and (b) Fc moieties.
Each of the at least two different epitope binding sites specifically binds to an individual epitope of the cytokine, whereby the plurality of individual epitopes of the cytokine to which the at least two different epitope binding sites bind. , Multiple epitopes that do not overlap,
An isolated multispecific anti-cytokine antibody or antigen-binding fragment thereof is provided.

Such multispecific anti-cytokine (preferably anti-GM-CSF) antibodies or antigen-binding fragments thereof according to the present invention mainly neutralize cytokines (particularly the targeting action of the cytokines) strongly (eg, for example). The multispecific antibody or antigen-binding fragment thereof according to the present invention mainly strongly neutralizes GM-CSF (particularly the targeting action of GM-CSF). Such neutralization is known to those of skill in the art and can be evaluated in neutralization evaluation methods as described below.

Preferably, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention contains a non-naturally occurring amino acid sequence.

As used herein, a "multispecific" antibody is one antibody molecule to at least two different epitopes (eg, at least two non-overlapping sites in a cytokine (particularly GM-CSF)). Refers to an antibody that can bind to an antibody. Multispecific anti-cytokine (preferably anti-GM-CSF) antibodies according to the invention, as opposed to the most known multispecific antibodies, which generally bind to at least two epitopes on different molecules. Alternatively, a single molecule of the antigen-binding fragment can bind to at least two different non-overlapping sites in a single cytokine molecule, especially a single GM-CSF molecule. Therefore, the multispecific antibody or antigen-binding fragment thereof according to the present invention is "multispecific" for a single cytokine (molecule).

Preferably, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention is bispecific, trispecific, quadrispecific or quintugenous. More preferably, the antibody or antigen-binding fragment thereof is bispecific, trispecific or quadruspecific, and more preferably, the antibody or antigen-binding fragment thereof is bispecific or trispecific. It is sex, and particularly preferably, the antibody or antigen-binding fragment thereof is trispecific. Thereby, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention is directed against a single cytokine (molecule) (particularly a single GM-CSF (molecule)). It is intended to be bispecific, triple specific, quadruple specific or quintuple specific.

Multiple epitopes to which the antibodies of the invention bind (ie, multiple sites in cytokines (particularly single cytokine molecules (eg, GM-CSF (particularly single GM-CSF molecules)))) are linear (continuous). Can be target) or stereoscopic (discontinuous). Preferably, the antibody and antibody fragment of the invention bind to a conformational epitope, more preferably the conformational epitope is present only in the non-reducing state. However, the antibodies and antibody fragments of the invention can also bind to linear epitopes, more preferably the linear epitopes are present in both non-reducing and reducing states.

The antibodies according to the invention contain at least two different epitope binding sites, each of which at least two different epitope binding sites specifically bind to an individual epitope of a cytokine, thereby at least said. The individual epitope of a cytokine to which two different epitope binding sites bind is a non-overlapping epitope, in particular a non-overlapping epitope of the primary sequence of the cytokine (eg, GM-CSF). Importantly, the different epitopes to which the antibodies of the invention bind (ie, multiple sites in cytokines (especially single cytokine molecules (eg, GM-CSF (especially single GM-CSF molecules))) are different (ie). That is, they are not the same) and do not overlap. In a single cytokine (eg, GM-CSF) molecule, multiple epitopes (also referred to as "sites") to which the multispecific anti-cytokine (preferably anti-GM-CSF) antibody according to the invention binds directly. Although they can be placed adjacent to each other or separated (eg, by a linker, other antibody domain, etc.), according to the invention, the plurality of epitopes do not necessarily overlap. Thereby, non-overlapping means that an amino acid in the amino acid sequence of a cytokine (eg, GM-CSF) is not used in more than one epitope / site to which the antibody according to the invention binds. In other words, each amino acid in the amino acid sequence of a cytokine (eg, GM-CSF) is part of one single epitope to which an antibody according to the invention binds, or any epitope to which an antibody according to the invention binds. It is not the part of.

Therefore, the antibody or antigen-binding fragment thereof according to the present invention can also be used to position an epitope to which they bind among cytokines (eg, GM-CSF).

An antibody or antigen-binding fragment thereof according to the present invention contains at least two different domains for specifically binding to at least two different non-overlapping sites in a cytokine (eg, GM-CSF). In other words, the antibody or antigen-binding fragment thereof according to the present invention has at least one first domain that specifically binds to a first site in a cytokine (eg, GM-CSF), and at least one second. Contains the domain. Here, the at least one second domain is different from the at least one first domain and specifically binds to a second site in a cytokine (eg, GM-CSF). Thereby, the second site in the cytokine (eg, GM-CSF) is different from and does not overlap with the first site in the cytokine (eg, GM-CSF).

As used herein, multiple domains of an antibody that specifically bind to a cytokine (eg, GM-CSF) are also referred to as "binding domains," "epitope binding domains," or "epitope binding sites." Preferably, such epitope binding sites of the antibody meet at least the minimum need to specifically bind to distinct epitopes (thus forming an "epitope binding site"), eg, monospecificity. It contains at least one, preferably three, and more preferably six CDRs that can be derived from the antibody. Thus, it is the six CDRs in which such epitope binding sites of the antibody form together with the epitope binding sites (eg, CDR1, CDR2 and CDR3 derived from the heavy chain of a monospecific antibody, and the same single. It is more preferable if the specific antibody contains CDR1, CDR2 and CDR3) derived from the corresponding light chain. More preferably, such epitope binding sites of multispecific anti-cytokine (preferably anti-GM-CSF) antibodies according to the invention can be derived (eg, from (identical) monospecific antibodies). It may include a heavy chain variable region and / or a (corresponding) light chain variable region.

In principle, each of the different epitope binding sites of the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention is used once or more (preferably, preferably) in the above antibody. 2 times, 3 times, 4 times, 5 times or 6 times or more) can appear. For example, natural IgG is divalent and unispecific because it contains the same two Fabs that both recognize the same epitope. Thus, a multispecific antibody or antigen-binding fragment thereof according to the present invention is at least divalent (ie, in the case of two different epitope binding sites that appear once in each antibody). Furthermore, the multispecific antibody or antigen-binding fragment thereof according to the present invention also appears, for example, in the case of three different epitope binding sites, each appearing once, or one appears once and the other appears twice. For different epitope binding sites, it may be trivalent; for example, for four different epitope binding sites that appear once and each appear once, or for two each appear once and a third epitope binding site. It may be tetravalent for three different epitope binding sites that appear twice, or for two different epitope binding sites that appear twice each, or one appears once and the other appears three times. May be pentavalent; may be hexavalent; may be 7 valent; may be octavalent; may be 9 valent; may be 10 valent; 11 It may be valent; it may be 12-valent; it may be 13-valent; it may be 14-valent or the like.

Preferably, each of the different epitope binding sites appears twice in the antibody molecule according to the invention. In other words, the antibody molecule according to the invention is exactly two copies of each of the different domains contained by the antibody or antigen-binding fragment thereof that specifically bind to at least two different, non-overlapping sites in the cytokine. Includes. Therefore, the multispecific antibody or antigen-binding fragment thereof according to the present invention is preferably tetravalent, hexavalent, 8-valent, 10-valent, 12-valent, 14-valent, or the like, and the antibody molecule is different accordingly. It contains exactly two copies of each of the epitope binding sites. More preferably, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention is a bispecific tetravalent antibody, a trispecific hexavalent antibody, or a quadruplex. It is a specific octavalent antibody, and more preferably, the multispecific antibody or the antigen-binding fragment thereof according to the present invention is a bispecific tetravalent antibody or a trispecific hexavalent antibody.

In general, the antibody according to the present invention or an antigen-binding fragment thereof is preferably a monoclonal antibody or an antigen-binding fragment of the monoclonal antibody. Monoclonal antibodies are usually produced by a single clone of B lymphocytes, for example by making hybrid antibody-producing cells, for example from fusion of non-secretory myeloma cells with immunospleen cells. In contrast to polyclonal antibodies, multispecific monoclonal antibodies bind to (pre-) predetermined epitopes. Therefore, unexpected binding, especially unexpected binding to undetermined epitopes, is largely avoided and monoclonal antibodies are considered to be safer compared to polyclonal antibodies.

Preferably, the antibody or antigen-binding fragment thereof according to the present invention is a human antibody, a monoclonal antibody, a human monoclonal antibody, or a purified antibody. The antibody according to the present invention or an antigen-binding fragment thereof may also be a single-chain antibody.

(Fc part)
The multispecific anti-antibody or antigen-binding fragment thereof according to the present invention contains an Fc moiety. Preferably, the Fc portion is derived from human origin, eg, human IgG1, IgG2, IgG3 and / or IgG4, whereby human IgG1 is particularly preferred.

As used herein, the term "Fc portion" refers to a sequence derived from a portion of an immunoglobulin heavy chain, wherein the portion of the immunoglobulin heavy chain is a papain cleavage site (eg, residue 216 in native IgG). It starts from the hinge region just upstream of (bringing the first residue of the heavy chain constant region to the 114th) and ends at the C end of the immunoglobulin heavy chain. Thus, the Fc portion may be a complete Fc portion or a portion thereof (eg, a domain). The complete Fc portion comprises at least a hinge domain, a CH2 domain, and a CH3 domain (eg, EU amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the C-terminus of the Fc portion, but is often cleaved from the mature antibody. Each amino acid position within the Fc region is numbered according to Kabat's technically approved EU numbering system. See “Sequences of Proteins of Immunological Interest” by Kabat et al., U.S. Dept. Health and Human Services, 1983 and 1987.

Preferably, in the context of the present invention, the Fc portion is at least one: hinge (eg, upper, middle, and / or lower hinge region) domain, CH2 domain, CH3 domain, or variant, portion, or fragment thereof. , Including. In a preferred embodiment, the Fc portion comprises at least a hinge domain, a CH2 domain or a CH3 domain. More preferably, the Fc portion is a complete Fc portion. The Fc moiety may also contain one or more amino acid insertions, amino acid deletions, or amino acid substitutions with respect to the naturally occurring Fc moiety. For example, at least one hinge domain, CH2 domain or CH3 domain (or a portion thereof) may be deleted. For example, the Fc portion may include or consist of: (i) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a CH3 domain. A hinge domain (or a portion thereof) fused to (or a part thereof), a CH2 domain (or a part thereof) fused to a (iii) CH3 domain (or a part thereof), and an (iv) hinge domain (or a part thereof). A part thereof), (v) CH2 domain (or a part thereof), or (vi) CH3 domain or a part thereof.

The Fc portion differs from the complete Fc portion of the naturally occurring immunoglobulin molecule in terms of amino acid sequence, and at the same time, the Fc moiety is said to maintain at least one desired function conferred by the naturally occurring Fc moiety. It will be appreciated by those skilled in the art that the Fc portion may be modified. Such functions include Fc receptor (FcR) binding, antibody half-life regulation, ADCC function, protein A binding, protein G binding, and complement fixation. Part of the naturally occurring Fc portion that carries out such a function and / or is essential for such a function is well known to those of skill in the art.

For example, to activate the complement cascade, C1q binds to at least two molecules of IgG1 or one molecule of IgM attached to an antigenic target (Ward, ES, and Ghetie, V. et al. , Ther. Immunol. 2 (1995) 77-94). Burton, D. R. explained that the heavy chain region containing amino acid residues 318-337 is involved in complement fixation (Mol. Immunol. 22 (1985) 161-206). Duncan, A.R. and Winter, G. reported that Glu318, Lys320 and Lys322 form a binding site for C1q using site-specific mutagenesis (Nature 332 (1988) 738-740). The role of Glu318, Lys320 and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated lysis.

For example, FcR binding can be mediated by the interaction of the Fc portion (of the antibody) with Fc receptors (FcRs), which are differentiated cell surface receptors on hematopoietic cells. The body. Fc receptors belong to the immunoglobulin superfamily, which are the elimination of antibody-coated pathogens by immune complex phagocytosis and antibody-dependent cellular cytotoxicity (ADCC; Van de Winkel). , JG, and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524) with the lysis of red blood cells and various other cell subjects (eg, tumor cells) coated with the corresponding antibody. It is shown to mediate both. FcRs are defined by the specificity of FcRs for a class of immunoglobulins; Fc receptors for IgG antibodies are referred to as FcγRs, FcεR for IgE, FcαR for IgA, and so on, and so on. Neonatal Fc receptors are referred to as FcRn. Fc receptor binding is described, for example, as follows: Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) ) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248 ..

Cross-linking of the receptor (FcγR) by the Fc domain of a native IgG antibody causes a wide variety of agonist functions, which are phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators. , And the removal of immune complexes and the regulation of antibody production. In humans, three classes of FcγR have been characterized: (i) FcγRI (CD64), which FcγRI (CD64) binds to monomeric IgG with high affinity. And expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32), which FcγRII (CD32) binds to IgG complexed with intermediate to low affinity. It is widely expressed, especially on leukocytes, and is known to be a central player in antibody-mediated immunity, and the FcγRII (CD32) can be divided into FcγRIIA, FcγRIIB and FcγRIIC. Yes, they perform different functions in the immune system, but bind to IgG-Fc with similar low affinity, and the external domains of these receptors are highly homologous; and (iii). FcγRIII (CD16), the FcγRIII (CD16) binds to IgG with medium to low affinity and there are two types: NK cells, macrophages, eosinophils, and some monocytes and T. FcγRIIIA found on cells, mediated by ADCC, as well as highly expressed FcγRIIIB on monocytes. FcγRIIA is found on many cells involved in lethality (eg macrophages, monocytes, neutrophils) and appears to be capable of activating the lethality process. FcγRIIB appears to play a role in the inhibitory process and is found on B cells, macrophages, and mast cells and eosinophils. On B cells, it appears to be a function to suppress the production of additional immunoglobulins and the switching of isotypes to approximately eg IgE classes. On macrophages, FcγRIIB acts to inhibit fagocytosis as mediated through FcγRIIA. On eosinophils and mast cells, b-form may help suppress activation of these cells through IgE that binds to its distinct receptor.

With respect to FcγRI binding, modification in at least one of E233-G236, P238, D265, N297, A327 and P329 in native IgG reduces binding to FcγRI. Substituted into IgG1 and IgG4, residue in IgG2 positions 233-236 are binding to Fc [gamma] RI, it is reduced to 10 ³ times to remove the response of human monocytes to the antibody sensitized erythrocytes (Armor, KL Et al. Eur. J. Immunol. 29 (1999) 2613-2624). With respect to FcγRII binding, reduced binding for FcγRIIA was found, for example, for at least one IgG mutation of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. With respect to FcγRIII binding, the reduced binding to FcγRIIIA is at least E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376. It was found for one mutation. To locate the binding site on human IgG1 for the Fc receptor, the above-mentioned mutation sites and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591. Explained in −6604.

With respect to binding to significant FcγRII, two regions of Fc of native IgG appear to be important for the interaction of FcγRIIs and IgGs, and the two regions are i.e. (i) the lower hinge of IgG Fc. Sites, especially those adjacent to the CH2 domain of amino acid residues L, L, G, G (234-237, EU numbered), and (ii) IgG Fc, especially the lower hinge region (eg, in the region of P331). Loops and strands in the upper CH2 domain (Wines, BD, et al., J. Immunol. 2000; 164: 5313-5318). Furthermore, FcRn and protein A bind to different sites on IgG Fc, whereas FcγRI appears to bind to the same site on IgG Fc, which is at the CH2-CH3 interface. Seems to be (Wines, BD, et al., J. Immunol. 2000; 164: 5313-5318).

For example, the Fc moiety may include, and is composed of, at least a portion of the Fc moiety that is technically known to be required for FcRn binding or an extended half-life. May be good. Alternatively or further, the Fc portion of the antibody of the invention comprises at least a portion of what is technically known to be required for protein A binding and / or of the antibody of the invention. The Fc portion comprises at least a portion of an Fc molecule that is technically known to be required for protein G binding. Preferably, the function maintained is the removal of cytokine-immune complexes, such as GM-CSF-immune complexes, which are hypothesized to be mediated by FcγR binding. Therefore, preferred Fc moieties include at least a portion that is technically known to be required for FcγR binding. As outlined above, preferred Fc moieties may therefore include at least the following (i) and (ii): (i) lower hinge sites of Fc in natural IgG, especially amino acids. Residues L, L, G, G (234-237, EU numbered), and (ii) adjacent regions of the CH2 domain of the natural IgG Fc, particularly adjacent to the lower hinge region (eg, in the region of P331). Loops and strands in the upper CH2 domain, eg, at least 3,4 in the upper CH2 domain around P331 of the native IgG Fc (eg, between amino acids 320 and 340 (EU numbering) of the native IgG Fc). Regions of 5, 6, 7, 8, 9, or 10 contiguous amino acids.

Preferably, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention contains an Fc region. As used herein, the term "Fc region" refers to a portion of immunoglobulin formed by two or more Fc moieties of an antibody heavy chain. For example, the Fc region may be a monomeric Fc region or a "single-strand" Fc region (ie, a scFc region). The single-stranded Fc region is composed of Fc moieties linked within a single polypeptide chain (eg, encoded in a single contiguous nucleic acid sequence). An exemplary scFc region is disclosed in WO 2008/143954A2. Preferably, the Fc region is a dimeric Fc region. "Dimer Fc region" or "dcFc" refers to a dimer formed by the Fc portion of two separate immunoglobulin heavy chains. The dimer Fc region may be a homodimer of two identical Fc moieties (eg, of the Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties. It may be a body.

The Fc portion of the Fc region may be the same or different class and / or subclass. For example, the Fc portion may be derived from an immunoglobulin of the IgG1, IgG2, IgG3 or IgG4 subclass (eg, human immunoglobulin). Preferably, the Fc portion of the Fc region is the same class and subclass. However, the Fc region (or one or more Fc moieties of the Fc region) may also be chimeric, where the chimeric Fc region comprises Fc moieties derived from different immunoglobulin classes and / or subclasses. You may be. For example, at least two Fc portions of a dimer or single chain Fc region may be from different immunoglobulin classes and / or subclasses. Further, or alternative, the chimeric Fc region may include one or more chimeric Fc moieties. For example, a chimeric Fc region or portion may comprise one or more portions derived from immunoglobulins of a first subclass (eg, IgG1, IgG2, or IgG3 subclass), as well as an Fc region or portion. The rest are different subclasses. For example, the Fc region or portion of an Fc polypeptide is a CH2 and / or CH3 domain derived from an immunoglobulin of a first subclass (eg, IgG1, IgG2, or IgG4 subclass), as well as a second subclass (eg, IgG3). It may include a hinge region from a subclass) immunoglobulin. For example, the Fc region or moiety is an immunogen, a hinge and / or CH2 domain derived from an immunoglobulin of the first subclass (eg, IgG4 subclass), and immunity of a second subclass (eg, IgG1, IgG2, or IgG3 subclass). It may contain a CH3 domain, from globulin. For example, the chimeric Fc region is an Fc portion from an immunoglobulin (eg, a complete Fc portion) for a first subclass (eg, IgG4 subclass) and a second subclass (eg, IgG1, IgG2, or IgG3 subclass). ) Fc portion from immunoglobulin, may be included. For example, the Fc region or moiety may include a CH2 domain from IgG4 immunoglobulin and a CH3 domain from IgG1 immunoglobulin. For example, the Fc region or moiety may include the CH1 and CH2 domains from an IgG4 molecule, as well as the CH3 domain from an IgG1 molecule. For example, the Fc region or portion may include a portion of the CH2 domain from a particular subclass of antibody, eg, EU positions 292-340 of the CH2 domain. For example, the Fc region or moiety is the amino acid position 292-340 of CH2 derived from the IgG4 moiety and the rest of CH2 derived from the IgG1 moiety (alternatively, 292-340 of CH2 which may be derived from the IgG1 moiety. And the rest of CH2 from the IgG4 moiety) may be included.

In addition, the Fc region or portion may (and / or alternativeally) include, for example, a chimeric hinge region. For example, the chimeric hinge may be, for example, partially derived from an IgG1, IgG2 or IgG4 molecule (eg, upper and lower middle hinge sequences) and partly from an IgG3 molecule (eg, middle hinge sequence). .. In another example, the Fc region or moiety may comprise a chimeric hinge, which is partly derived from an IgG1 molecule and partly derived from an IgG4 molecule. In another embodiment, the chimeric hinge may include upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such chimeric hinges may be made, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the central hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may comprise an amino acid at EU positions 233-236, the amino acid being from an IgG2 antibody and / or a Ser228Pro mutation, where the remaining amino acids of the hinge are from an IgG4 antibody. (For example, the chimeric hinge of the sequence ESKYGPCPPCPAPPVAGP). Further, a chimeric hinge that may be used in the Fc portion of an antibody according to the invention is described in US Publication No. 2005/0163783A1.

Specifically included in the definition of "Fc region" is "aglycosylated Fc region". The term "glycated Fc region" lacks covalently linked oligosaccharides or glycans at the N-glycosylation site at EU position 297, for example, in one or more Fc moieties of the Fc region. Refers to the Fc region. For example, the glycated Fc region may be completely glycated, that is, the entire Fc portion of the Fc region may be deficient in carbohydrates. Alternatively, the glycated Fc region may be partially glycated (ie, hemi-glycosylated). The glycated Fc region may be a deglycosylated Fc region, i.e., an Fc region from which Fc carbohydrates have been removed, eg, chemically or enzymatically. Alternatively or further, the glycosylated Fc region is nonglycosylated (ie, unglycosylated) (ie, without Fc carbohydrates (eg, glycosylation pattern (eg, EU position 297 or 299)). Mutation of one or more residues encoding the N-glycosylation site in (eg, expression in organisms (eg, bacteria) that do not naturally bind carbohydrates to proteins, or glycosylation mechanisms are genetic manipulations or glycosylation inhibitors (eg,) Antibodies expressed (by expression in host cells or organisms that are deficient by the addition of (glycosylation enzyme inhibitors)) are obtained. Alternatively, the Fc region is a "glycosylated Fc region", i.e., the Fc region is fully glycosylated at all effective glycosylation sites.

In the present invention, the Fc moiety or Fc region preferably has or consists of an amino acid sequence derived from a sequence of human immunoglobulin (eg, Fc region or Fc moiety from a human IgG molecule). However, the polypeptide may contain one or more amino acids from another mammalian species. For example, the Fc portion of a primate or the binding site of a primate may be included in the polypeptide of interest. Alternatively, one or more mouse amino acids may be present in the Fc portion or in the Fc region.

Preferably, the multispecific anti-cytokine (preferably anti-GM-CSF) antibody according to the present invention contains, in particular, other moieties in addition to the Fc moieties as described above. Is derived from a constant region, particularly a constant region of IgG, preferably a constant region of IgG1, more preferably a constant region of human IgG1. More preferably, the multispecific antibody according to the present invention is particularly preferably all other parts of the constant region, particularly all other parts of the constant region of IgG, in addition to the Fc moiety as described above. Contains all of the other parts of the constant region of IgG1, more preferably all of the other parts of the constant region of human IgG1.

As outlined above, a particularly preferred multispecific antibody according to the invention comprises a (complete) Fc region derived from human IgG. More preferably, the multispecific antibody according to the invention particularly comprises a (complete) Fc region derived from human IgG and all other parts of the constant region of IgG, preferably the constant region of IgG1. It also includes all of the other parts, more preferably all of the other parts of the constant region of human IgG1.

(Cytokine)
The multispecific anti-cytokine (preferably anti-GM-CSF) antibody or antigen-binding fragment thereof according to the present invention binds to a cytokine, preferably GM-CSF. Cytokines are usually small proteins (~ 5-20 kDa) that are important in cellular signaling. The cytokine is released by the cell and affects the behavior of other cells, sometimes affecting the behavior of the cell releasing the cytokine itself. Cytokines include chemokines, interferons, interleukins, lymphocains, tumor necrosis factors, monokines and colony-stimulating factors, but generally hormone-free. Cytokines are produced by a wide range of cells, including immune cell-like macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various interstitial cells, thereby predetermining. Cytokines may be produced by more than one type of cell.

Chemokines mediate cell-cell chemical attraction (chemotactic). Cytokine proteins are classified as chemokines based on their behavioral and structural characteristics. In addition to being known to mediate chemotaxis, chemokines are all about 8-10 kDa in size and are important for forming the three-dimensional shape of the chemokine at the conserved position. It has four cysteine residues. These proteins have historically been known under several other names, such as the SIS family of cytokines, the SIG family of cytokines, the SCY family of cytokines, and the platelet factor-4 superfamily. Or contains interclean. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC.

CC chemokines (or β-chemokines) proteins have two adjacent cysteines (amino acids) near their amino terminus. For mammals, at least 27 identifiable group members belonging to this subgroup have been reported and are called CC chemokine ligands (CCL) -1 to -28; CCL10 is identical to CCL9. This subfamily of chemokines usually contains 4 cysteines (C4-CC chemokines), whereas a few CC chemokines carry 6 cysteines (C6-CC chemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23 and CCL28. CC chemokines cause migration of monocytes and other cell types such as NK cells and dendritic cells. Examples of CC chemokines include monocyte chemotactic protein-1 (MCP-1 or CCL2), which causes monocytes to move away from the bloodstream into surrounding tissues. Enters and causes to become tissue macrophages. CCL5 (or RANTES) attracts cells such as T cells, eosinophils and basophils that express the receptor CCR5. Increased CCL11 levels in plasma are associated with aging (and decreased neurogenesis) in mice and humans. CC chemokines include, for example, CCL1-CCL28.

The two N-terminal cysteines of CXC chemokines (or α-chemokines) are separated by an amino acid, which is represented by this name using "X". There are 17 different CXC chemokines described in mammals, which have been subdivided into two categories, which are glutamate-leucine-arginine (or ELR for short) immediately prior to the first cysteine of the CXC motif. ) Has a specific amino acid sequence (or motif) (ELR-positive), and does not have an ELR motif (ELR-negative). ELR-Positive CXC chemokines specifically induce neutrophil migration and interact with chemokine receptors CXCR1 and CXCR2. An example of an ELR-positive CXC chemokine is interleukin-8 (IL-8), which causes neutrophils to leave the bloodstream and enter the surrounding tissue. Other CXC chemokines lacking the ELR motif, such as CXCL13, tend to be chemically attracted to lymphocytes. CXC chemokines bind to CXC chemokine receptors and seven CXC chemokine receptors have been discovered to date and are represented as CXCR1-7. CXC chemokines include, for example, CXCL1-CXCL17.

A third group of chemokines is known as C chemokines (or γ chemokines), the third group being the only two cysteines in the third group; one N-terminal cysteine and one downstream. It differs from all other chemokines in that it has cysteine. Two chemokines have been described for this subgroup, the two chemokines being called XCL1 (phosphotactin-α) and XCL2 (phosphotactin-β).

The fourth group have also been found, the group member has the three amino acids between the two cysteines, referred to as CX 3 _C chemokines (or d- chemokines). To date, only one CX ₃ C chemokine has been discovered and is called a fractal kine (or CX ₃ CL1). The single CX ₃ C chemokine is both secreted and tethered to the surface of the cell expressing the single CX ₃ C chemokine, resulting in the single CX ₃ C chemokine. Serves as both a chemical migration and an adhesion molecule.

Interferons (IFNs) are a group of cytokines that are produced and released in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. In a typical scenario, virus-infected cells will release interferon, which causes nearby cells to enhance their antiviral defenses. More than 20 identifiable IFN genes and proteins have been identified in animals, including humans. Human interferon is classified into three main types based on the type of receptor: type I IFN, type II IFN, and type III IFN, and the human interferon communicates through the receptor. IFNs, which belong to all three categories, are important for combating viral infections and for the regulation of the immune system.

All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which is the IFNAR 1 chain and the IFNAR 1 chain. It consists of two IFNAR chains. The type I interferons present in humans are IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. In general, type I interferon is produced when the body recognizes that the virus has invaded the body. Type I interferon is produced by fibroblasts and monocytes. However, the production of type I IFN-α can be hampered by another cytokine known as interleukin-10. Once activated, type I interferon can produce molecules that prevent the virus from producing and replicating the viral RNA and DNA. Overall, IFN-α has been proposed to be used to treat hepatitis B and hepatitis C infections, while IFN-β has been proposed to treat multiple sclerosis. It has been proposed to be used.

Interferon type II is also known as immune interferon and is specifically activated by interleukin-12. In addition, type II interferon is released by helper T cells, especially type 1. However, type II interferon can block the proliferation of helper T cell type 2. So far, the result is inhibition of the Th2 immune response and further induction of the Th1 immune response, which leads to the progression of debilitating diseases such as multiple sclerosis. IFN type II binds to IFNGR, which consists of one IFNGR chain and two IFNGR chains. An exemplary INF type II in humans is IFN-γ.

Interferon type III communicates through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although more recently discovered than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of viral infections.

Interleukins are a group of cytokines, first understood to be expressed by white blood cells (leukocytes). The functioning of the immune system is largely dependent on interleukins, some rare interleukin deficiencies have been described, and all of the interleukins characterize autoimmune diseases or immunodeficiencies. The majority of interleukins are synthesized by helper CD4T lymphocytes and through monocytes, macrophages, and endothelial cells. Interleukins promote the development and differentiation of T and B lymphocytes, as well as hematopoietic cells. Examples of interleukins are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. ,including.

Lymphocains are a subpopulation of cytokines, which are produced by lymphocytes. Lymphocaines are protein mediators that are generally produced by T cells and are responsible for directing the immune system response through signal transduction between the cells. Lymphocains have many roles, in order to attract other immune cells, including macrophages and other lymphocytes, to the infected site, and to initiate the subsequent immune response. Includes their activation, to prepare them. Circulating lymphocytes can detect very low concentrations of lymphocaine, after which the circulating lymphocytes can elevate the concentration gradient to a concentration gradient that requires an immune response. is there. Lymphocain helps B cells produce antibodies. Important lymphocines secreted by helper T cells include: IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, and interferon-gamma.

Tumor necrosis factor (or TNF family) refers to a group of cytokines that can cause cell death (apoptosis). 19 proteins as part of the TNF family based on sequence, function, and structural similarities (tumor necrosis factor (TNF), also known as cassette or TNF alpha, a cytokine with various functions) Has been identified). For example, tumor necrosis factor can cause cell lysis of certain tumor cell lines, is involved in the induction of malaise, and is a potent cause of heat either by direct action or by stimulation of interleukin-1 secretion. It is a pyrogen and can stimulate cell proliferation and induce cell differentiation under certain circumstances. The 19 proteins described above are two related cytokines, phosphorus photoxin-alpha (LT-alpha) and phosphorus photoxin, which are cytotoxic to a wide range of tumor cells in vitro and in vivo and are produced by lymphocytes. -Beta (LT-beta); T cell antigen gp39 (CD40L), a cytokine that appears to be important for B cell development and activation; T cells that play a role in T cell activation and are co-stimulated. CD27L, a cytokine that induces the proliferation of cytotoxic T cells and improves the production of cytotoxic T cells, CD30L, a cytokine that induces the proliferation of T cells, FASL; T cells, which are cell surface proteins involved in cell death. 4-1BBL, an inducible T cell surface molecule that contributes to stimulation; OX40L, a cell surface protein that co-stimulates T cell proliferation and cytokine production; and TNF-related apoptosis, a cytokine that induces apoptosis. Inducing ligand (TRAIL), including.

All these TNF family members appear to form a complex of homotrimers (or heterotrimers in the case of LT-alpha / beta) recognized by their specific receptors. .. Strong hydrogen bonds between the monomers stabilize the tertiary structure. One example is the Asn34-Arg82 hydrogen bond in TNF alpha of M. musculus. The PROSITE pattern for this family is located in beta-strands in the central portion of the protein, which is conserved throughout all its members. All members of the TNF family are type II transmembrane proteins that project from immune cells, with the exception of secreted phosphorus photoxins and growth-inducing ligands (APRIL). Such membrane-bound TNF ligands often signal the original immune cell when the membrane-bound TNF ligand contacts and binds to their syngeneic receptors on other cells. Examples of members of the TNF family are CD40LG (TNFSF5); CD70 (TNFSF7); EDA; FASLG (TNFSF6); LTA (TNFSF1); LTB (TNFSF3); TNF, TNFSF4 (OX40L); TNFSF8 (CD153); Includes TNFSF10 (TRAIL); TNFSF11 (RANKL); TNFSF12 (TWEAK); TNFSF13; TNFSF13B; TNFSF14; TNFSF15; TNFSF18.

Monokines are cytokines, and monokines are produced primarily by monocytes and macrophages. Examples of monokines include IL-1, TNF alpha, interferon alpha and interferon beta, and colony stimulating factor.

Colony-stimulating factors (CSFs) are secreted glycoproteins that bind to receptor proteins on the surface of hematopoietic stem cells, causing the cells to proliferate and differentiate into certain types of blood cells. Obtain, activate intracellular signal transduction pathways. In contrast to other membrane-binding substances in the hematopoietic microenvironment, colony-stimulating factors are usually soluble. This is sometimes used as a definition of CSFs. Colony-stimulating factor is transmitted by signaling paracrine, endocrine, or autoclines. Examples of colony stimulating factors are CSF1 (also known as "macrophage colony stimulating factor"), CSF2 ("granulocyte macrophage colony stimulating factor"; also known as GM-CSF and sargramostim), CSF3 (" Granulocyte colony stimulating factor (also known as G-CSF and filgrastim), and synthetic CSFs such as promegapoetin.

In the context of the present invention, a cytokine to which a multispecific, anti-cytokine (preferably anti-GM-CSF) antibody, which has two different domains that bind to two different, non-overlapping sites in the cytokine, particularly binds. Is preferably a colony stimulating factor or interferon. Among the colony stimulating factors, naturally occurring CSFs are preferable (particularly CSF1, CSF2 (GM-CSF) and CSF3 (G-CSF)), and GM-CSF is more preferable. Among the interferons, type I and type II interferons are preferable, type I interferons are more preferable, and interferon β is even more preferable. Therefore, the cytokine is preferably GM-CSF or interferon beta, and the more preferred cytokine is GM-CSF.

(Linker)
Preferably, the antibody or antigen-binding fragment thereof according to the present invention further comprises: (c) at least one linker.

In general, the linkage between the two components of a multispecific anti-cytokine (preferably anti-GM-CSF) antibody according to the invention may be direct or indirect. Good. That is, the two components may be directly adjacent, or the two components may be linked by additional components of the complex, such as by a linker. In particular, some components of the multispecific antibody according to the invention may be directly linked, whereas other components are linked by a linker. Preferably, the multispecific antibody according to the invention comprises a linker in, for example, two VH sequences, two VL sequences, and / or a heavy chain between the VH and VL sequences (“VH sequence””. Is derived from the heavy chain of a monospecific antibody and is therefore referred to as "VH" even if it may be present in the heavy or light chain of the multispecific antibody according to the invention. Also, the "VL sequence" is derived from the light chain of the monospecific antibody, so it may be present even within the heavy or light chain of the multispecific antibody according to the invention. If not, it is called "VL"). Thus, in the light chain, the linker may preferably be present between two VH sequences, two VL sequences, and / or VH and VL sequences. Furthermore, it is also preferred that the linker is present in the heavy chain of the multispecific antibody according to the invention between constant domains such as CH3 (eg in IgG CH1-CH2-CH3) and the VH / VL sequence. ..

As used herein, the terms "concatenated," "fused," or "fused" may be used interchangeably. These terms refer to the binding of two or more elements or components, by any means, including chemical binding or recombinant means. Methods of chemical bonding (eg, chemical bonding using a heterobifunctional cross-linking agent) are technically known. Preferably, the components of the antibody according to the invention are, for example, by covalent linkage or attachment of two or more proteins, polypeptides, or fragments thereof, eg, via their individual peptide backbones, eg, of them. Linked through expression of a single protein molecule encoding a component or peptide fragment. A preferred fusion is in-frame. That is, at the level of the encoding nucleic acid molecule, two or more open reading frames (ORFs) are fused and continuous longer ORFs in a way that maintains the correct reading frame of the original multiple ORFs. To form. Thus, the resulting recombinant fusion protein will contain two or more protein fragments that correspond to the polypeptides encoded by the original multiple ORFs, which are not naturally bound in that way. It is a single polypeptide containing. Although the reading frame is thus continuously produced throughout the fused gene fragment, the protein fragment may be physically or spatially separated, for example, by a (in-frame) peptide linker. ..

Therefore, the linker linking the two components of the multispecific antibody may be a peptide linker. Alternatively, the linker may also be non-peptide, eg, a cross-linking agent, but peptide linkers are preferred.

Non-peptidic spacers can include esters, thioesters, and disulfides, or may be esters, thioesters, and disulfides. Crosslinking agents for cross-linking peptides or proteins are, for example, (i) cross-linking agents for amines and amines, eg, NHS-esters and imide esters for the selective binding of primary amines. Homobifunctional amine-specific protein cross-linking reagent based on reactive groups; short-term and long-term effective, cleaveable, irreversible, membrane-permeable, and cell surface diverse; (ii) ) Crosslinking agents between crosslinks and carbohydrates, eg, maleimide and hydrazide reactive groups-based crosslinking reagents for the binding and formation of covalent crosslinks; (iii) A crosslinker, eg, based on maleimide or pyridyldithiol reactive groups for selective and covalent crosslinking of proteins and peptide thiols (reduced cysteines) to form stable thioether bonds. Homobifunctional sulfidelyl-specific cross-linking reagent; (iv) a photoreactive cross-linking agent, eg, proteins, nucleic acids and others involved in a receptor-ligand interaction complex via two-step activation. With aryl azide, diazilin, and other photo-reactive (photo-active) chemically heterobifunctional cross-linking reagents for binding the molecular structure of (v) amines and sulfideryl. There are, for example, heterobifunctional protein cross-linking reagents for binding between primary amine (lysine) and sulfideryl (cysteine) groups of proteins and other molecules; different lengths and types of spacer arms. Available; as well as (vi) amine-to-amine cross-linking agents, eg, carboxyl-amine cross-linking agents, eg, carboxyl groups (glutamate, aspartate, C-terminal) are primary. Carbodiimide cross-linking reagents for binding to amines (lysine, N-terminal), DDC and EDC (EDAC), and also N-hydroxysuccinimide (NHS) for stable activation of carboxylates for amine binding. ..

Peptidic linkers are preferably composed of about 1-30 amino acids, whereby "short linkers" are preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. It is composed of amino acids, more preferably from about 1, 2, 3, 4, 5, or 6 amino acids, more preferably from 4 to 6 amino acids, particularly preferably from 5 amino acids, which is preferably composed of SEQ ID NO: 143 or a functional sequence variant thereof may be followed. In contrast, a "long linker" is preferably composed of about 10-30 amino acids, more preferably about 12-25 amino acids, and even more preferably about 14-20 amino acids. A particularly preferred long linker has 15 to 17 amino acids, preferably 16 amino acids, more preferably according to SEQ ID NO: 144 or a functional sequence variant thereof. In the multispecific antibody according to the invention, the long linker is preferably the VH sequence and the corresponding VL sequence (“corresponding” is herein the VH and the VH forming the epitope binding site together. VL sequence), eg, a VH sequence and a VL sequence derived from the same monospecific antibody are linked. The short linker may preferably ligate the VH / VL sequence with an "unmatched" VH / VL sequence, eg, from a different monospecific antibody, and / or preferably a constant domain. For example, CH3 (eg, in IgG CH1-CH2-CH3) may be linked to the VH / VL sequence.

Preferred linkers contain or consist of an amino acid sequence according to SEQ ID NO: 143 or SEQ ID NO: 144 or a functional sequence variant thereof.

Alternatively, the amino acid sequence of the peptidic linker may be identical to the amino acid sequence of the N-terminal or C-terminal flanking region. Alternatively, the peptidic linker can be composed of an unnatural amino acid sequence, such as the amino acid sequence resulting from the conserved amino acid substitution of the natural flanking region. In certain embodiments, the peptidic spacer does not contain any Cys (C) residues. In a preferred embodiment, the linker sequence contains at least 20%, more preferably at least 40%, more preferably at least 50%, and particularly preferably at least 70% of the Gly (G) or β-alanine residue (A). , Includes. More preferably, the linker sequence contains at least 20%, more preferably at least 40%, more preferably at least 50%, and particularly preferably at least 70% of Gly (G) residues. Suitable linker sequences can be readily selected and prepared by those skilled in the art. The linker sequence may be composed of D and / or L amino acids.

(Antibody composition and construction type)
In principle, a multispecific antibody according to the invention is one of at least two different domains in which the antibody specifically binds to at least two different, non-overlapping sites in a cytokine (eg, GM-CSF). And any antibody form may be used as long as it contains an Fc portion.

For example, the antibody may be a multispecific antibody fragment having an Fc portion. In particular, examples of bispecific antibody fragments with Fc moieties include tandem scFv-Fc, scFv-Fc, scFv-Fc knob-into-holes, scFv-Fc-scFv, and scDiabody. -Fc, which are shown, for example, in Figure 3b of Chan, AC and Carter, PJ (2010) Nat Rev Immu 10: 301-316, and are also described in the above literature.

Antibodies according to the present invention may be based on any immunoglobulin class (eg IgA, IgG, IgM, etc.) and subclasses (eg, IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, etc.). Preferably, the multispecific antibody according to the invention is based on IgG (also referred to as "IgG type"). Within the IgG class, the antibody may be based on the IgG1, IgG2, IgG3, or IgG4 subclass, and thus an antibody based on IgG1 (also referred to as "IgG1 type") is preferred. Preferably, the antibody of the invention may have a κ light chain or a λ light chain.

Multispecific antibody forms based on IgG are well known to those of skill in the art. Preferred IgG-based antibody forms are, for example, hybrid hybridomas, knob-into-holes with common light chains, various IgG-scFv forms, various scFv-IgG forms, two-in-one IgG (two-). Includes in-one IgG), double (or multiple, eg, 3 times, 4 times, etc.) V-domain IgG, IgG-V, and V-IgG, which are based on bispecific IgG. Antibodies, or any combination thereof resulting in IgG-type multispecific antibodies, are shown in Figure 3c of Chan, AC and Carter, PJ (2010) Nat Rev Immu 10: 301-316, and the above literature. Explained in. Other preferred IgG-based antibody forms include, for example, DAF, cross-Mab, IgG-dsscFv, DVD, IgG-dsFV, IgG-scFab, scFab-dsscFv and Fv2-Fc, which are Weidle UH et al. 2013) Cancer Genomics and Proteomics 10: 1-18, shown in Figure 1A and also explained in the above literature.

Preferably, the multispecific antibody or antigen-binding fragment thereof according to the present invention is IgG type, preferably IgG1 type, and more preferably IgG1 CH1-CH2-CH3 type heavy chain constant region and IgG GK type. , And more preferably IgG1 CH1-CH2-CH3 type, which comprises or comprises an amino acid sequence according to SEQ ID NO: 140 or a functional sequence variant thereof. It comprises a heavy chain constant region and a light chain constant region of IgG GK type, which comprises or consists of an amino acid sequence according to SEQ ID NO: 141 or a functional sequence variant thereof.

Since IgG has two natural epitope binding sites, additional epitope binding sites, i.e. "non-natural" epitope binding sites, are CH3 of one or both heavy chains that are constructing the IgG antibody form. It is preferably bound to the domain, preferably to the C-terminal of the CH3 domain. Alternatively, additional epitope binding sites, i.e. "non-natural" epitope binding sites, are preferably light chain variable regions relative to one or both of the light chain variable regions of the natural epitope binding sites of IgG antibody form. For one or both N-terminals of, and / or for one or both of the heavy chain variable regions of the natural epitope binding site of the IgG antibody form, preferably for one or both of the heavy chain variable regions. It is preferably bound to the N-terminal of. Alternatively, an additional epitope binding site, i.e., an "unnatural" epitope binding site, is preferably the C-terminal of the CH3 domain relative to the CH3 domain of one or both heavy chains constructing the IgG antibody form. And for one or both of the light chain variable regions of the natural epitope binding site of the IgG antibody form, preferably for the N-terminal of one or both of the light chain variable regions. To be combined. Alternatively, an additional epitope binding site, i.e., an "unnatural" epitope binding site, is preferably the C-terminal of the CH3 domain relative to the CH3 domain of one or both heavy chains constructing the IgG antibody form. And for one or both of the heavy chain variable regions of the natural epitope binding site of the IgG antibody form, preferably for the N-terminal of one or both of the heavy chain variable regions. To be combined.

In any such attachment site of the IgG antibody form, preferably in the light and / or heavy chain variable regions of the natural epitope binding site, and / or in the CH3 domain of the heavy chain, one or more, eg, 2 One, three, four, or more (s) of non-natural epitope binding sites may be attached, whereby one or two (s) of non-natural epitope binding sites are attached. Preferably, attachment of one non-natural epitope binding site is more preferred.

For example, if the antibody according to the invention is an IgG-type bispecific antibody, one "specificity" is preferably provided by the natural epitope binding site and the other "specificity" is preferably. , Provided by a non-natural epitope binding site. In particular, the non-natural epitope binding site may then be attached to any of the above-mentioned attachment sites, preferably the light chain variable region of the natural epitope binding site, the heavy chain variable of the natural epitope binding site. It may be attached to a region, or a heavy chain CH3 domain in the form of an IgG antibody.

For example, if the antibody according to the invention is a trispecific antibody in the form of an IgG antibody, one "specificity" ("specificity 1") is preferably provided by the natural epitope binding site and the other. The two "specificities" ("specificity 2" and "specificity 3") are preferably provided by non-natural epitope binding sites. Thereby, the non-natural epitope binding site for "specificity 2" may preferably be attached to any of the aforementioned attachment sites, preferably the light chain variable region of the natural epitope binding site. It may preferably be attached to the heavy chain variable region of the natural epitope binding site, or to the CH3 domain of the heavy chain in the form of an IgG antibody. On the other hand, the non-natural epitope binding site for "specificity 3" is preferably to any other aforementioned attachment site, i.e., the non-natural epitope binding site for "specificity 2". May be attached to any attachment site to which is not attached. Alternatively, non-natural epitope binding sites for the other two "specificities" ("specificity 2" and "specificity 3") may preferably be attached to the same attachment site. Well, for example, the non-natural epitope binding sites for the other two "specificities" ("specificity 2" and "specificity 3") are both in the light chain variable region of the natural epitope binding site. Non-natural epitope binding sites that are bound to or for two other "specificities" ("specificity 2" and "specificity 3") are both the weight of the natural epitope binding site. Non-natural epitope binding sites that bind to the chain variable region or for two other "specificities" ("specificity 2" and "specificity 3") are both IgG antibody forms. It is bound to the CH3 domain of the heavy chain of. In this case, i.e., the non-natural epitope binding sites for the other two "specificities" ("specificity 2" and "specificity 3") are both bound to the same attachment site. In the case, the non-natural epitope binding site for "specificity 2" and the non-natural epitope binding site for "specificity 3" are preferably arranged contiguously, i.e. one unnatural. Only the epitope binding site of is attached directly (or via a linker as described herein) to the attachment site, while the other non-natural epitope binding site is preferably. , Linked to the first non-natural epitope binding site.

In particular, the antibody according to the invention preferably comprises two copies of each of the epitope binding sites, whereby the two copies correspond to the first and second heavy chains of the IgG-based antibody. It is preferably adhered at the position. For example (i) one copy is bound to the heavy chain variable region of the natural epitope binding site of the first heavy chain of IgG antibody form and the other copy is of the second heavy chain of IgG antibody form. , Is bound to the heavy chain variable region of the natural epitope binding site; or (ii) one copy is to the light chain variable region of the natural epitope binding site of the first light chain in the form of an IgG antibody. And the other copy is bound to the light chain variable region of the natural epitope binding site of the second light chain in IgG antibody form; or (iii) one copy is in IgG antibody form. It is preferred that the first heavy chain is bound to the CH3 domain and the other copy is bound to the CH3 domain of the first light chain in IgG antibody form.

Preferred antibody forms for multispecific antibodies according to the present invention, and their construction, are described in US Publication No. US2009 / 0155275A1 which specifically comprises the Fc region of the constant domain of the antibody. It is associated with a multispecific epitope-binding protein. Therefore, the antibody form disclosed in US Publication No. US2009 / 0155275A1 is preferably used for the multispecific antibody according to the present invention.

Exemplary constructive types of antibodies according to the invention include constructive types "Bs1", "Bs2", "Bs3", "Ts1", "Ts2" and "Ts3", which are shown in FIG. Therefore, the multispecific antibody or antigen-binding fragment thereof according to the present invention is preferably based on a construct type selected from the group containing Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3. More preferably, the multispecific antibody or antigen-binding fragment thereof according to the present invention is based on constructive Ts3, and the preferred antibody or antigen-binding fragment thereof is a trispecific antibody based on constructive Ts3.

Constructed "Bs1" is an IgG-based, preferably IgG1-based, bispecific, tetravalent antibody form. The heavy chain of Bs1 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy or light chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the first unispecific antibody. Variable area;
(B) The corresponding heavy chain derived from the corresponding second variable region, eg, the first monospecific antibody, forming the first epitope binding site with the first variable region (a). Or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the first monospecific antibody;
(C) For example, a third variable region derived from the heavy chain of the second unispecific antibody; preferably a VH sequence derived from the second unispecific antibody, i.e., the heavy chain variable region; And (d) IgG CH1-CH2-CH3.

Preferably, the components (a) and (b) are linked by a long linker, the components (b) and (c) are linked by a short linker, and the components (c) and (d) are directly linked. Be connected. The light chain of Bs1 contains (in this order from N-terminus to C-terminus):
(E) The corresponding light chain variable region derived from the fourth variable region, eg, the second unispecific antibody, forming the second epitope binding site with the third variable region (c). VL sequences, i.e., light chain variable regions, preferably derived from a second monospecific antibody; and (f) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (e) and (f) are directly linked. Since Bs1 is based on IgG, Bs1 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody and where the CDR / variable regions (components (a) and (b)) forming the first epitope binding site in Bs1 are located is. The position referred to as "position A" and in the antibody, where the CDR / variable region (components (c) and (d)) forming the second epitope binding site in Bs1 is located is It is referred to as "position B" (see FIG. 4). In construction, the heavy and light chain variable regions from the (second) monospecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, said (second) single The specific antibody may serve as a "scaffold". A preferred embodiment of the constructive "Bs1" and the construct of the constructive "Bs1" are described in US Publication No. US2009 / 0155275A1, FIGS. 4E and 4F and the corresponding description.

Constructed "Bs2" is an IgG-based, preferably IgG1-based, bispecific, tetravalent antibody form. The heavy chain of Bs2 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain variable region, derived from the first unispecific antibody; And (b) IgG CH1-CH2-CH3.

Preferably, the components (a) and (b) are directly connected. The light chain of Bs2 contains (in this order from N-terminus to C-terminus):
(C) Second variable region, eg, derived from the heavy or light chain of the second unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the second unispecific antibody. Variable area;
(D) The corresponding heavy chain derived from the corresponding third variable region, eg, the second unispecific antibody, forming the first epitope binding site with the second variable region (c). Or the light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the second monospecific antibody;
(E) A fourth variable region that forms a second epitope binding site with the first variable region (a), eg, a corresponding light chain variable region derived from the first monospecific antibody. VL sequences, i.e. light chain variable regions, preferably derived from the first monospecific antibody; and (f) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (c) and (d) are linked by a long linker, the components (d) and (e) are linked by a short linker, and the components (e) and (f) are directly linked. Be connected. Since Bs2 is based on IgG, Bs2 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDR / variable regions (components (c) and (d)) forming the first epitope binding site in Bs2 are located, is The position referred to as "position A" and in the antibody where the CDR / variable region (components (a) and (e)) forming the second epitope binding site in Bs2 is located is It is referred to as "position B" (see FIG. 4). In construction, the heavy and light chain variable regions of the (first) monospecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, the (first) monospecific The sex antibody may serve as a "scaffold". Preferred embodiments of constructive "Bs1" and constructive constructive "Bs2" are described in US Publication US2009 / 0155275A1, FIGS. 4C and 4D and the corresponding descriptions.

Constructed "Bs3" is an IgG-based, preferably IgG1-based, bispecific, tetravalent antibody form. The heavy chain of Bs3 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain variable region, derived from the first unispecific antibody;
(B) IgG CH1-CH2-CH3;
(C) Second variable region, eg, derived from the heavy or light chain of the second unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the second unispecific antibody. Variable regions; and (d) derived from the corresponding third variable region, eg, a second monospecific antibody, forming a first epitope binding site with the second variable region (c). The corresponding heavy or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the second monospecific antibody.

Preferably, the components (a) and (b) are directly linked, the components (b) and (c) are linked by a short linker, and the components (c) and (d) are linked by a long linker. Be connected. The light chain of Bs3 contains (in this order from N-terminus to C-terminus):
(E) A fourth variable region that forms a second epitope binding site with the first variable region (a), eg, a corresponding light chain variable region derived from the first monospecific antibody. VL sequences, i.e. light chain variable regions, preferably derived from the first monospecific antibody; and (f) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (e) and (f) are directly linked. Since Bs3 is based on IgG, Bs3 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody and where the CDR / variable regions (components (c) and (d)) forming the first epitope binding site in Bs3 are located is. The position referred to as "position C" and in the antibody where the CDR / variable region (components (a) and (e)) forming the second epitope binding site in Bs3 is located is It is referred to as "position B" (see FIG. 4). In construction, the heavy and light chain variable regions of the (first) monospecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, the (first) monospecific The sex antibody may serve as a "scaffold". The principle of constructive "Bs3" (but not based on the present invention, constructive "Bs3" having other "specificity", i.e., an epitope binding site), and the construction of such constructive "Bs3" are also Dimasi, N., Gao, C., Fleming, R., Woods, RM, Yao, X.-T., Shirinian, L., Kiener, PA, and Wu, H. (2009). The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators. J Mol Biol 393, 672-692: illustrated in FIG. 1 (d) (“Bs3Ab”) and the corresponding description.

Constructed "Ts1" is an IgG-based, preferably IgG1-based, trispecific, hexavalent antibody form. The heavy chain of Ts1 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain variable region, derived from the first unispecific antibody;
(B) IgG CH1-CH2-CH3;
(C) Second variable region, eg, derived from the heavy or light chain of the second unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the second unispecific antibody. Variable area;
(D) The corresponding heavy chain derived from the corresponding third variable region, eg, the second unispecific antibody, forming the first epitope binding site with the second variable region (c). Or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the second monospecific antibody;
(E) A fourth variable region, eg, derived from the heavy or light chain of a third unispecific antibody; preferably a VH sequence, i.e., a heavy chain, derived from the third unispecific antibody. Variable regions; and (f) derived from a corresponding fifth variable region, eg, a third unispecific antibody, forming a second epitope binding site with a fourth variable region (e). The corresponding heavy or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from a third monospecific antibody.

Preferably, the components (a) and (b) are directly linked, the components (b) and (c) and the components (d) and (e) are linked by a short linker, and the components ( c) and (d) and the components (e) and (f) are linked by a long linker. The light chain of Ts1 contains (in this order from N-terminus to C-terminus):
(G) A sixth variable region that forms a third epitope binding site with the first variable region (a), eg, a corresponding light chain variable region derived from the first monospecific antibody. The VL sequence, i.e., the light chain variable region, preferably derived from the first monospecific antibody; and (h) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (g) and (h) are directly linked. Since Ts1 is based on IgG, Ts1 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDR / variable regions (components (c) and (d)) forming the first epitope binding site in Ts1 are located, and as well. , The position in the antibody where the CDR / variable region (components (e) and (f)) forming the second epitope binding site in Ts1 is located is referred to as "position C". And the position in the antibody where the CDR / variable region (components (a) and (g)) forming the third epitope binding site in Ts1 is located is referred to as "position B". (See Figure 4). In construction, the heavy and light chain variable regions of the (first) monospecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, the (first) monospecific The sex antibody may serve as a "scaffold". A preferred embodiment of the constructive "Ts1" and construction of the constructive "Ts1" are described in US Publication US2009 / 0155275A1, FIGS. 3A and 3B and the corresponding description.

Constructed "Ts2" is an IgG-based, preferably IgG1-based, trispecific, hexavalent antibody form. The heavy chain of Ts2 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy or light chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the first unispecific antibody. Variable area;
(B) The corresponding heavy chain derived from the corresponding second variable region, eg, the first monospecific antibody, forming the first epitope binding site with the first variable region (a). Or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the first monospecific antibody;
(C) For example, a third variable region derived from the heavy chain of the second unispecific antibody; preferably a VH sequence derived from the second unispecific antibody, i.e., the heavy chain variable region; And (d) IgG CH1-CH2-CH3.

Preferably, the components (a) and (b) are linked by a long linker, the components (b) and (c) are linked by a short linker, and the components (c) and (d) are directly linked. Be connected. The light chain of Ts2 contains (in this order from N-terminus to C-terminus):
(E) A fourth variable region, eg, derived from the heavy or light chain of a third unispecific antibody; preferably a VH sequence, i.e., a heavy chain, derived from the third unispecific antibody. Variable area;
(F) The corresponding heavy chain derived from the corresponding fifth variable region, eg, the third unispecific antibody, forming the second epitope binding site with the fourth variable region (e). Or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from a third unispecific antibody;
(G) The corresponding light chain variable region derived from the sixth variable region, eg, the second unispecific antibody, forming the third epitope binding site with the third variable region (c). The VL sequence, i.e., the light chain variable region, preferably derived from the second monospecific antibody; and (h) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (e) and (f) are linked by a long linker, the components (f) and (g) are linked by a short linker, and the components (g) and (h) are directly linked. Be connected. Since Ts2 is based on IgG, Ts2 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDR / variable regions (components (a) and (b)) forming the first epitope binding site in Ts2 are located, and , The position in the antibody where the CDR / variable region (components (e) and (f)) forming the second epitope binding site in Ts2 is located is referred to as "position A". And the position in the antibody where the CDR / variable region (components (c) and (g)) forming the third epitope binding site in Ts2 is located is referred to as "position B". (See Figure 4). In the construction, the heavy and light chain variable regions of the (second) unispecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, the (second) unispecific. The sex antibody may serve as a "scaffold". A preferred embodiment of the constructive "Ts2" and the construct of the constructive "Ts1" are described in US Publication US2009 / 0155275A1, FIGS. 4G and 4H and the corresponding description.

Constructed "Ts3" is an IgG-based, preferably IgG1-based, trispecific, hexavalent antibody form. The heavy chain of Ts3 contains (in this order from N-terminus to C-terminus):
(A) First variable region, eg, derived from the heavy or light chain of the first unispecific antibody; preferably the VH sequence, i.e., the heavy chain, derived from the first unispecific antibody. Variable area;
(B) The corresponding heavy chain derived from the corresponding second variable region, eg, the first monospecific antibody, forming the first epitope binding site with the first variable region (a). Or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from the first monospecific antibody;
(C) For example, a third variable region derived from the heavy chain of the second unispecific antibody; preferably a VH sequence derived from the second unispecific antibody, i.e., the heavy chain variable region;
(D) IgG CH1-CH2-CH3;
(E) A fourth variable region, eg, derived from the heavy or light chain of a third unispecific antibody; preferably a VH sequence, i.e., a heavy chain, derived from the third unispecific antibody. Variable regions; and (f) derived from a corresponding fifth variable region, eg, a third monospecific antibody, forming a second epitope binding site with a fourth variable region (e). Corresponding heavy or light chain variable region; preferably the VL sequence, i.e. the light chain variable region, derived from a third monospecific antibody.

Preferably, the components (a) and (b) and the components (e) and (f) are linked by a long linker, and the components (b) and (c) and the components (d) and (e) are short. It is linked by a linker, and components (c) and (d) are directly linked. The light chain of Ts3 contains (in this order from N-terminus to C-terminus):
(E) A corresponding light chain variable region derived from a sixth variable region, eg, a second unispecific antibody, forming a third epitope binding site with the third variable region (c). VL sequences, i.e., light chain variable regions, preferably derived from a second monospecific antibody; and (f) IgG CK or IG CL; preferably Ig CK.

Preferably, the components (e) and (f) are directly linked. Since Ts3 is based on IgG, Ts3 contains two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDR / variable regions (components (a) and (b)) forming the first epitope binding site in Ts3 are located, is The position in the antibody, referred to as "position A", where the CDR / variable region (components (e) and (f)) forming the second epitope binding site in Ts3 is located is "position A". The position referred to as "Position C" and in the antibody where the CDR / variable region (components (c) and (d)) forming the third epitope binding site in Ts3 is located is "Position C". It is referred to as "position B" (see FIG. 4). In the construction, the heavy and light chain variable regions of the (second) unispecific antibody are used for the heavy and light chains of the multispecific antibody, respectively, the (second) unispecific. The sex antibody may serve as a "scaffold".

For further information on the construction of constructive Bs1, Bs2, Bs3, Ts1, and Ts2, see Literature, US Publication No. 2009/015275A1 and Dimasi, N., Gao, C., Fleming, R., Woods. , RM, Yao, X.-T., Shirinian, L., Kiener, PA, and Wu, H. (2009): The design and characterization of oligospecific antibodies forsimultaneous targeting of multiple disease mediators; J Mol Biol 393, 672− 692 may be used. The construction principles outlined in these documents may be adapted to the new construction Ts3 and may subsequently be applied.

(Neutralization)
Preferably, the multispecific antibody or antigen-binding fragment thereof according to the present invention neutralizes the target effect of cytokines (particularly GM-CSF) under the following conditions.
(I) Strict conditions with an IC ₉₀ of 150 ng / ml or less, preferably 120 ng / ml or less, more preferably 100 ng / ml or less, even more preferably 50 ng / ml or less, particularly preferably 10 ng / ml or less;
(Ii) 20 ng / ml or less, preferably 15 ng / ml or less, more preferably 10 ng / ml or less, even more preferably 5 ng / ml or less, particularly preferably 1 ng / ml or less, with an IC ₉₀ of a lower degree. Strict conditions;
(Iii) More rigorous conditions with IC ₉₀ of 160 ng / ml or less, preferably 130 ng / ml or less, more preferably 100 ng / ml or less, even more preferably 50 ng / ml or less, particularly preferably 10 ng / ml or less. ; And / or
(Iv) Very rigorous with IC ₉₀ of 1000 ng / ml or less, preferably 500 ng / ml or less, more preferably 250 ng / ml or less, even more preferably 100 ng / ml or less, particularly preferably 50 ng / ml or less. conditions.

Here, "IC _{90 of} xng / ml or less" represents the concentration (xng / ml or less) of the antibody or antigen-binding fragment thereof according to the present invention. The above concentration is the concentration required for 90% neutralization (IC ₉₀ ) of the target effect of cytokines (eg, GM-CSF).

In general, antibody functionality is assessed by its ability to neutralize significant effects (“target effects”) of cytokines (eg, GM-CSF). In the neutralization assay, for example, the target effect of a cytokine (eg, GM-CSF) or the concentration of antibody required to neutralize the infectivity of the virus can be determined. Various neutralization assays are known to those of skill in the art. And usually, one of ordinary skill in the art will select a neutralization assay depending on the cytokine (eg, GM-CSF) and its targeting effect. Targeting effects of cytokines (eg, GM-CSF) useful in neutralization assays include, for example, cytokine-induced proliferation. The cytokine-induced proliferation is, for example, index cell line, cytokine-induced cytokine production, lethality of TNF-α-induced L929 cell line, and IFN-γ-protection against viral infection of L929 cell line and A549 cell line. Is a cytokine-induced proliferation.

Below are non-limiting examples of neutralization assays to illustrate the principles of neutralization assays. (1) Different concentrations of antibody to be tested (eg, dilution series) are prepared, for example, in a microtiter plate (or any other suitable container);
(2) A target amount of cytokine (ie, a predetermined amount of cytokine (eg, GM-CSF)) is added to the antibody and incubated together under appropriate conditions (eg, room temperature or 37 ° C. for 1 hour);
(3) Wells containing co-incubated antibody-cytokines (eg, co-incubated antibodies-GM-CSF) in appropriate culture target cells (eg, target cells (eg, monolayer target cells)). Move in). Then, incubate for a predetermined period (for example, 1, 2, 3, 4, 5, 6 or 7 days) at room temperature or 37 ° C.;
(4) After that, the target effect can be quantitatively analyzed to determine the IC ₉₀ .

The effect measured is usually quantity-dependent. That is, the higher the antibody titer of the antibody, the higher the degree of neutralization of the target effect. The value of IC ₉₀ changes depending on the neutralizing property of the antibody. For example, an antibody having a large neutralizing property needs to be added in a smaller amount (antibody) in order to neutralize the target effect to the same extent in the above assay, for example.

As used herein, "lower strict conditions" refers to a final concentration of about 50 pg / ml cytokine (eg, GM-CSF), and about 1000 cells / well. "Strict conditions" represent a final concentration of about 50 pg / ml cytokine (eg, GM-CSF), and about 10,000 cells / well. "Stricter conditions" represent a final concentration of about 500 pg / ml cytokine (eg, GM-CSF), and about 1000 cells / well. "Very strict conditions" represent a final concentration of about 500 pg / ml cytokine (eg, GM-CSF), and about 10,000 cells / well.

Examples of suitable indicator cell lines for a neutralization assay of cytokine-induced proliferation or cytokine-induced cytokine production are TF-1, MC / 9, L929, D10, CTLL-2, B9, splenocytes. , NIH / 3T3, COLO205, A549, hPBMC, NHDF and Nag7 / 8. TF-1 cells are preferably used to neutralize the cytokines GM-CSF, IL-13, IL-4 and IL-5. MC / 9 cells are preferably used to neutralize the cytokines GM-CSF, IL-5 and IL-10. L929 cells are preferably used to neutralize the cytokines IFN-gamma and TNF-alpha. D10 cells are preferably used to neutralize the cytokines IL-1alpha and IL-1beta. CTLL-2 cells are preferably used to neutralize the cytokines IL-2, IL-4 and IL-5. B9 cells are preferably used to neutralize the cytokines IL-6 and IL-21. Spleen cells are preferably used to neutralize the cytokines IL-12 / IL-23p40 and IL-23. NIH / 3T3 cells are preferably used to neutralize the cytokine IL-17A. COLO205 cells are preferably used to neutralize the cytokine IL-22. A549 cells are preferably used to neutralize the cytokines IFN-gamma and INF beta. hPBMC is preferably used to neutralize the cytokines IL-12 / IL23p40. NHDF cells are preferably used to neutralize the cytokine IL-17A. Nag7 / 8 cells are preferably used to neutralize the cytokine TSLP.

When assessing the ability of an antibody to neutralize the cytokine GM-CSF, especially the ability to neutralize GM-CSF-induced proliferation, the cells that are favorably dispersed are TF-1 cells. A preferred GM-CSF neutralization assay comprises the following steps:
(1) Different concentrations of antibody to be tested (eg, dilution series) are prepared, for example, in a microtiter plate (or any other suitable container);
(2) A target amount of GM-CSF (eg, 100 pg / ml) is added to the antibody and incubated together under appropriate conditions (specifically, at 37 ° C. for 1 hour);
(3) Transfer the co-incubated antibody-GM-CSF into TF-1 cells (specifically, into wells containing 1000 or 10000 TF-1 cells per well). Then, incubate, for example, at 37 ° C. for 3-4 days (eg, 72 hours); (4) then quantitative analysis of growth neutralization can be performed to determine IC ₉₀ . For example, the neutralization of GM-CSF can be calculated by the following formula as the rate at which the growth of TF-1 is inhibited.
[1-{(CCPM in one well)-(mean CCPM for control cell growth (without GM-CSF))} / {(mean CCPM for control cell growth (with GM-CSF))-(control cell growth (GM) -Average CCPM (without CSF)}]] x 100
(CCPM = corrected count / min) For all samples, IC90 (μg / ml) can be calculated by non-linear regression analysis (eg, using GraphPad Prism 5 software).

In the above assay, the lower rigorous conditions represent a final concentration of about 50 pg / ml of GM-CSF and about 1000 cells / well of TF-1. Strict conditions represent a final concentration of about 50 pg / ml of GM-CSF and about 10000 cells / well of TF-1. More stringent conditions represent a final concentration of about 500 pg / ml of GM-CSF and about 1000 cells / well of TF-1. Very strict conditions represent a final concentration of GM-CSF of about 500 pg / ml and TF-1 of about 10000 cells / well.

(Variable region and CDR)
The antibody or antigen-binding fragment thereof according to the present invention preferably contains one or more complementarity determining regions (CDRs). Generally, complementarity determining regions (CDRs) are hypervariable regions that are present in the heavy and light chain variable domains of an antibody. The antibody or antigen-binding fragment thereof according to the present invention preferably contains at least three CDRs on the heavy chain and at least three CDRs on the light chain. It is more preferable that the antibody or antigen-binding fragment thereof according to the present invention contains at least 6 CDRs on the heavy chain and at least 3 CDRs on the light chain. The antibody or antigen-binding fragment thereof according to the present invention (1) contains at least 9 CDRs on the heavy chain and contains at least 3 CDRs on the light chain, or (2) heavy. It is even more preferred to have at least 6 CDRs on the chain and at least 6 CDRs on the light chain.

Usually, the domain of an antibody that specifically binds to an epitope of an antigen (eg, to a specific site of a cytokine molecule (particularly GM-CSF)) is also referred to as an "antigen receptor" or "epitope binding site". The domain of the antibody (ie, the antigen receptor / epitope binding site) is usually the three CDRs of the heavy chain and the three CDRs of the linked light chain, especially in the natural monospecific IgG antibody. Is formed by. In other words, especially in natural monospecific IgG antibodies, the antigen receptor / epitope binding site is usually composed of two variable domains, so that each antibody receptor has six CDRs (heavy chain). : CDRH1, CDRH2 and CDRH3; Light chain: CDRL1, CDRL2 and CDRL3). One antibody, particularly one natural monospecific IgG antibody, usually has two (identical) antigen receptors and therefore contains 12 CDRs (ie 2x6). CDR).

However, the multispecific antibody or antigen-binding fragment thereof according to the present invention contains at least two different domains. The at least two different domains specifically bind to at least two different non-overlapping sites of cytokines (particularly GM-CSF). One molecule of the multispecific antibody according to the present invention or an antigen-binding fragment thereof preferably contains two identical domains for each different domain. Each of the above different domains specifically binds to at least two different non-overlapping sites of cytokines. It is also preferable that one molecule of the multispecific antibody according to the present invention or an antigen-binding fragment thereof contains two heavy chains and two light chains. One molecule of the multispecific antibody according to the present invention or an antigen-binding fragment thereof contains two heavy chains and two light chains, and (1) the above two heavy chains are preferably at least 80%. Has 85%, more preferably 90%, even more preferably 95%, particularly preferably 100% sequence identity and / or (2) the two light chains are at least 80. %, More preferably 85%, more preferably 90%, even more preferably 95%, and particularly preferably 100% sequence identity. Therefore, one molecule of the multispecific antibody according to the present invention or an antigen-binding fragment thereof contains (1) two identical domains for each different domain, and (2) the different domains are cytokines (eg, for example. When specifically binding to at least two different non-overlapping sites of GM-CSF), one of the same domains is composed of a first heavy chain and a first light chain of the multispecific antibody. The other is preferably composed of the second heavy chain and the second light chain of the multispecific antibody.

The heavy and / or light chains of the multispecific antibody according to the invention, or antigen-binding fragments thereof, have more than three CDRs (particularly, different epitope binding sites) due to their "multispecificity (ie, different epitope binding sites)". More than three different CDRs) may be included (each). For example, a multispecific antibody or antigen-binding fragment thereof according to the present invention contains at least two different domains that specifically bind to at least two different non-overlapping sites of cytokines (particularly GM-CSF). Good. Here, each of the at least two different domains is derived from a different monospecific antibody (eg, of the IgG type). Such monospecific antibodies usually form an antigen receptor / epitope binding site by including three CDRs in the heavy chain and three CDRs in the light chain. Therefore, the multispecific antibody according to the present invention includes (1) three CDRs of the heavy chain of the first antibody, three CDRs of the light chain of the first antibody, and (2) the heavy chain of the second antibody. Three CDRs and three CDRs of the light chain of the second antibody, (3) optionally three CDRs of the heavy chain of the third antibody and three CDRs of the light chain of the third antibody, etc., in particular. May include. Therefore, the number of CDRs contained in the heavy chain and / or light chain of the multispecific antibody according to the present invention is preferably a multiple of 3 (for example, 3, 6, 9, 12, etc.). Therefore, it is also preferable that the total CDR contained in both the heavy chain and the light chain of the multispecific antibody according to the present invention is a multiple of 6 (for example, 6, 12, 18, etc.).

In particular, in a multispecific antibody or antigen-binding fragment thereof according to the present invention, the heavy chain may also contain a CDR or variable region derived from the light chain of the monospecific antibody. For example, in the multispecific antibody according to the present invention, the heavy chain is (1) a heavy chain variable region (VH) and a light chain variable region (VL) derived from the first monospecific antibody, and (2) a second. It may include a heavy chain variable region (VH), which is derived from two monospecific antibodies (different from the first monospecific antibody above). On the other hand, the light chain variable region (VL) derived from the second monospecific antibody is contained in the light chain of the multispecific antibody according to the present invention. In such a multispecific antibody, the second monospecific antibody may be used particularly as a "scaffold". Therefore, in the multispecific antibody according to the present invention, the heavy chains are (1) one or more (preferably all three) heavy chain CDRs derived from the first monospecific antibody and one or more (preferably all three). Is a weight of one or more (preferably all three) derived from (all three) light chain CDRs and (2) a second monospecific antibody (different from the first monospecific antibody above). Chain CDR, may be included. On the other hand, one or more (preferably all three) light chain CDRs derived from the second monospecific antibody are contained in the light chain of the multispecific antibody according to the present invention. In such a multispecific antibody, the second monospecific antibody may be used particularly as a "scaffold".

Usually, three CDRs (CDR1, CDR2 and CDR3) are arranged discontinuously in the variable domain, especially in the natural monospecific IgG antibody. In other words, the CDRs in the heavy and / or light chain may be separated, for example, by a framework region. Here, the framework region (FR) is a region in a variable domain that is less "variable" than a CDR. For example, in an antibody according to the invention, the variable region (or each variable region) may preferably include four framework regions separated by three CDRs.

As described above, in the multispecific antibody according to the invention, a single strand (preferably a heavy strand) exceeds three CDRs (CDR1, CDR2 and CDR3) and / or one of the above. It may include a variable region. Usually, an "antigen receptor" is characterized by CDRs (ie, CDRH1, CDRH2 and CDRH3, and CDRL1, CDRL2 and CDRL3). Therefore, in the multi-characteristic antibody according to the present invention, the CDR preserves the ability to specifically bind to a specific site of an antigen (eg, a cytokine, particularly GM-CSF) in order to conserve an "antigen receptor". It is preferred that the order (eg, CDRH1, CDRH2 and CDRH3, and / or CDRL1, CDRL2 and CDRL3) derived from the same monospecific antibody is maintained. That is, for example, it is preferable that the order of CDRH1, CDRH2 and CDRH3 derived from the first monospecific antibody is not blocked by any CDR derived from the second monospecific antibody in the amino acid chain. Further, when one strand (preferably heavy strand) of the multispecific antibody according to the present invention contains a CDR derived from the heavy chain and the light chain of the first monospecific antibody, the heavy chain CDR , Preferably placed next to the light chain CDR derived from the same monovalent antibody. For example, the sequences described above are: -CDRH1 (a) -CDRH2 (a) -CDRH3 (a) -CDRL1 (a) -CDRL2 (a) -CDRL3 (a) -CDRH1 (b) -CDRH2 (b) -CDRH3 ( b)-Can be-. In the above sequence, (a) and (b) represent different monospecific antibodies from which the respective CDRs are derived. Also, the CDRs are usually arranged discontinuously. That is, the CDRs may be separated by any non-CDR amino acid sequence (eg, framework region and / or linker). Importantly, if the multispecific antibody according to the invention contains an epitope binding site (antigen receptor) derived from at least two different monospecific antibodies, the CDR or variable region of the monospecific antibody. Are arranged as follows in the multispecific antibody according to the present invention. That is, a particular site of an antigen (eg, a cytokine, especially GM-CSF) such that the "antigen receptor" of each monospecific antibody from which the CDR (or variable region) is derived is conserved. (To preserve the ability to specifically bind to).

In this specification, the positions of CDR amino acids are defined according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. Et al. (2009) Nucleic Acids Res. 37, D1006-D1012). Encoding the above-mentioned CDR, heavy chain, light chain, (1) examples of sequences of CDR, heavy chain, light chain, and (2) the above-mentioned CDR, heavy chain, light chain. Examples of sequences of nucleic acid molecules are disclosed in the Sequence Listing. The CDRs of the multispecific antibody according to the present invention, which are derived from the heavy chain CDR of the monospecific antibody, are also referred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the multispecific antibody according to the invention, which are derived from the light chain CDR of the monospecific antibody, are also referred to as CDRL1, CDRL2 and CDRL3, respectively. Thus, for example, CDRL1, CDRL2 and CDRL3 may also be present on the heavy chain of the multispecific antibody according to the invention. Therefore, the variable region of the multispecific antibody according to the invention, which is derived from the heavy chain variable region of the monospecific antibody, is also referred to as VH. In addition, the variable region of the multispecific antibody according to the present invention, which is derived from the light chain variable region of the monospecific antibody, is also referred to as VL. Thus, for example, VL may also be present on the heavy chain of the multispecific antibody according to the invention.

The antibody or antigen-binding fragment thereof according to the present invention comprises (1) a heavy chain containing at least one CDRH1, at least one CDRH2 and at least one CDRH3, and (2) at least one CDRL1, at least one CDRL2 and It preferably contains a light chain, which contains at least one CDRL3. Here, (1) the at least one heavy chain CDRH3 contains an amino acid sequence according to SEQ ID NO: 3, 51, 69 or 107, or a functional sequence variant thereof, and (2) preferably at least one of the above. One heavy chain CDRH3 contains an amino acid sequence according to SEQ ID NO: 3 or 69, or a functional sequence variant thereof. It is also preferred that the heavy chain comprises at least two CDRH1, at least two CDRH2 and at least two CDRH3. Here, one CDRH1, one CDRH2 and one CDRH3 are derived from the first monospecific antibody, and one CDRH1, one CDRH2 and one CDRH3 are the second monospecific antibody (the above-mentioned first). It is derived from (different from the monospecific antibody of 1).

A heavy chain in which the multispecific antibody or antigen-binding fragment thereof of the present invention contains (1) at least one CDRH1, at least one CDRH2 and at least one CDRH3, and (2) at least one CDRL1, at least one. It is also preferred to include a light chain, which contains CDRL2 and at least one CDRL3. Here, the at least one heavy chain CDRH3 is composed of SEQ ID NO: 3, 51, 69 or 107 (preferably SEQ ID NO: 3 or 69) and at least 90% (for example, 90%, 91%, 92%, 93%). 94%, 95%, 96%, 97%, 98%, 99% or 100%) contain identical amino acid sequences.

Table 1 shows the SEQ ID NOs for the amino acid sequences of the six CDRs. The CDR is derived from a representative example of a monospecific antibody, and is used in a representative example of the multispecific antibody of the present invention.

Variant antibodies are also included within the scope of the invention. Therefore, variants of the sequences listed in this application are also included within the scope of the invention. Such variants include (1) natural variants produced in vivo by somatic mutations during an immune response, or (2) natural variants produced in vitro in culture of immortalized B cell clones. It has been. Alternatively, the alteration of the genetic information may cause a variant, or a transcription or translation error may cause a variant.

In addition, variants of the antibody sequences with improved affinity and / or antibody titers may be obtained using known methods. Such variants are also included within the scope of the invention. For example, amino acid substitutions may be used to obtain antibodies with improved affinities. Alternatively, codon optimization of nucleotide sequences may be used to improve transcriptional efficiency in the expression system for antibody production. In addition, polynucleotides containing sequences optimized for antibody specificity or neutralization activity by applying directed evolution methods to any nucleic acid sequence of the invention are also included within the scope of the invention. Will be done.

Preferably, the variant antibody sequence is 70% or more (ie, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97) relative to the sequences listed herein. %, 98%, 99% or more) may have amino acid sequence identity. Such variants are usually more homologous to the sequences described herein in the above CDRs of the heavy and light chain variable regions ( _VH ) and light chain variable regions ( _VL ) than in the framework region. Has sex. As is known to those of skill in the art, mutations are more tolerated (ie, lack of function (eg, specificity or neutralizing ability)) in the framework regions than in the CDRs, or are absent. ).

Therefore, the present invention includes an antibody or an antigen-binding fragment thereof. Here, mutations from the sequences provided herein are preferably located in the framework region of the antibody or in nucleic acid residues encoding the framework region of the antibody.

In the present invention, such (variant) antibodies are preferred in that they can reduce the number of somatic mutations (ie, "germlined" antibodies: reverted to the "germline" form). Germline sequences of antibodies are, for example, by reference to the IMGT database (eg, according to IMGT's decoding of VDJ and VJ allocation and rearrangement: http://www.imgt.org/; cf. Lefranc, M. .-P. Et al. (2009) Nucleic Acids Res. 37, D1006-D1012) May be determined. “Germline” antibody variants may be produced, for example, by gene synthesis or site-specific mutagenesis. Low levels of somatic mutations reduce the potential risk of antibody immunogenicity. The number of somatic mutations is preferably reduced in the framework region (FR) (ie, "germlined framework region" antibodies, also referred to herein as FR-GL variants. ). (Variant) antibodies that do not contain any somatic mutations in the framework region (FR), or antigen-binding fragments thereof and FR-GL variants, respectively, are more preferred. Such (variant) antibodies, or antigen-binding fragments thereof and FR-GL variants, with as few somatic mutations as possible, while containing (more) somatic mutations, reference antibodies / fragments. Those in which the neutralizing activity is not impaired (compared to) are particularly preferable. Such antibodies, on the other hand, do not impair their neutralizing activity. Therefore, it exhibits a very high antibody titer and range. On the other hand, the potential risk of antibody immunogenicity is significantly reduced.

In a preferred embodiment, the multispecific antibody or antibody fragment of the invention comprises at least one CDR. The sequence of at least one CDR has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111.

The multispecific antibody or antibody fragment of the present invention preferably contains more than one CDR. The sequences of more than one CDR have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111.

The antibody or antigen-binding fragment thereof preferably contains two CDRs. The sequences of the two CDRs have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Therefore, the antibody or antigen-binding fragment thereof preferably comprises: (i) having at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105: CDRH1 and CDRL1 having at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108; (ii) of SEQ ID NOs: 2, 50, 68 and 106. CDRH2 has at least 95% sequence identity to any one, and at least 95 to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110. % Sequence identity of CDRL2; or (iii) CDRH3 having at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, and. CDRL3 having at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

The antibody or antigen-binding fragment thereof preferably contains three CDRs. The sequences of the three CDRs have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Therefore, the antibody or antigen-binding fragment thereof preferably comprises: (i) having at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105: CDRH1 which has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, and any one of SEQ ID NOs: 3, 51, 69 and 107. CDRH3 has at least 95% sequence identity to one; or (ii) has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108. CDRL1 which has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, and SEQ ID NOs: 7, 55. , 73 and 111, CDRL3 having at least 95% sequence identity to any one.

The antibody or antigen-binding fragment thereof preferably contains four CDRs. The sequences of the four CDRs have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Therefore, the antibody or antigen-binding fragment thereof preferably comprises: (i) having at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105: To any one of CDRH2, SEQ ID NOs: 3, 51, 69 and 107 having at least 95% sequence identity to any one of CDRH1, SEQ ID NOs: 2, 50, 68 and 106 CDRH3 has at least 95% sequence identity relative to, and at least 95% sequence identity to any one of SEQ ID NOs: 4-7, 52-55, 70-73 or 108-111. (Ii) CDRL1, SEQ ID NOs: 5, 6, 53, 54 having at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108. , 71, 72, 109 and 110 have at least 95% sequence identity to any one of CDRL2, SEQ ID NOs: 7, 55, 73 and 111. CDRL3 having the sequence identity of, and at least 95% sequence identity to any one of SEQ ID NOs: 1-3, 49-51, 67-69 and 105-107. CDRH. Of these, CDRH3 having at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107 is particularly preferred; (iii) SEQ ID NOs: 1, 49, 67 and 105. Which has at least 95% sequence identity to any one of CDRH1, which has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108. CDRH2 having at least 95% sequence identity to any one of CDRL1, SEQ ID NOs: 2, 50, 68 and 106, and SEQ ID NOs: 5, 6, 53, 54, 71, 72, CDRL2 having at least 95% sequence identity for any one of 109 and 110; (iv) at least 95% of the sequence for any one of SEQ ID NOs: 1, 49, 67 and 105. Of CDRH1, SEQ ID NOs: 3, 51, 69 and 107 having at least 95% sequence identity to any one of CDRH1, SEQ ID NOs: 4, 52, 70 and 108 having identity. CDRH3 has at least 95% sequence identity for any one, and at least 95% sequence identity for any one of SEQ ID NOs: 7, 55, 73 and 111. CDRL3; or (v) CDRH2, SEQ ID NOs: 5, 6, 53, 54, which have at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106. At least 95% for any one of CDRL2, SEQ ID NOs: 3, 51, 69 and 107 having at least 95% sequence identity for any one of 71, 72, 109 and 110. CDRH3 having sequence identity and CDRL3 having at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

The antibody or antigen-binding fragment thereof preferably contains 5 CDRs. The sequences of the five CDRs have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Therefore, the antibody or antigen-binding fragment thereof preferably comprises 5 CDRs selected from the following group: at least 95% relative to any one of SEQ ID NOs: 1, 49, 67 and 105. CDRH1 having sequence identity of; CDRH2 having at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106; SEQ ID NOs: 3, 51, 69 and CDRH3 having at least 95% sequence identity to any one of 107; having at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108 CDRL1; CDRL2 having at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110; and SEQ ID NOs: 7, 55. , 73 and 111, CDRL3 having at least 95% sequence identity to any one.

The antibody or antigen-binding fragment thereof preferably contains 6 CDRs. The sequences of the above six CDRs have at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Therefore, the antibody or antigen-binding fragment thereof preferably comprises 6 CDRs selected from the following groups: at least 95% relative to any one of SEQ ID NOs: 1, 49, 67 and 105. CDRH1 having sequence identity of; CDRH2 having at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106; SEQ ID NOs: 3, 51, 69 and CDRH3 having at least 95% sequence identity to any one of 107; having at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108 CDRL1; CDRL2 having at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110; and SEQ ID NOs: 7, 55. , 73 and 111, CDRL3 having at least 95% sequence identity to any one. More preferably, the antibody or antigen-binding fragment thereof comprises:
(I) (1) according to SEQ ID NOs: 1-5 and 7, or their functional sequence variants, or (2) according to SEQ ID NOs: 1-4 and 6-7, or their functional sequence variants, respectively. Amino acid sequences of chains CDRH1, CDRH2 and CDRH3, and amino acid sequences of light chain CDRL1, CDRL2 and CDRL3;
(Ii) (1) according to SEQ ID NOs: 49-53 and 55, or their functional sequence variants, or (2) according to SEQ ID NOs: 49-52 and 54-55, or their functional sequence variants, respectively. Amino acid sequences of chains CDRH1, CDRH2 and CDRH3, and amino acid sequences of light chain CDRL1, CDRL2 and CDRL3;
(Iii) (1) according to SEQ ID NOs: 67-71 and 73, or their functional sequence variants, or (2) according to SEQ ID NOs: 67-70 and 72-73, or their functional sequence variants, respectively. The amino acid sequences of the chains CDRH1, CDRH2 and CDRH3, and the amino acid sequences of the light chains CDRL1, CDRL2 and CDRL3; and / or
(Iv) (1) according to SEQ ID NOs: 105-109 and 111, or their functional sequence variants, or (2) according to SEQ ID NOs: 105-108 and 110-111, or their functional sequence variants, respectively. The amino acid sequences of the chains CDRH1, CDRH2 and CDRH3, as well as the amino acid sequences of the light chains CDRL1, CDRL2 and CDRL3.

Having the above-mentioned antibody of the present invention or an antigen-binding fragment thereof (as described above, having at least one CDR, that is, one, two, three, four, five, six or more CDRs. Among the embodiments, the following embodiments of antibodies or antigen-binding fragments thereof are preferable. That is, an embodiment of an antibody or antigen-binding fragment thereof comprising CDRH3 having at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107 is preferred.

Also preferred are isolated antibodies or antigen-binding fragments of the invention that include: heavy chain CDR1, sequences having the amino acid sequences of SEQ ID NOs: 1, 49, 67 and 105 or functional sequence variants thereof. Has heavy chain CDR2 having the amino acid sequences of Nos. 2, 50, 68 and 106 or functional sequence variants thereof, and amino acid sequences of SEQ ID NOs: 3, 51, 69 and 107 or functional sequence variants thereof. Heavy chain CDR3. In certain embodiments, the antibodies or antibody fragments provided herein comprise a heavy chain comprising the following amino acid sequences: (i) CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2. , And (iii) CDRH1 of SEQ ID NO: 49, CDRH2 of SEQ ID NO: 50, and CDRH3 of SEQ ID NO: 21; (iii) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and SEQ ID NO: 69. CDRH3; and / or (iv) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 106, and CDRH3 of SEQ ID NO: 107.

The antibodies or antigen binding fragments of the invention preferably comprise: a light chain having an amino acid sequence according to any of SEQ ID NOs: 4, 52, 70 and 108 or a functional sequence variant thereof. CDR1; light chain CDR2; and / or SEQ ID NO: 7 having an amino acid sequence or a functional sequence variant thereof according to any of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110. , 55, 73, and 111, the light chain CDR3 having an amino acid sequence or a functional sequence variant thereof. In certain embodiments, the antibodies or antibody fragments provided herein include a light chain comprising the following amino acid sequences: (i) CDRL1, SEQ ID NO: 5 or 6 of SEQ ID NO: 4. CDRL2, and CDRL3 of SEQ ID NO: 7; (ii) CDRL1 of SEQ ID NO: 52, CDRL2 of SEQ ID NO: 53 or 54, and CDRL3 of SEQ ID NO: 55; (iii) CDRL1 of SEQ ID NO: 70, CDRL2 of SEQ ID NO: 71 or 72. , And / or CDRL3 of SEQ ID NO: 73; and / or (iv) CDRL1 of SEQ ID NO: 108, CDRL2 of SEQ ID NO: 109 or 110, and CDRL3 of SEQ ID NO: 111.

In another embodiment of the invention, the invention comprises an isolated antibody or antigen-binding fragment thereof comprising: SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. For any 6 of the amino acid sequences of, at least 80% (eg, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, respectively. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) The amino acid sequences of heavy chain CDR1, CDR2 and CDR3, and , Amino acid sequences of light chain CDR1, CDR2 and CDR3.

Table 2 shows the sequence numbers of the amino acid sequences and the nucleic acid sequences encoding them in the heavy chain variable region and the light chain variable region. The heavy chain variable region and the light chain variable region are derived from typical examples of monospecific antibodies, and are used as typical examples of the multispecific antibody of the present invention.

Whether the above sequence is derived from the heavy chain of a monospecific antibody (eg, GCA7, GCA21, GCB59 or GCE536) or the light chain of a monospecific antibody (eg, GCA7, GCA21, GCB59 or GCE536) Correspondingly, it is expressed as "VH sequence" or "VL sequence", respectively. Thus, in the multispecific antibody according to the invention, for example, the heavy chain is also (eg, in addition to one or more VH sequences) one or more VL sequences (the monospecific antibody from which the VL sequence is derived). (Contained in the light chain of) may also be included.

The isolated antibody or antigen-binding fragment according to the present invention is about 70%, about 75%, about 80%, about 85%, relative to any one of the sequences according to SEQ ID NOs: 37, 63, 95 and 130. Includes VH sequences having amino acid sequences that are about 88%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical. It is preferable to be. In other embodiments, the isolated antibody or antigen binding fragment is about 70%, about 75%, about 80%, about any one of the sequences according to SEQ ID NOs: 38, 64, 96, 131. VL having an amino acid sequence that is 85%, about 88%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical. Contains an array.

The antibody or antigen-binding fragment according to the present invention preferably contains:
(I) VH amino acid sequence according to SEQ ID NO: 37 or its functional sequence variant, and VL amino acid sequence according to SEQ ID NO: 38 or its functional sequence variant; and / or
(Ii) A VH amino acid sequence according to SEQ ID NO: 63 or its functional sequence variant, and a VL amino acid sequence according to SEQ ID NO: 64 or its functional sequence variant; and / or.
(Iii) A VH amino acid sequence according to SEQ ID NO: 95 or its functional sequence variant, and a VL amino acid sequence according to SEQ ID NO: 96 or its functional sequence variant; and / or.
(Iv) A VH amino acid sequence according to SEQ ID NO: 130 or its functional sequence variant, and a VL amino acid sequence according to SEQ ID NO: 131 or its functional sequence variant.

Further, the heavy chain of the antibody according to the present invention, or an antigen-binding fragment thereof, comprises a VL amino acid sequence selected from an amino acid sequence according to SEQ ID NOs: 38, 64, 96 and 131 or a functional sequence variant thereof. Is preferable. It is more preferred that the heavy chain of the antibody or its antigen-binding fragment comprises a VL amino acid sequence selected from the amino acid sequences according to SEQ ID NOs: 38 and 96 or functional sequence variants thereof. It is even more preferred that the heavy chain of the antibody or its antigen-binding fragment comprises a VL amino acid sequence according to SEQ ID NO: 96 or a functional sequence variant thereof.

In the multispecific antibody or antigen-binding fragment thereof according to the present invention, the following is preferable:
(I) The antibody or antigen-binding fragment is a construct type selected from the group consisting of construct type Bs1, constructive Bs2, constructive Bs3, constructive Ts1, constructive Ts2 and constructive Ts3; and (ii). ) The antibody or antigen-binding fragment contains a CDRH1 amino acid sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid sequence, a CDRL1 amino acid sequence, a CDRL2 amino acid sequence, and a CDRL3 amino acid sequence selected from the group consisting of the following (a) to (h). Being out. (A) Amino acid sequences according to SEQ ID NOs: 1-5 and 7 or their functional sequence variants; (b) Amino acid sequences according to SEQ ID NOs: 1-4 and 6-7 or their functional sequence variants; (c). ) Amino acid sequences according to SEQ ID NOs: 67-71 and 73, or functional sequence variants thereof; (d) Amino acid sequences according to SEQ ID NOs: 67-70 and 72-73, or functional sequence variants thereof; (e) Sequence Amino acid sequences according to numbers 49-53 and 55, or functional sequence variants thereof; (f) Amino acid sequences according to SEQ ID NOs: 49-52 and 54-55, or functional sequence variants thereof; (g) SEQ ID NO: 105 An amino acid sequence according to ~ 109 and 111, or a functional sequence variant thereof; and (h) an amino acid sequence according to SEQ ID NOs: 105-108 and 110-111, or a functional sequence variant thereof.

The multispecific antibody or antigen-binding fragment thereof according to the present invention is more preferably as follows:
(I) Being a construct type selected from the group consisting of construct type Bs1, construct type Bs2, construct type Bs3, construct type Ts1, construct type Ts2 and construct type Ts3; and (ii) the following (a) to ( The CDRH1 amino acid sequence, the CDRH2 amino acid sequence, the CDRH3 amino acid sequence, the CDRL1 amino acid sequence, the CDRL2 amino acid sequence and the CDRL3 amino acid sequence selected from the group consisting of d) are contained at any of positions A and / or C. (A) Amino acid sequences according to SEQ ID NOs: 1-5 and 7 or their functional sequence variants; (b) Amino acid sequences according to SEQ ID NOs: 1-4 and 6-7 or their functional sequence variants; (c). ) Amino acid sequences according to SEQ ID NOs: 67-71 and 73, or functional sequence variants thereof; and (d) Amino acid sequences according to SEQ ID NOs: 67-70 and 72-73, or functional sequence variants thereof.

The multispecific antibody or antigen-binding fragment thereof according to the present invention is even more preferably:
(I) Being a construct type selected from the group consisting of construct type Bs1, construct type Bs2, construct type Bs3, construct type Ts1, construct type Ts2 and construct type Ts3; and (ii) (1) SEQ ID NO: 67 to CDRH1 amino acid sequence, CDRH2 amino acid sequence, according to 71 and 73, or a functional sequence variant thereof, or (2) according to SEQ ID NOs: 67-70 and 72-73, or a functional sequence variant thereof. , CDRH3 amino acid sequence, CDRL1 amino acid sequence, CDRL2 amino acid sequence and CDRL3 amino acid sequence at any of positions A and / or C.

The multispecific antibody or antigen-binding fragment thereof according to the present invention is particularly preferably as follows:
(I) Being a construct type selected from the group consisting of construct type Bs1, construct type Bs2, construct type Bs3, construct type Ts1, construct type Ts2 and construct type Ts3; and (ii) (1) SEQ ID NO: 67 to CDRH1 amino acid sequence, CDRH2 amino acid sequence, according to 71 and 73, or a functional sequence variant thereof, or (2) according to SEQ ID NOs: 67-70 and 72-73, or a functional sequence variant thereof. , CDRH3 amino acid sequence, CDRL1 amino acid sequence, CDRL2 amino acid sequence and CDRL3 amino acid sequence at position A.

The multispecific antibody or antigen-binding fragment thereof according to the present invention is gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1b, gBs2GC1c, gBs2GC1b, gBs2GC1c, gBs2GC1d, gB. Is preferably according to gTs3GC2d or gBs1GC3a. The above multispecific antibody or its antigen-binding fragment is Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs3GC2b, Bs3GC2b Alternatively, it is more preferably Bs1GC3a.

We have designed and constructed 16 multispecific antibodies. In the present specification, these are referred to as Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs1GC3 reference). The above antibodies Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2d, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs1GC3a , GCB59, GCE536) (based on a combination of VH and VL sequences), as used herein, the terms gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1a, gBs3GC1a, gBs3GC1a, gBs3GC1a, gBs3GC1a gBs3GC2b, gBs1GC3a, gBs3GC3b, gBs3GC4 and gBs3GC5 mean their respective "generic" antibodies or antigen-binding fragments thereof. The "general" antibody comprises a particular VH amino acid sequence and a VL amino acid sequence (encoded by a particular nucleotide sequence, as described below).

In Table 7 (Example 5), the antibodies Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC2a, Bs3GC2b, Bs1GC2b The SEQ ID NO: and the SEQ ID NO of the amino acid sequence of the light chain are shown respectively (referred to as "complete sequence" in Table 7). In addition, the nucleotide sequences encoding these are also shown. Each sequence is shown in the "Table of Sequences and SEQ ID NOs" in [Example] below.

As used herein, "gTs1GC1" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) Includes a VH amino acid sequence according to SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38, a VH amino acid sequence according to SEQ ID NO: 95, and a VL amino acid sequence according to SEQ ID NO: 96. Heavy chain; as well as a light chain comprising the VL amino acid sequence according to (ii) SEQ ID NO: 64.

gTs1GC1 is preferably IgG1 type. Here, (1) the light chain of gTs1GC1 further comprises an IgG1 CK sequence according to SEQ ID NO: 141, and (2) the heavy chain of gTs1GC1 according to SEQ ID NO: 140 further contains an IgG1 CH1-CH2-CH3 sequence. And optionally, it further comprises one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker is placed between (1) the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37, and (2) the said SEQ ID NO: 38. It may be placed between the VL amino acid sequence according to the above and the VH amino acid sequence according to SEQ ID NO: 95. Also preferably, the long linker is (1) between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38, and (2) the VH amino acid sequence according to SEQ ID NO: 95. It may be placed between the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs1GC1 is preferably Ts1.

As used herein, "gTs1GC2a" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) Includes a VH amino acid sequence according to SEQ ID NO: 130, a VH amino acid sequence according to SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38, a VH amino acid sequence according to SEQ ID NO: 95, and a VL amino acid sequence according to SEQ ID NO: 96. Is, heavy chain; as well
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 131.

gTs1GC2a is preferably IgG1 type. Here, (1) the light chain of gTs1GC2a further comprises an IgG1 CK sequence according to SEQ ID NO: 141, and (2) the heavy chain of gTs1GC2a according to SEQ ID NO: 140 further contains an IgG1 CH1-CH2-CH3 sequence, And optionally, it further comprises one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker is placed between (1) the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37, and (2) the said SEQ ID NO: 38. It may be placed between the VL amino acid sequence according to the above and the VH amino acid sequence according to SEQ ID NO: 95. Also preferably, the long linker is (1) between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38, and (2) the VH amino acid sequence according to SEQ ID NO: 95. It may be placed between the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs1GC2a is preferably Ts1.

As used herein, "gTs2GC2b" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, and a VH amino acid sequence according to SEQ ID NO: 130;
(Ii) A light chain comprising a VH amino acid sequence according to SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38, and a VL amino acid sequence according to SEQ ID NO: 131.

gTs2GC2b is preferably IgG1 type. Here, the light chain of gTs2GC2b further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, (1) preferably a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 38 and the VL amino acid sequence according to SEQ ID NO: 131, and (2) preferably a long linker. May be placed between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. Also, the heavy chain of gTs2GC2b further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, (1) preferably a short linker may be placed on the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 130, and (2) preferably a long linker may be placed above. It may be placed between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs2GC2b is preferably Ts2.

As used herein, "gTs2GC2c" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) Heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, and a VH amino acid sequence according to SEQ ID NO: 37; and (ii) VH amino acid according to SEQ ID NO: 130. A light chain comprising a sequence, a VL amino acid sequence according to SEQ ID NO: 131, and a VL amino acid sequence according to SEQ ID NO: 38.

gTs2GC2c is preferably IgG1 type. Here, the light chain of gTs2GC2c further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, (1) preferably a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 131 above and the VL amino acid sequence according to SEQ ID NO: 38 above, and (2) preferably a long linker. May be placed between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. The heavy chain of gTs2GC2c also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, (1) preferably a short linker may be placed on the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 37, and (2) preferably a long linker may be placed above. It may be placed between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs2GC2c is preferably Ts2.

As used herein, "gTs3GC2d" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) Includes a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, a VH amino acid sequence according to SEQ ID NO: 130, a VH amino acid sequence according to SEQ ID NO: 37, and a VL amino acid sequence according to SEQ ID NO: 38. Is, heavy chain; as well
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 131.

gTs3GC2d is preferably IgG1 type. Here, the light chain of gTs3GC2d further comprises an IgG1 CK sequence according to SEQ ID NO: 141. The heavy chain of gTs3GC2d also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, the short linker is (1) between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37, and (2) to SEQ ID NO: 96. It may be placed between the VL amino acid sequence according to the above and the VH amino acid sequence according to SEQ ID NO: 130. Also preferably, the long linker is (1) between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38, and (2) the VH amino acid sequence according to SEQ ID NO: 95. , May be placed between the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs3GC2d is preferably Ts3.

As used herein, "gTs3GC2e" represents a trispecific antibody or antigen-binding fragment thereof, including:
(I) Includes a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, a VH amino acid sequence according to SEQ ID NO: 37, a VH amino acid sequence according to SEQ ID NO: 130, and a VL amino acid sequence according to SEQ ID NO: 131. Is, heavy chain; as well
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 38.

gTs3GC2e is preferably IgG1 type. Here, the light chain of gTs3GC2e further comprises an IgG1 CK sequence according to SEQ ID NO: 141. The heavy chain of gTs3GC2e also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, the short linker is (1) between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130, and (2) to SEQ ID NO: 96. It may be placed between the VL amino acid sequence according to the above and the VH amino acid sequence according to SEQ ID NO: 37. Also preferably, the long linker is (1) between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131, and (2) the VH amino acid sequence according to SEQ ID NO: 95. , May be placed between the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gTs3GC2e is preferably Ts3.

As used herein, "gBs3GC1a" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 130, a VH amino acid sequence according to SEQ ID NO: 37, and a VL amino acid sequence according to SEQ ID NO: 38;
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 131.

gBs3GC1a is preferably IgG1 type. Here, the light chain of gBs3GC1a further comprises an IgG1 CK sequence according to SEQ ID NO: 141. Also, the heavy chain of gBs3GC1a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. The construction type of gBs3GC1a is preferably Bs3.

As used herein, "gBs3GC1b" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 37, a VH amino acid sequence according to SEQ ID NO: 130, and a VL amino acid sequence according to SEQ ID NO: 131;
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 38.

gBs3GC1b is preferably IgG1 type. Here, the light chain of gBs3GC1b further comprises an IgG1 CK sequence according to SEQ ID NO: 141. The heavy chain of gBs3GC1b also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. The construction type of gBs3GC1b is preferably Bs3.

As used herein, "gBs2GC1c" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising the VH amino acid sequence according to SEQ ID NO: 130;
(Ii) A light chain comprising a VH amino acid sequence according to SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38, and a VL amino acid sequence according to SEQ ID NO: 131.

gBs2GC1c is preferably IgG1 type. Here, the light chain of gBs2GC1c further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 38 and the VL amino acid sequence according to SEQ ID NO: 131. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. In addition, the heavy chain of gBs2GC1c further contains an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140. The construction type of gBs2GC1c is preferably Bs2.

As used herein, "gBs2GC1d" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising the VH amino acid sequence according to SEQ ID NO: 37;
(Ii) A light chain comprising a VH amino acid sequence according to SEQ ID NO: 130, a VL amino acid sequence according to SEQ ID NO: 131, and a VL amino acid sequence according to SEQ ID NO: 38.

gBs2GC1d c is preferably IgG1 type. Here, the light chain of gBs2GC1d further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 131 and the VL amino acid sequence according to SEQ ID NO: 38. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. In addition, the heavy chain of gBs2GC1d further contains an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140. The construction type of gBs2GC1d is preferably Bs2.

As used herein, "gBs1GC2a" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, and a VH amino acid sequence according to SEQ ID NO: 130;
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 131.

gBs1GC2a is preferably IgG1 type. Here, the light chain of gBs1GC2a further comprises an IgG1 CK sequence according to SEQ ID NO: 141. Also, the heavy chain of gBs1GC2a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 130. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gBs1GC2a is preferably Bs1.

As used herein, "gBs3GC2b" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VH amino acid sequence according to SEQ ID NO: 130, and a VL amino acid sequence according to SEQ ID NO: 131;
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 96.

gBs3GC2b is preferably IgG1 type. Here, the light chain of gBs3GC2b further comprises an IgG1 CL sequence according to SEQ ID NO: 142. The heavy chain of gBs3GC2b also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. The construction type of gBs3GC2b is preferably Bs3.

As used herein, "gBs1GC3a" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96, and a VH amino acid sequence according to SEQ ID NO: 37; and (ii) a VL amino acid according to SEQ ID NO: 38. A light chain that contains a sequence.

gBs1GC3a is preferably IgG1 type. Here, the light chain of gBs1GC3a further comprises an IgG1 CK sequence according to SEQ ID NO: 141. Also, the heavy chain of gBs1GC3a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 37. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. The constructed type of gBs1GC3a is preferably Bs1.

As used herein, "gBs3GC3b" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 95, a VH amino acid sequence according to SEQ ID NO: 37, and a VL amino acid sequence according to SEQ ID NO: 38; and (ii) a VL amino acid according to SEQ ID NO: 96. A light chain that contains a sequence.

gBs3GC3b is preferably IgG1 type. Here, the light chain of gBs3GC3b further comprises an IgG1 CL sequence according to SEQ ID NO: 142. The heavy chain of gBs3GC3b also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. The construction type of gBs3GC3b is preferably Bs3.

As used herein, "gBs3GC4" refers to a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 130, and a VL amino acid sequence according to SEQ ID NO: 131;
(Ii) A light chain comprising a VL amino acid sequence according to SEQ ID NO: 64.

gBs3GC4 is preferably IgG1 type. Here, the light chain of gBs3GC4 further comprises an IgG1 CK sequence according to SEQ ID NO: 141. The heavy chain of gBs3GC4 also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. The construction type of gBs3GC4 is preferably Bs3.

As used herein, "gBs3GC5" represents a bispecific antibody or antigen-binding fragment thereof, including:
(I) A heavy chain comprising a VH amino acid sequence according to SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 37, and a VL amino acid sequence according to SEQ ID NO: 38; and (ii) a VL amino acid according to SEQ ID NO: 64. A light chain that contains a sequence.

gBs3GC5 is preferably IgG1 type. Here, the light chain of gBs3GC5 further comprises an IgG1 CK sequence according to SEQ ID NO: 141. The heavy chain of gBs3GC5 also further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and optionally one or more linker sequences (eg, according to SEQ ID NO: 143 or 144). Here, preferably, a short linker may be placed between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37. Also, preferably, a long linker may be placed between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. The construction type of gBs3GC5 is preferably Bs3.

The multispecific isolated antibody or antigen-binding fragment according to the present invention preferably contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs3GC2d, respectively. Alternatively, the multispecific isolated antibody or antigen-binding fragment according to the present invention preferably contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs1GC3a, respectively. Table 3 below shows which CDR (sequence number of the amino acid sequence) contains which VH sequence or VL sequence (sequence number of the amino acid sequence). At the same time, a representative example of a monospecific antibody from which the above VH sequence or VL sequence is derived is also shown. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs1GC1 respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs1GC2a, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs2GC2b, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs2GC2c, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs2GC2c, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs3GC2d, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs3GC2d, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gTs3GC2e, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC1a, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC1b, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs2GC1c, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs2GC1d, respectively. Alternatively, the multispecific isolated antibody or antigen-binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs1GC2a, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC2b, respectively. Alternatively, the multispecific isolated antibody or antigen-binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs1GC3a, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC3b, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC4, respectively. Alternatively, the multispecific isolated antibody or antigen binding fragment according to the present invention contains all the CDRs contained in the total VH sequence and the total VL sequence of gBs3GC5, respectively.

The multispecific antibody or antigen-binding fragment thereof according to the present invention may be used as a medicine. In particular, the multispecific antibody or antigen-binding fragment thereof according to the present invention may be used for the prevention, treatment or weakening of inflammatory diseases and / or autoimmune diseases. More details regarding the above-mentioned uses will be described later, for example in relation to pharmaceutical compositions and medical uses.

The present invention further includes antibodies or fragments thereof. The antibody or fragment thereof can be used as an antibody or antigen-binding fragment of the present invention or as an antibody that competes with the antibody or antigen-binding fragment of the present invention against the same epitope (ie, the same "cytokine (particularly GM-CSF)). (For at least two different non-overlapping sites).

The antibody according to the present invention or an antigen-binding fragment thereof, which binds to the same epitope as the above-mentioned antibody, preferably also contains an Fc moiety.
A multispecific antibody according to the invention or antigen binding thereof comprising (a) at least two different domains that specifically bind to at least two different non-overlapping sites of a cytokine; and (b) an Fc moiety. With respect to fragments, the preferred embodiments described above can also be applied to antibodies according to the invention or antigen-binding fragments thereof that bind to the same epitope as the antibodies described above.

Antibodies of the invention also include multispecific hybrid antibody molecules, including the following (a) and (b) described above.
(A) At least two different domains that specifically bind to at least two different non-overlapping sites of cytokines; and (b) Fc moieties The multispecific hybrid antibody molecule described above is (1) the antibody of the invention. It contains one or more CDRs from which it is derived, and (2) one or more CDRs from other antibodies. The other antibodies are specifically antibodies against the same epitope or additional epitope on the same cytokine (particularly GM-CSF), or other antigens (eg, other cytokines, cancer-related epitopes, T cell epitopes, B cell epitopes, etc.). Is. In one embodiment, the hybrid antibody is derived from (1) three CDRs derived from the antibody of the invention, and (2) another antibody (eg, an antibody against the same or different epitopes, as described above) 3 Includes one CDR. Typical examples of hybrid antibodies are (i) three heavy chain CDRs derived from the present invention, and three light chain CDRs derived from other antibodies (antibodies to the same or different epitopes as described above), or (ii). ) Includes three light chain CDRs from the present invention and three heavy chain CDRs from other antibodies (antibodies to the same or different epitopes, as described above).

(Nucleic acid molecule)
In another aspect, the invention also includes a nucleic acid molecule comprising a polynucleotide encoding an antibody according to the invention or an antigen-binding fragment thereof as described above. Nucleic acid sequences encoding some or all of the light, heavy and CDRs of the antibodies of the invention are preferred. Therefore, preferably, nucleic acid sequences encoding some or all of the light and heavy chains (particularly VH and VL sequences) and CDRs of the exemplary antibodies of the invention are provided herein. .. The SEQ ID NOs of the nucleic acid sequences that are derived from the monospecific antibody and that encode the VH and VL sequences used in the antibodies of some examples of the invention can be found in Table 7. Table 4 below presents the SEQ ID NOs of the nucleic acid sequences encoding the CDRs used in the antibodies of some examples of the invention. Due to genetic code duplication, the invention also includes variants of these nucleic acid sequences encoding similar amino acid sequences.

Therefore, the present invention also includes a nucleic acid molecule containing a polynucleotide encoding an antibody according to the present invention or an antigen-binding fragment thereof.

The nucleic acid molecule is a molecule containing a nucleic acid component (preferably a molecule composed of the nucleic acid component). The term nucleic acid molecule preferably represents a DNA or RNA molecule. In particular, it is used synonymously with the term "polynucleotide". Preferably, the nucleic acid molecule is a polymer containing or composed of nucleotide monomers covalently bonded to each other by phosphate diester bonds in the sugar / phosphate backbone. The term "nucleic acid molecule" also includes a modified nucleic acid molecule (eg, a DNA molecule or RNA molecule that has been subjected to base modification, sugar modification, skeletal modification, etc.).

Preferably, the sequences of the nucleic acid molecules according to the present invention are SEQ ID NOs: 8-36, 39-48, 56-62, 65-66, 74-94, 97-104, 112-129, 132-139, 152, 154. , 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 and 190 or any one of their functional sequence variants. Containing or consisting of the corresponding nucleic acid sequence.

In addition, the nucleic acid sequence according to the present invention is at least 70%, at least 75%, at least 80%, and at least 85% of the nucleic acid encoding the VH sequence and / or the VL sequence used in the antibody according to the present invention. , At least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of nucleic acid sequences having the same identity. Thus, a nucleic acid molecule comprises a nucleic acid sequence whose polynucleotide sequence follows any one of SEQ ID NOs: 39-48, 65-66, 97-104 and 132-139 or their functional sequence variants. It preferably consists of the nucleic acid sequence. More preferably, the nucleic acid molecule according to the invention comprises or consists of a nucleic acid sequence encoding one complete heavy chain or complete light chain of an exemplary antibody according to the invention. Therefore, the nucleic acid molecule has a polynucleotide sequence of SEQ ID NO: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188. And 190, or preferably comprises or consists of a nucleic acid sequence according to any one of their functional sequence variants.

In other embodiments, the nucleic acid sequence of the invention comprises a sequence of nucleic acids encoding the heavy or light chain CDRs of an exemplary antibody of the invention. For example, the nucleic acid sequences according to the present invention include SEQ ID NOs: 8-36, 39-48, 56-62, 65-66, 74-94, 97-104, 112-129, 132-139, 152, 154, 156, Nucleic acid sequences of 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 and 190 and at least 75%, at least 80%, at least 85%, Contains or consists of sequences that are at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical.

In general, the nucleic acid molecule may be engineered to insert, delete or modify a particular nucleic acid sequence. Changes made by such operations include changes to introduce restriction sites, changes to improve codon usage, changes to add or optimize transcriptional and / or translational regulatory sequences, and the like. However, it is not limited to these. Nucleic acids can also be modified to alter the encoded amino acids. For example, one or more amino acid substitutions in the amino acid sequence of an antibody (eg, 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, etc.) , Amino acid deletion and / or amino acid insertion may be useful. Such point mutations may alter effector function, antigen binding affinity, post-translational modification, immunogenicity, etc., may introduce amino acids for binding covalent groups (eg, labels), and may introduce tags (eg, labels). A tag for purification purposes) may be introduced. Mutations may be introduced at specific sites or may be randomly introduced following selection (eg molecular evolution). For example, one or more nucleic acids encoding either the CDR regions, VH sequences or VL sequences or heavy or light chains of the (exemplary) antibodies of the invention are randomly or directly mutated. , Different properties may be introduced into the encoded amino acids. Such changes may be the result of an iterative process in which the initial changes are maintained and new changes are introduced at other nucleotide sites. In addition, changes achieved in independent processes may be combined. The different properties introduced into the encoded amino acid may include, but are not limited to, improved affinity.

(vector)
Further, a vector containing the nucleic acid sequence according to the present invention (for example, an expression vector) is included in the scope of the present invention. Preferably, the vector comprises a nucleic acid molecule according to the invention (eg, a nucleic acid molecule as described above).

The term "vector" refers to a nucleic acid molecule (preferably an artificial nucleic acid molecule (ie, a non-naturally occurring nucleic acid molecule)). The vector according to the invention is suitable for incorporating or incorporating the desired nucleic acid sequence. Such a vector may be a storage vector, an expression vector, a cloning vector, an introduction vector or the like. The storage vector is a vector capable of suitable storage of nucleic acid molecules. Therefore, the vector may also contain, for example, a sequence corresponding to the desired antibody or antibody fragment thereof according to the present invention. Expression vectors may be used in the production of expression products (eg, RNA (eg, mRNA), peptides, polypeptides or proteins). For example, the expression vector may contain the sequences required for sequence stretch of the vector (eg, promoter sequence). The cloning vector is typically a vector containing a cloning site and may be used to integrate the nucleic acid sequence into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector. The introduction vector may be a vector suitable for introducing a nucleic acid molecule into a cell or an organism (for example, a viral vector). The vector according to the present invention may be, for example, an RNA vector or a DNA vector. Preferably, the vector is a DNA molecule. For example, a vector in the sense herein contains a cloning site, a selectable marker (eg, an antibiotic resistance factor) and a sequence suitable for replication of the vector (eg, a replication origin). Preferably, the vector according to the present specification is a plasmid vector.

〔cell〕
Cells transformed with such a vector are also included in the scope of the present invention. Examples of such cells include, but are not limited to, eukaryotic cells (eg, yeast cells, animal cells or plant cells). In one embodiment, the cells are mammalian cells (eg, human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, myeloma cells or hybridoma cells). Therefore, the present invention also provides cells expressing the antibody or antigen-binding fragment thereof according to the present invention; or cells containing the vector according to the present invention.

In particular, the cells may be transfected with the vector according to the invention (preferably an expression vector). The term "transfection" refers to the introduction of a nucleic acid molecule (eg, a DNA molecule or an RNA (eg, mRNA) molecule) into a cell (preferably a eukaryotic cell). With respect to the present invention, the term "transfection" includes any method of introducing a nucleic acid molecule into a cell (preferably a eukaryotic cell (eg, mammalian cell)) known to those of skill in the art. Such methods include, for example, electroporation, lipofection (eg, cationic lipid and / or liposome-based lipofection), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, etc. Alternatively, transfections based on cationic polymers (eg, DEAE dextran or polyethyleneimine) are included. Preferably, the introduction is non-viral.

(Polypeptide)
The present invention also comprises a polypeptide (eg, an isolated or purified immunogenic polypeptide) comprising at least two epitopes that specifically bind to an antibody according to the invention or an antigen-binding fragment thereof. ). For example, the (immunogenic) polypeptide according to the present invention can be used in the diagnosis and / or antibody production, purification and / or verification steps and / of the disease as described herein. Alternatively, it may be used as a vaccine in quality control. Preferably, the (immunogenic) polypeptide according to the invention comprises at least two epitopes that specifically bind to the antibody or antigen-binding fragment thereof according to the invention, wherein the polypeptide is a recombinant polypeptide. (That is, a polypeptide that does not occur naturally).

Monoclonal and recombinant antibodies are particularly useful in the identification and purification of individual polypeptides or other antigens against them. Therefore, the antibody of the present invention is further useful in that it can be used as a reagent in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). Have. In these uses, the antibody may be labeled with an analytically detectable reagent (eg, a radioisotope, fluorescent molecule or enzyme). The above antibodies may also be used for molecular identification and characterization (epitope mapping) of antigens.

Therefore, the polypeptide that binds to the antibody of the invention can have many uses. The polypeptides in purified or synthetic form and their polypeptide variants may be used to elicit an immune response (ie, as a vaccine or for the production of antibodies for other uses), epitopes or these. May be used for screening sera for antibodies that immunoreact with mimotopes. In one embodiment, such a polypeptide or polypeptide variant or an antigen comprising such a polypeptide or polypeptide variant comprises an antibody of the same quality as the antibody described in the present invention, immune. It may be used as a vaccine to provoke a response.

In addition, the polypeptide that binds to the antibody of the present invention may be suitable for screening a ligand that binds to the above-mentioned polypeptide. Such ligands include antibodies (eg, antibodies from camels, sharks and other species), antibody fragments, peptides, phage display technology products, aptamers, adonectins or fragments of other viral or cellular proteins. However, but not limited to these, such ligands can inhibit the epitope and thereby prevent infection. Such ligands are within the scope of the invention.

[Any additional feature of the antibody]
The antibodies of the invention may, for example, bind to a drug for delivery to a treatment site or may bind to a detectable label to facilitate imaging of the site containing the desired cells. Methods of binding an antibody to a drug and a detectable label are well known to those of skill in the art as imaging methods using a detectable label. Labeled antibodies can be used in different measurement methods with different labels. Detection of the form of an antibody-antigen complex between an antibody of the present invention and a desired epitope in a cytokine (particularly GM-CSF) can be easily carried out by binding a detectable substance to the antibody. Suitable detection methods include labels (eg, radioactive nuclei, enzymes, coenzymes, fluorescent agents, chemical luminescent agents, chromogens, enzyme substrates or enzyme cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, etc. Use of particles, dyes, etc.) can be mentioned. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable complementing molecular family complexes include streptabin / biotin and avidin / biotin; suitable. Fluorescent substances include umbelliferone, fluorescein, fluorescein isothianate, rhodamine, dichlorotriazinylamine fluorescein, dansil lolide or ficoeritrin; an example of a luminescent substance is luminol; an example of a bioluminescent substance is luciferase. , Luciferin and aequorin; suitable radioactive substances include 125I, 131I, 35S or 3H. Such labeled reagents may be used in a variety of well-known metrics such as radioimmunoassay, enzyme-linked immunosorbent assay (eg ELISA), fluorescence immunoassay, and the like. Therefore, labeled antibodies according to the invention are measured as described, for example, in US3,766,162; US3,791,932; US3,817,837; and US4,233,402. May be used in law.

Antibodies according to the invention may bind to therapeutic moieties (eg, cytotoxins, therapeutic agents or radiometal ions or radioisotopes). Examples of radioisotopes are I-131, I-123, I-125, Y-90, Re-188, Re-186, At-111, Cu-67, Bi-212, Bi-213, Pd-109. , Tc-99, In-111 and the like, but are not limited thereto. Such antibody conjugation may be used to alter a particular biological response; the drug moiety is not constructed to be confined to typical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins (eg, abrin, ricin A, sucrose exotoxin or diphtheria venom).

Techniques for binding such therapeutic moieties to antibodies are well known. For example, Arnon et al. (1985) “Monoclonal Antibodies for Immunotargeting of Drugs in CancerTherapy,” in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed Hellstrom et al. (1987) “Antibodies for Drug Delivery,” in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) "Antibody Carriers" of Cytotoxic Agents in Cancer Therapy: A Review, "in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. Pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985);" Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, ”in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62: 119-158.

Alternatively, the antibody or antibody fragment thereof may bind to a second antibody or antibody fragment thereof to form a heterocomplex antibody as described in US 4,676,980. Further, for example, as described in US 4,831,175, a linker may be used between the label and the antibody of the present invention. Antibodies or antigen-binding fragments thereof are directly labeled with radioactive iodine, radioactive iodine, radioactive yttrium or other radioactive particles known to those of skill in the art, for example, as described in US 5,595,721. May be good. The treatment may consist, for example, a combination of treatments with bound and unbound antibodies administered simultaneously or thereafter, as described in WO 00/52031; WO 00/52473.

The antibodies of the invention can also bind to solid supports. In addition, the antibodies of the invention or functional antibody fragments thereof may be chemically modified by covalent bonds with polymers, for example to increase the circulating half-life. Examples of polymers and methods for binding them to peptides are set forth in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. In some embodiments, the polymer may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and can be of the general formula: R (O-CH2--CH2) nO-R (where R is a hydrogen or protecting group (eg, an alkyl or alkanol group) ). Preferably, the protecting group may have between 1 and 8 carbons. For example, the protecting group is methyl. The symbol n is a positive integer. In one embodiment, n is between 1 and 1,000. In other embodiments, n is between 2 and 500. Preferably, PEG has an average molecular weight between 1,000 and 40,000, and more preferably, PEG has an average molecular weight between 2,000 and 20,000. , Even more preferably, PEG has an average molecular weight between 3,000 and 12,000. Further, the PEG may have at least one hydroxy group, for example, the PEG may have a terminal hydroxy group. For example, this may be a terminal hydroxy group that is activated to react with the free amino group in the inhibitor. However, it will be appreciated that the type and amount of reactive groups may be varied to achieve covalently bound PEG / antibodies of the invention.

Water-soluble polyoxyethylated polyols are also useful in the present invention. Examples thereof include pooxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) and the like. In one embodiment, POG is used. Without being bound by any theory, this branch is because the glycerol backbone of polyoxyethylated glycerol is the same backbone that naturally occurs in monoglycerides, diglycerides, triglycerides, for example in animals and humans. , Will not always be considered foreign in the body. POG may have a molecular weight similar to that of PEG. Another drug delivery system that can be used to increase the circulating half-life is liposomes. Methods of preparing a liposome delivery system are known to those of skill in the art. Other drug delivery systems are known in the art and are referenced and described, for example, in Poznansky et al. (1980) and Poznansky (1984).

The antibodies of the invention may be provided in purified form. Typically, the antibody is present in a composition that is substantially free of other polypeptides (eg, less than 90 (w)%), and generally less than 60% of the composition, and more. Generally, less than 50% of the composition is composed of other polypeptides.

The antibodies of the invention may be immunogenic in a non-human (or heterologous) host (eg, mouse). In particular, the antibody may have an idiotope that is immunogenic in a non-human host but not immunogenic in a human host. Antibodies of the invention for use in humans cannot be readily isolated from hosts (eg, mice, goats, rabbits, rats, non-primate mammals, etc.) and are generally by humanization or from xenomouse. It contains antibodies that cannot be obtained.

(Antibody production)
The antibody according to the present invention may be produced by any method known to those skilled in the art. For example, a general methodology for producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C ,. 1975; Kozbar et al. 1983). In one embodiment, an alternative EBV immobilization method described in WO2004 / 0766777 is used.

The methods described in WO2004 / 0766777 can be used to transform B cells producing the antibodies of the invention with EBV and polyclonal B cell activators. Further stimulants of cell growth and differentiation may be optionally added during the transformation phase to further increase efficiency. These stimulants may be cytokines (eg, IL-2 and IL-15). In one aspect, IL-2 is added during the immortalization phase to further increase the efficiency of immortalization, but its use is not mandatory. Immortalized B cells produced using these methods may then be cultured using methods known in the art and antibodies isolated from them.

The method described in WO2010 / 046775 allows plasma cells to be cultured in a limited number or single plasma cells in a microwell culture plate. Antibodies can be isolated from plasma cell media. In addition, RNA can be extracted from plasma cell media and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, and then cloned into an expression vector that is transfected into HEK293T cells or other host cells. Any method known to those skilled in the art can be used to clone the nucleic acid in the expression vector, transfect the host cell, culture the transfected host cell and isolate the produced antibody.

If desired, the antibody may be further purified using filtration, centrifugation and various chromatographic methods (eg, HPLC or affinity chromatography). Techniques for purifying antibodies (eg, monoclonal antibodies) (eg, techniques for producing pharmaceutical grade antibodies) are well known in the art.

Fragments of the antibodies of the invention can be obtained from the above antibodies by methods involving digestion with an enzyme (eg, pepsin or papain) and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, a fragment of the antibody can be obtained by cloning and expression of a portion of the heavy or light chain sequence. The antibody "fragment" includes a Fab fragment, a Fab'fragment, an F (ab') 2 fragment and an Fv fragment. The present invention also includes single chain Fv fragments (scFv) derived from the heavy and light chains of the antibodies of the invention. For example, the present invention includes scFv containing CDRs derived from the antibodies of the present invention. Not only single chain antibodies (eg, single chain Fv in which the heavy and light chain variable domains are linked by a peptide linker), but also heavy or light chain monomers and dimers, single domain heavy chain antibodies, single. It also contains domain light chain antibodies.

The antibody fragments of the present invention may impart monospecific or multispecific interactions and may be included in various structures as described above. For example, an scFv molecule may be synthesized to produce a trivalent "trispecific antibody" or a tetravalent "quadrispecific antibody". The scFv molecule may contain a domain of the Fc region that gives rise to divalent minibodies. Furthermore, the sequences of the invention may be components of a multispecific molecule in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv and bispecific antibodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126- 1136).

Standard techniques of molecular biology may be used to produce the DNA sequences encoding the antibodies or antibody fragments of the invention. Oligonucleotide synthesis techniques may be used to synthesize the desired DNA sequence completely or partially. Site-specific mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell / vector system may be used for the expression of the DNA sequences encoding the antibody molecules of the invention or fragments thereof. Due to the expression of antibody fragments (eg, Fab fragment and F (ab') 2 fragments, in particular Fv fragment and single chain antibody fragment (eg, single chain Fv)), bacteria (eg E. coli) and other bacterial systems , May be partially used. A eukaryotic (eg, mammalian) host cell expression system may be used for the production of larger antibody molecules (eg, complete antibody molecules). Suitable mammalian host cells include CHO cells, HEK293T cells, PER. Examples include, but are not limited to, C6 cells, NS0 cells, myeloma cells or hybridoma cells.

The present invention relates to a step of culturing a host cell containing a vector encoding the nucleic acid of the present invention under conditions suitable for expression of a protein from DNA encoding the antibody molecule of the present invention, and the above antibody. Also provided is a process for the production of antibody molecules according to the invention, which comprises the step of isolating the molecule.

The antibody molecule may contain only the heavy or light chain polypeptide, in which case only the heavy or light chain polypeptide encoding the sequence is to transfect the host cell. Needs to be used. For the production of products containing both heavy and light chains, two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. The cell line may be transfected. Alternatively, a single vector may be used, which contains the sequences encoding the light chain and heavy chain polypeptides. Alternatively, the antibody according to the present invention may be produced by (i) expressing the nucleic acid sequence according to the present invention in a host cell and (ii) isolating the antibody product to be expressed. Alternatively, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for antibodies of the desired specificity or function they produce.

The screening step identifies any immunoassay (eg, ELISA), staining of tissues or cells (eg, transfected cells), neutralization assays, or the desired specificity or function known in the art. It may be done by one of various other methods for. The measurement method may be selected based on the mere recognition of one or more antigens (eg, the properties of the target cell to select an antibody that is neutralized over an antibody that is simply bound to the antigen. (For example, these signal cascades, their morphology, their growth rate, their ability to affect other cells, their response to the effects of other cells or other reagents or changes in circumstances, their differentiation status, etc.) It may be selected on the basis of further desired function (for selecting an antibody capable of altering).

Individual transformed B cell clones may then be made from positive transformed B cell medium. The cloning process for separating individual clones from a mixture of positive cells uses single cell deposition by limiting dilution, microscopy, cell sorting or other methods known in the art. It may be done.

Nucleic acids derived from cultured plasma cells may be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.

Immortalized B cell clones or transfected host cells of the invention can be used in a variety of ways (eg, as a source of monoclonal antibody, as a source of nucleic acid (DNA or mRNA) encoding the desired monoclonal antibody. , For research, etc.) may be used.

The present invention also provides a composition comprising an immortalized B memory cell or a transfected host cell that produces the antibody according to the invention.

Immortalized B cell clones or cultured plasma cells of the present invention may also be used as a source of nucleic acids for cloning antibody genes for subsequent recombinant expression. For example, expression from recombinant sources is more common for pharmaceutical purposes than expression from B cells or hybridomas for reasons such as stability, productivity, ease of culture and the like.

Therefore, the present invention is: (i) obtaining one or more nucleic acids (eg, heavy chain mRNA and / or light chain mRNA) from a B cell clone or cultured plasma cell encoding the desired antibody; Recombinant cells are prepared, comprising the steps of ii) inserting the nucleic acid into the expression vector and (iii) transfecting the vector into the host cell to allow expression of the desired antibody in the host cell. It also provides a way to do this.

Similarly, the invention is: (i) sequencing one or more nucleic acids from a B cell clone or cultured plasma cell encoding the desired antibody; and (ii) expression of the desired antibody in host cells. To provide a method for producing recombinant cells, which comprises the step of using the sequence information of step (i) to produce nucleic acid for insertion into the host cell. The nucleic acids are engineered between steps (i) and (ii) to introduce restriction sites, alter codon usage, and / or optimize transcriptional and / or translational regulatory sequences. It may be, but it does not need to be manipulated.

In addition, the present invention also provides a method for making a transfected host cell, comprising transfecting the host cell with one or more nucleic acids encoding the desired antibody. In this method, the nucleic acid is a nucleic acid derived from an immortalized B cell clone or cultured plasma cell of the present invention. Therefore, the procedure of first preparing the one or more nucleic acids and then using it to transfect host cells is performed at different times by different people in different places (eg different countries). can do.

These recombinant cells of the invention may then be used for expression and culture purposes. These are particularly useful for the expression of antibodies for large-scale pharmaceutical production. In addition, these may be used as an active ingredient of a pharmaceutical composition. Any suitable culture technique (eg, static culture, roller bottle culture, ascites, hollow fiber bioreactor cartridge, modular mini fermenter, stirring tank, microcarrier culture, ceramic core perfusion, etc., but is limited thereto). Not done) may be used.

Methods of obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (see, eg, Chapter 4 of Kuby Immunology, 4th edition, 2000).

Transfected host cells include eukaryotic cells (eg, plant cells as well as yeast and animal cells, especially mammalian cells (eg, CHO cells, NS0 cells, human cells (eg, PER.C6 cells or HKB-)). It may be 11 cells) or melanoma cells)). A preferred expression host allows the antibodies of the invention to be saccharified (particularly in a sugar chain structure that is not immunogenic in humans). In one embodiment, the transfected host cells can grow in a serum-free medium. In a further embodiment, the transfected host cells can grow in a culture medium that does not contain animal-derived products. In addition, the transfected host cells may be cultured to obtain a cell lineage.

The present invention comprises (i) a step of producing an immortalized B cell clone or cultured plasma cell according to the present invention and (ii) a nucleic acid encoding a desired antibody from the B cell clone or cultured plasma cell. Also provided are methods for producing one or more nucleic acid molecules (eg, heavy and light chain genes) encoding the desired antibody, comprising the step of obtaining. Further, the present invention encodes (i) a step of producing an immortalized B cell clone or cultured plasma cell according to the present invention and (ii) a desired antibody derived from a B cell clone or cultured plasma cell. Provided is a method for obtaining a nucleic acid sequence encoding a desired antibody, which comprises the step of sequencing a nucleic acid.

The present invention further provides a method of producing one or more nucleic acid molecules encoding a desired antibody, comprising the step of obtaining nucleic acids obtained from the transformed B cells or cultured plasma cells of the present invention. .. Therefore, the procedure of first obtaining a B cell clone or cultured plasma cell and then obtaining one or more nucleic acids from a B cell clone or cultured plasma cell is performed by different people in different places (eg, different countries). It can be carried out at different times.

The present invention (i) obtains one or more nucleic acids (eg, heavy and light chain genes) from selected B cell clones or cultured plasma cells expressing the desired antibody and / or Sequencing step; (ii) Inserting the one or more nucleic acids into or using one or more nucleic acid sequences to prepare an expression vector; (iii). ) Transfection of host cells capable of expressing the desired antibody; (iv) Culturing or sub-culture of the transfected host cells under conditions in which the desired antibody is expressed. ) Steps; and optionally (v) methods for producing antibodies according to the invention (eg, antibodies for pharmaceutical use), including the step of purifying the desired antibody.

The present invention also comprises a step of culturing or sub-culturing a transfected host cell population under conditions in which the desired antibody is expressed, and optionally purifying the desired antibody, the method of producing the antibody. provide. The transfected host cell population comprises (i) one or more nucleic acids encoding the desired antibody of choice produced by the B cell clone or cultured plasma cells produced as described above. The steps of providing, (ii) inserting the one or more nucleic acids into an expression vector, (iii) transfecting the vector in a host cell capable of expressing the desired antibody, and (iv) the desired antibody. It is produced by the step of culturing or sub-culturing transfected host cells that contain the nucleic acid inserted for production. Therefore, the procedure of first preparing a recombinant host cell and then culturing it to express the antibody should be performed at very different times by different people in different places (eg different countries). Can be done.

(Pharmaceutical composition)
The present invention also provides a pharmaceutical composition comprising one or more of the following (i)-(v):
(I) An antibody or antibody fragment according to the present invention;
(Ii) Nucleic acid encoding an antibody or antibody fragment according to the present invention;
(Iii) A vector encoding a nucleic acid according to the present invention;
(Iv) A cell expressing an antibody according to the present invention or a cell containing a vector according to the present invention; or (v) an immunogenic polypeptide recognized by the antibody according to the present invention or an antigen-binding fragment thereof.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier, diluent and / or excipient. Preferably, the pharmaceutical composition according to the present invention comprises one or more of the following (i)-(v):
(I) An antibody or antibody fragment according to the present invention;
(Ii) Nucleic acid encoding an antibody or antibody fragment according to the present invention;
(Iii) A vector encoding a nucleic acid according to the present invention;
(Iv) A cell expressing an antibody according to the present invention or a cell containing a vector according to the present invention; or (v) an immunogenic polypeptide recognized in the antibody according to the present invention or an antigen-binding fragment thereof. And pharmaceutically acceptable excipients, diluents and / or carriers.

The carrier or excipient can facilitate administration, but should not encourage the individual receiving the composition to produce an antibody that is harmful to itself. Also, it must not be toxic. Suitable carriers are macromolecules with large molecules that are slowly metabolized (eg, proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymerized amino acids, amino acid copolymers and inert viral particles). May be good.

Pharmaceutically acceptable salts are, for example, mineral salts (eg, hydrochlorides, hydrobromates, phosphates and sulfates) or salts of organic acids (eg, acetates, propionates, malonates). And benzoate) may be used.

The pharmaceutically acceptable carrier in the therapeutic composition may further comprise liquids (eg, water, saline, glycerol and ethanol). In addition, auxiliary substances (eg, surfactants or emulsifiers or pH buffering substances) may be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for inoculation by the subject.

The compositions of the present invention may be prepared in various forms. For example, the composition may be prepared as a solution or suspension, or as an injectable material. Solid form suitable as a solution or suspension in a liquid solvent prior to injection (eg, lyophilized compositions for reconstitution in sterile water containing preservatives (eg, Synagis ^TM and Herceptin ^TM )) May be prepared. The composition may be prepared for topical administration (eg, as an ointment, cream or powder). The composition may be prepared for oral administration (eg, as a tablet or capsule, as a spray, or as a syrup (optionally flavored syrup)). The composition may be prepared for pulmonary administration (eg, as an inhalant using a fine powder or spray). The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, ear or ocular administration (eg, as an infusion). The composition may be in the form of a kit designed to reconstitute the mixed composition immediately prior to administration to the subject. For example, the lyophilized antibody may be provided in the form of a kit with sterile water or sterile buffer.

The active ingredient in the composition is an antibody molecule, an antibody fragment or variant and a derivative thereof, and in particular, the active ingredient in the composition is an antibody, an antibody fragment or variant according to the present invention and a derivative thereof. Is preferable. Therefore, it may be susceptible to digestion in the gastrointestinal tract. Therefore, when the composition is administered by a gastrointestinal route, the composition comprises a drug that prevents the degradation of the antibody but releases the antibody when absorbed from the gastrointestinal tract. It may be a composition that is used.

A detailed discussion of pharmaceutically acceptable carriers can be found in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

The pharmaceutical compositions of the present invention generally have a pH between 5.5 and 8.5, and in some embodiments this may be between 6 and 8 and others. In the embodiment, it is about 7. The pH may be maintained by the use of buffer. The composition is sterile and / or does not contain pyrogens. The composition is isotonic for humans. In one embodiment, the pharmaceutical composition of the present invention is supplied in a closed container.

To the extent of the invention, the compositions are present in several forms of administration; such forms are suitable for parenteral administration (eg, by injection or infusion (eg, by bolus injection or continuous infusion)). These forms include, but are not limited to. If the product is suitable for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or water soluble medium, which may be a formulation (eg, suspending agent, preservative). Agents, stabilizers and / or dispersants) may be included. Alternatively, the antibody molecule may be in dry form for pre-use reconstruction with a suitable sterile liquid. It is generally understood that the vehicle is a substance suitable for storing, transporting and / or administering a compound, such as a pharmaceutically active compound (particularly an antibody according to the invention). For example, the medium may be a physiologically acceptable liquid, which is suitable for storing, transporting and / or administering pharmaceutically active compounds, especially antibodies according to the invention. Immediately after being formulated, the compositions of the invention may be administered directly to the subject. In one embodiment, the composition is suitable for administration to mammals (eg, human subjects).

The pharmaceutical composition of the present invention comprises any number of pathways (eg, oral pathway, venous pathway, intramuscular pathway, intraarterial pathway, intramedullary pathway, intraperitoneal pathway, intrathecal pathway, etc. These include, but are limited to, intraventricular, transdermal, transcutaneous, local, subcutaneous, intranasal, translingual, sublingual, intravaginal or rectal routes. May not be administered). Hypospray can also be used to administer the pharmaceutical compositions of the present invention. Preferably, the pharmaceutical composition may be prepared for oral administration (eg, as a tablet, capsule, etc.), may be prepared for topical administration, and as an injectable substance (eg, a liquid solution). Alternatively, it may be prepared (as a suspension). Solid forms suitable for solutions or suspensions in liquid solvents prior to injection may be prepared.

For injections (eg, intravenous, intradermal or subcutaneous injections, or injections in painful areas), the active ingredient is preferably pyrogen-free and has suitable pH, isotonicity and stability. , In the form of a pharmaceutically acceptable aqueous solution. One of ordinary skill in the art can successfully prepare a water-soluble solution using an isotonic medium (eg, sodium chloride injection, Ringer injection, Lactated Ringer injection). Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, if desired. Whether this is a polypeptide, peptide, nucleic acid molecule, or other pharmaceutically useful compound of the invention that is to be given to an individual, administration is (possibly) "prophylactically" It is preferably an "effective amount" or a "therapeutically effective amount", which is sufficient for the benefit to the individual to appear. The actual amount administered and the rate of administration and the time course of administration are determined by the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the invention may be provided, for example, in a prefilled syringe.

Also, the pharmaceutical compositions of the invention as described above include, but are limited to, any orally acceptable dosage form (eg, capsules, tablets, water-soluble suspensions or solutions). It may be administered orally. For tablets for oral use, commonly used carriers include lactose and cornstarch. Typically, a lubricant (eg, magnesium stearate) is also added. Diluents useful for oral administration in capsule form include lactose and dried cornstarch. When a water-soluble suspension is required for oral use, the active ingredient (ie, the transporter cargo conjugate molecule as described above) is mixed with the emulsifier and suspending agent. Will be done. In addition, specific sweeteners, flavors or colorings may be added as required.

Also, in particular, when the target of treatment includes an area or tissue that is readily reachable by topical use, such as a skin disorder or any other reachable epithelial tissue disorder, the present invention. The pharmaceutical composition may be administered topically. Suitable topical formulations can be readily prepared for each of these regions or organs. For topical use, the pharmaceutical composition of the present invention may be prepared in a suitable ointment containing the pharmaceutical composition of the present invention (particularly the components as described above), one or more carriers. It may be suspended or dissolved in. Carriers for topical administration include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified wax and water. Alternatively, the pharmaceutical compositions of the present invention may be formulated in suitable lotions or creams. Suitable carriers for the present invention include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. ..

The prescribing treatment may be a single dose plan or a multiple dose plan, and a multiple dose plan is preferred with respect to the present invention. Known antibody-based drugs, especially those based on anti-cytokine antibodies (eg, anti-GM-CSF antibodies), provide guidance on the frequency of administration (eg, drugs are delivered daily, weekly, monthly). Should be etc.) is provided. The frequency and dosage may also be determined by the severity of the symptoms.

For example, the pharmaceutical compositions according to the invention are daily, eg, once or several times a day (eg, once, twice, three or four times a day), preferably once a day. It may be administered once or twice, more preferably once a day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st, or more (eg, 1 month, 2 months, 3) May be administered daily for months, 4 months, 5 months and 6 months). Preferably, the pharmaceutical composition according to the invention is weekly, eg, once or twice, preferably once a week, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks. , 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks, or more (For example, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or 2 years, 3 years, 4 years or 5 years May be administered (weekly during).

In particular, for single doses (eg, daily, weekly or monthly), especially for weekly doses, the amount of antibody or antigen-binding fragment thereof in the pharmaceutical composition according to the invention is 150 mg. It does not exceed, preferably does not exceed 100 mg, more preferably does not exceed 50 mg, even more preferably does not exceed 20 mg, and particularly preferably does not exceed 10 mg. This amount of antibody preferably represents a single dose as described above, which is preferably administered, eg, daily, weekly, etc., as described above. Such a low amount of the antibody according to the invention is in stable form (eg, lyophilized formulations (eg, in previous studies, monoclonal antibodies stored by lyophilization are at 40 ° C. for 33 months, 50 ° C.). Has been shown to be stable for 5 months)) and can be manufactured and formulated at a reasonable cost.

The pharmaceutical composition typically comprises an effective amount of one or more of the antibodies of the invention and / or the epitopes that bind the antibodies of the invention (ie, the desired disease or Sufficient amount to treat, ameliorate, weaken or prevent the condition, or to exhibit a detectable therapeutic effect). Therapeutic effects also include suppression or weakening of disease intensity or physiological symptoms. The exact effective amount for any particular subject may also be determined by the size and weight of the subject, as well as the health condition, nature and extent of the condition, and the treatment or combination of treatments selected for administration. Good. The effective amount in a particular situation is determined by regular experimentation and is within the judgment of the clinician. For the purposes of the present invention, effective amounts are generally from about 0.005 mg / kg to about 100 mg / kg, preferably about 100 mg / kg, relative to the body weight (eg kg) of the individual to whom the pharmaceutical composition is administered. , About 0.0075 mg / kg to about 50 mg / kg, more preferably about 0.01 mg / kg to about 10 mg / kg, even more preferably about 0.02 mg / kg to about 5 mg / kg, particularly preferably about. It may be 0.03 mg / kg to about 1 mg / kg of the antibody of the invention (eg, the amount of antibody in the pharmaceutical composition).

Furthermore, the pharmaceutical composition according to the present invention may also contain at least two antibodies or antigen-binding fragments thereof according to the present invention. The two antibodies or antigen-binding fragments thereof specifically bind to different sets of non-overlapping epitopes in cytokines (particularly GM-CSF). For example, the pharmaceutical composition according to the present invention may contain a first antibody or an antigen-binding fragment thereof according to the present invention and a second antibody or an antigen-binding fragment thereof according to the present invention. The first antibody or antigen-binding fragment thereof specifically binds to a first set of non-overlapping epitopes in cytokines (particularly GM-CSF), which is bound by the second antibody or antigen-binding fragment thereof. It differs from the set of non-overlapping epitopes in the cytokines (especially GM-CSF). In the present specification, "a different set of non-overlapping epitopes in cytokines (particularly GM-CSF)", for example, the first set comprises epitopes I and II in cytokines and a second (different) set. Means that it contains Epitope I and Epitope III in the cytokine. Therefore, the different sets may overlap (ie, may contain the same epitope), but they are always different at least one epitope. Preferably, however, the different sets of epitopes do not overlap (ie, the first antibody binds to at least two different sites in a cytokine (eg, GM-CSF)). Each of the above two different sites is different from at least two different sites on a cytokine (eg, GM-CSF) to which the second antibody binds. For example, the pharmaceutical composition according to the present invention comprises two antibodies or antigen-binding fragments thereof according to the present invention, and the first antibody or antigen-binding fragment thereof is a non-overlapping site in a cytokine (particularly GM-CSF). When specifically binding to I and II, the second antibody or antigen-binding fragment thereof may specifically bind to non-overlapping sites III and IV in cytokines (particularly GM-CSF). Thus, all (non-overlapping) sites on cytokines (eg, GM-CSF) may be covered by the antibodies according to the invention.

Further, the above-mentioned pharmaceutical composition also contains two or more (for example, three, four, five, six, etc.) antibodies according to the present invention, and at least two containing antibodies, preferably. Two or more contained antibodies, more preferably all contained antibodies, bind to different sets of epitopes of cytokines (eg, GM-CSF).

Preferably, the two antibodies according to the present invention are present in equimolar amounts in the pharmaceutical composition, preferably as an equimolar mixture.

Preferably, the composition comprises two or more (eg, 2, 3, 4, 5, etc.) antibodies of the invention to provide additional therapeutic or therapeutic synergistic effects. You may be. The term "synergistic" is used to describe the effect of a combination of two or more active ingredients, which is greater than or equal to the sum of the effects of each active ingredient. Therefore, when the effect of a combination of two or more agents is an "synergistic inhibition" of activity or process, it is intended that the inhibition of that activity or process is greater than the sum of the inhibitory effects of each active ingredient. .. The term "therapeutic synergy" is used in a combination of two or more treatments where the therapeutic effect (measured by a number of parameters) is greater than or equal to the sum of the therapeutic effects seen in each treatment. Represents the therapeutic effect seen.

In other embodiments, the composition comprises one or more (eg, two, three, etc.) antibodies of the invention and one or more (eg, two, three, etc.) antibodies. It contains additional antibodies against cytokines (particularly GM-CSF). Furthermore, administration of the antibodies of the invention with antibodies specific for other cytokines (more generally other antigens) is within the scope of the invention. The antibodies of the invention may be mixed / administered simultaneously or may be administered at a different time than antibodies specific for other cytokines (more generally other antigens).

Examples of antibodies of the invention against cytokines (particularly GM-CSF) include Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs2GC1d, Bs1GC2a, Bs1GC2a However, it is not limited to these.

In addition, pharmaceutical compositions containing antibodies according to gTs1GC1 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions comprising an antibody according to gTs1GC2a or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions containing antibodies according to gTs2GC2b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibodies according to gTs2GC2c or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibodies according to gTs3GC2d or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions comprising an antibody according to gTs3GC2e or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions comprising antibodies according to gBs3GC1a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions comprising an antibody according to gBs3GC1b or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions containing an antibody or antigen-binding fragment thereof according to gBs2GC1c, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions comprising antibodies according to gBs2GC1d or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing an antibody according to gBs1GC2a or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions comprising an antibody according to gBs3GC2b or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier are also preferred. Pharmaceutical compositions comprising antibodies according to gBs1GC3a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibodies according to gBs3GC3b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibodies according to gBs3GC4 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibodies according to gBs3GC5 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred.

In addition, pharmaceutical compositions containing antibody Ts1GC1 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing the antibody Ts1GC2a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing the antibody Ts2GC2b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing the antibody Ts2GC2c or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing the antibody Ts3GC2d or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing the antibody Ts3GC2e or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs3GC1a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs3GC1b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs2GC1c or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs2GC1c or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs2GC1d or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs1GC2a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs3GC2b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs1GC3a or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs1GC3b or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs1GC4 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred. Pharmaceutical compositions containing antibody Bs1GC5 or antigen-binding fragments thereof, and pharmaceutically acceptable carriers are also preferred.

In one embodiment, the composition of the invention may comprise an antibody of the invention, which antibody is at least 50% by weight (eg, 60% by weight, 70% by weight) of the total protein in the composition. , 75% by weight, 80% by weight, 85% by weight, 90% by weight, 95% by weight, 97% by weight, 98% by weight, 99% by weight or more). In such compositions, the antibody is preferably in purified form.

The present invention comprises the steps of (i) preparing an antibody of the invention; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers. A method of preparation is also provided.

In other embodiments, the method of preparing a pharmaceutical composition comprises the step of mixing the antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is the transformed B cell of the invention or It is a monoclonal antibody obtained from cultured plasma cells. Therefore, first, the procedure for obtaining a monoclonal antibody and the procedure for preparing a drug from it can be performed at very different times by different people in different places (eg different countries).

As an alternative to delivery of antibodies or B cells for therapeutic purposes, a nucleic acid (typically DNA) encoding the desired monoclonal antibody (or active fragment thereof) derived from B cells or cultured plasma cells is targeted. The nucleic acid can be expressed in a subject in situ to provide the desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known to those of skill in the art.

The composition may include an antibacterial agent, especially if packaged in a multidrug dosage form. These may contain a surfactant (eg Tween (polysorbate) (eg Tween 80)). Surfactants are generally present at low levels (eg less than 0.01%). The composition may also contain a sodium salt (eg, sodium chloride) to impart tonicity. For example, a NaCl concentration of 10 ± 2 mg / ml is typical.

Further, in particular, if the composition contains a material that has been lyophilized or reconstituted from the lyophilized material, the composition may be a sugar alcohol (eg, mannitol) or a disaccharide (eg, scroll or). Trehalose) may be included, for example, at about 15-30 mg / ml (eg 25 mg / ml). The pH of the composition for lyophilization may be adjusted between 5-8, 5.5-7, or about 6.1 prior to lyophilization.

The compositions of the present invention may also contain one or more immunomodulators. In one embodiment, one or more immunomodulators include an adjuvant.

[Medical treatment and use]
In a further aspect, the invention is an antibody or antibody according to the invention in (i) prevention, treatment or weakening of inflammatory and / or autoimmune disease; or (ii) diagnosis of inflammatory and / or autoimmune disease. Provided are the use of an antigen-binding fragment thereof, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention, an immunogenic polypeptide according to the present invention or a pharmaceutical composition according to the present invention.

The inflammatory disease may be due to various causes. With respect to the present invention, preferably, such an immune disorder may be treated, weakened and / or prevented, and such an immune disorder may have a physical cause (eg, burn, frost, injury, blunting or hypersensitivity). (Blunt or penetrating), foreign bodies (eg, debris, dust and dust), trauma and ionizing radiation; biological causes (eg, pathogen infection, immune response and stress due to hypersensitivity); and chemical causes (eg, eg) Due to chemical irritants, toxins and alcohol).

Inflammatory diseases (also referred to as inflammatory disorders) include the following diseases, and as described below, with respect to the present invention, inflammatory diseases are preferably vasculitis, vasculitis, colitis, vasculitis, dermatomyositis. Flame, venous inflammation, RSD / CRPS, rhinitis, tendonitis, tonsillitis, vasculitis, Alzheimer's disease, tonic spondylitis, arthritis (degenerative arthritis, rheumatic arthritis (RA), psoriatic arthritis, asthma, atheroscleritis) Sclerosis, Crohn's disease, colitis, dermatomyositis, vasculitis, vasculitis, hepatitis, irritable bowel syndrome (IBS), systemic erythematosus (SLE), nephritis, Parkinson's disease, ulcerative colitis, vulgaris Acne, autoinflammatory disease, celiac disease, prostatic inflammation, alveolar proteinosis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, Qualitative cystitis, inflammatory myopathy, encephalomyositis (especially acute diffuse encephalomyositis), spondylitis (especially rigid spondylitis), anti-ARS antibody syndrome, dermatomyositis (especially atopic dermatomyositis or contact dermatomyositis) ), Hepatitis (especially autoimmune hepatitis), autoimmune peripheral neuropathy, pancreatitis (especially autoimmune pancreatitis), Bechette's disease, Bickerstaff type, brain stem encephalitis, Blau syndrome, Celiac's disease, Shagas' disease, polyneuropathy ( Especially chronic inflammatory demyelinating polyneuropathy), myelitis (especially chronic recurrent multiple myelitis), Churg-Strauss syndrome, Cogan syndrome, giant cell arteritis, CREST syndrome, vasculitis (especially dermatomyositis) (Cutaneous small-vessel vasculitis) and urticaria-like vasculitis), herpes dermatomyositis, dermatomyositis, systemic sclerosis, Dressler syndrome, drug-induced lupus, columnar erythematosus, tendonitis, eosinophilia Myositis, eosinophilic gastroenteritis, nodular erythema, idiopathic pulmonary fibrosis, vasculitis, Graves' disease, Gillan Valley syndrome, Hashimoto thyroiditis, Henoch-Schoenlein purpura, sweat gland abscess, idiopathic inflammatory prolapse Myelopathy, myositis (especially inclusion myositis), dermatomyositis (especially interstitial dermatomyositis), Kawasaki disease, squamous lichen, rupoid hepatitis, Majeed syndrome, Meniere's disease, microscopic polyvasculitis, mixed connective tissue disease, spinal cord Inflammation (especially optic neuromyositis), thyroiditis (especially Ord's thyroiditis), rheumatism (especially recurrent rheumatism), personage-turner syndrome, vesculitis vulgaris, perivenous encephalitis, nodular polyarteritis , Polymyositis (especially rheumatic polymyositis), polymyositis, liver cirrhosis (especially primary) Biliary cirrhosis), cholangitis (especially primary sclerosing cholangitis), progressive inflammatory neuropathy, Rasmussen encephalitis, recurrent polychondritis, arthritis (especially reactive arthritis (Reiter's disease) and rheumatoid arthritis) ), Rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, serum disease, spondyloarthritis, Takayasu's arthritis, Troza-Hunt syndrome, transversal myelitis and Wegener's granulomatosis.

Autoimmune disorders (also referred to as autoimmune diseases) include, therefore, with respect to the present invention, autoimmune diseases are preferably CNS autoimmune diseases, autoimmune diseases, Celiac's disease; Schegren's syndrome. , Systemic erythematosus, Blau syndrome, bullous vesicles, cancer, Castleman's disease, Celiac's disease, Shagas' disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent polymyelitis, chronic obstructive pulmonary disease , Charg-Strauss Syndrome, Scarring Syndrome, Cogan Syndrome, Cold Aggregation Disease, Complementary Second Component Deficiency, Contact Dermatitis, Head Artitis, CREST Syndrome, Crohn's Disease, Cushing Syndrome, Darkham Disease, Herpes dermatitis, dermatomyitis, type 1 diabetes, diffuse systemic rigidity, Dressler syndrome, lupus, columnar erythematosus, eczema, acute diffuse encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, muscle Atrophic lateral sclerosis (Lou Gehrig's disease; also called motor neuron disease), tonic spondylitis antiphospholipid antibody syndrome, anti-ARS antibody syndrome, atopic dermatitis, autoimmune poor regeneration anemia, autoimmune Cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune internal ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome ), Autoimmune progesterone syndrome, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune vasculitis, Barrow disease / Barrow concentric sclerosis, Bechet's disease, Berger's disease, Bickerstaff-type brain stem Encephalitis, endometriosis, attachment inflammation-related arthritis, eosinophilia gastroenteritis, acquired epidermal vesicular disease, fetal erythrocytosis, Evans syndrome, osteofibrotic dysplasia, fibrotic aneuritis ( Or idiopathic pulmonary fibrosis), gastrointestinal inflammation, glomerulonephritis, Good Pasture syndrome, Graves disease, Gillan Valley syndrome, Hashimoto encephalopathy, Hashimoto thyroiditis, gestational herbitis, sweat gland abscess, hypogamma globulinemia, idiopathic Sexual thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgG nephritis, ocular scarring scoliosis, inclusion myopathy, rheumatoid arthritis, chronic inflammatory rheumatic fever, demyelinating polyneuropathy, sarcoidosis , Recurrent rheumatism, interstitial cystitis, juvenile idiopathic schizophrenia, PANDAS (pediatric autoimmune lytic bacterium-related psychiatric neuropathy), Schmidt syndrome, other forms of neuropsychiatric Kawasaki disease of APS (neur) opsychiatric Kawasaki's disease another form of APS), Schnitzler syndrome, paraneoplastic cerebral degeneration syndrome, leukoblastic serum disease, squamous lichen, Sjogren's syndrome, sclerosing lichen, personality turner, linear IgA disease, stale disease, Psoriasis vulgaris, Lupoid hepatitis, autoimmune hepatitis, Stiffperson syndrome, malignant anemia, subacute bacterial endocarditis (SBE), POEMS syndrome, erythema plaque, Sweet syndrome, sympathetic eye inflammation, Meniere's disease, Systemic lupus, primary biliary cirrhosis, Miller-Fisher syndrome, hyperan arteritis, cholangitis, progressive inflammatory neuropathy, Much-Habermann's disease, psoriasis, psoriatic arthritis, necrotizing pyoderma, multiple sclerosis Disease, erythroblast, Rasmussen encephalitis, severe myasthenia, transverse myelitis, Reynaud phenomenon, microscopic colitis, ulcerative colitis, myitis, idiopathic inflammatory bowel disease (IBD), neuromyelitis optica, Devic It may be selected from the group consisting of disease and neuromyelitis.

Typically, autoimmune diseases result from the body's abnormal immune response (autoimmunity) to substances and tissues normally present in the body. It may be limited to specific organs (eg, autoimmune thyroiditis) or may be associated with specific tissues at different sites (eg, goodpasture that can affect the basement membranes of both lungs and kidneys) Disease). Autoimmune diseases may be classified according to the corresponding type of hypersensitivity: type I (eg, autologous serum-induced urticaria), type II, type III or type IV.

In particular, at least some autoimmune diseases may be inflammatory diseases and vice versa.

Autoimmune and / or inflammatory diseases are preferably treated, prevented and / or weakened with respect to the present invention, and autoimmune and / or inflammatory diseases include multiple sclerosis, pulmonary alveolar proteinosis, arthritis. Includes (especially rheumatoid arthritis) and asthma.

Within the scope of the present invention, with respect to pharmaceutical compositions, the administration of some of the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, immunogenic polypeptides or pharmaceutical compositions as described above. Morphology and route of administration are included. It is also used with respect to the use of antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, immunogenic polypeptides as described herein (especially with respect to preferred forms of administration and routes of administration). To.

Methods of diagnosis include contacting an antibody or antibody fragment with a sample. Such samples may be isolated from the subject, eg, nasal cavity, nasal passage, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries, pituitary glands, adrenal glands, thyroid glands It may be, for example, an isolated tissue sample obtained from the brain, skin or blood (preferably serum). The diagnostic method may also include detection of the antigen / antibody complex (particularly, contact of the sample with the antibody or antibody fragment as shown below). Such diagnostic steps are typically performed in the laboratory (ie, without touching the human or animal body at all). Examples of detection methods include, for example, ELISA (enzyme-linked immunosorbent assays).

The present invention relates to (i) the present invention in (a) the manufacture of agents for the treatment or weakening of inflammatory and / or autoimmune diseases or (b) the diagnosis of inflammatory and / or autoimmune diseases. Also provided are the use of antibodies, antibody fragments or variants and derivatives thereof, (ii) immortalized B cell clones according to the invention, (iii) nucleic acids or vectors according to the invention or (iv) pharmaceutical compositions of the invention. To do.

The present invention also provides the compositions of the present invention for use as agents for the prevention or treatment of inflammatory and / or autoimmune diseases. It also provides the use of the antibodies of the invention and / or proteins containing epitopes to which such antibodies bind in the treatment of the subject and / or the manufacture of agents for diagnosis in the subject. It also provides a method of treating a subject, comprising the step of administering the composition of the invention to the subject. In some embodiments, the subject may be a human. One method of confirming the effectiveness of a therapeutic treatment involves observing disease symptoms after administration of the compositions of the invention. The treatment may be a single dose plan or a multiple dose plan.

In one embodiment, an antibody, antibody fragment, immortalized B cell clone or pharmaceutical composition according to the invention is administered to a subject in need of such treatment. Such subjects include, but are not limited to, those at particular risk of inflammatory and / or autoimmune disease or those who are particularly susceptible to inflammatory and / or autoimmune disease.

Antibodies and fragments thereof as described in the present invention may be used in kits for the diagnosis of inflammatory and / or autoimmune diseases. In addition, at least two epitopes capable of binding the antibodies of the invention, especially epitopes of cytokines (eg, GM-CSF), detect the presence of anti-cytokine protective antibodies (particularly anti-GM-CSF protective antibodies) or anti-cytokines. It may be used in kits for observing the effectiveness of the procedure by determining the antibody titer of a protective antibody, especially an anti-GM-CSF protective antibody.

The present invention also provides a method of preparing a medicament, comprising mixing a monoclonal antibody with one or more pharmaceutically acceptable carriers. The monoclonal antibody is a monoclonal antibody obtained from the transfected host cell of the present invention. Therefore, the procedure of first obtaining the monoclonal antibody (eg, expressing it and / or purifying it) and then mixing it with one or more pharmaceutical carriers can be performed at various locations (eg, for example). It can be carried out at very different times by different people in different countries).

In order to immobilize the antibody expressed by the transformed B cells or the cultured plasma cells, including the transformed B cells or the cultured plasma cells of the present invention, in order to immobilize the antibody expressed by the transformed B cells or the cultured plasma cells, the culture step, the subculture step, Various steps such as a cloning step, a subcloning step, a sequencing step, and a nucleic acid preparation step may be performed. In one embodiment, the methods described above further include optimization techniques used for nucleic acids encoding antibodies (eg, affinity maturation and optimization). The present invention includes all of the cells, nucleic acids, vectors, sequences, antibodies, etc. used and prepared during these steps.

In all these methods, the nucleic acid used in the expression host may be engineered to insert, delete or alter a particular nucleic acid sequence. Changes made by such operations include changes to introduce restriction sites, changes to improve codon usage, changes to add or optimize transcriptional and / or translational regulatory sequences, and the like. However, it is not limited to these. Nucleic acids can also be modified to alter the encoded amino acids. For example, one or more amino acids (eg, one, two, three, four, five, six, seven, eight, nine, ten, etc.) to the amino acid sequence of an antibody. Substitution, amino acid deletion and / or amino acid insertion may be useful. Such point mutations may alter effector function, antigen binding affinity, post-translational modification, immunogenicity, etc., may introduce amino acids for binding covalent groups (eg, labels), and may introduce tags (eg, labels). A tag for purification purposes) may be introduced. Mutations may be introduced at specific sites or may be randomly introduced following selection (eg molecular evolution). For example, one or more nucleic acids encoding any of the CDR regions, heavy chain variable regions or light chain variable regions of the antibodies of the invention are mutated randomly or directly to differ into the encoded amino acids. Properties may be introduced. Such changes may be the result of an iterative process in which the initial changes are maintained and new changes are introduced at other nucleotide sites. In addition, changes achieved in independent processes may be combined. The different properties introduced into the encoded amino acid may include, but are not limited to, improved affinity.

The scope of the present invention is not limited by the particular embodiments described herein. Indeed, various modifications of the invention, as well as the modifications described herein, will be apparent to those skilled in the art from the description and accompanying drawings. Such changes are included in the appended claims.

Although not otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The same or corresponding methods and materials as described herein may be used in the practice or testing of the present invention, but suitable methods and materials are described herein. All references, patent specifications, patents and other references referred to herein are hereby incorporated by reference in their entirety. In case of dispute, this specification containing the definition is restricted.

The drawings, sequence listings and examples below are intended to further illustrate the invention. These are not intended to limit the object of the present invention to these.

[Explanation of drawings]
FIG. 1 shows (A) to (C). (A) SPR cross-competition between GCA21, GCA7 and GCB59, (B) multi-channel chips coated with GCA21, GCA7 and site IV GCB59 are soaked by GM-CSF and into excess amounts of the same antibody. Continuously exposed. (C) Samples containing three non-cross-competitive antibodies, either alone or with GM-CSF added at equimolar concentrations (1: 1) or 10-fold excess of antibody (10: 1). SEC-HPLC profile.

FIG. 2 shows the strong in vitro neutralization of GM-CSF by a combination of three antibodies. A fixed amount of GM-CSF (final concentration 50 pg ml-1) was incubated with a serial dilution of one or more antibodies and added to TF1 cells (10000 / well) and cell proliferation was performed on day 3 by uptake of thymidine. Measured to. (A) Schematic diagram of the TF-1 bioassay. (B) Serial dilutions of a single monoclonal antibody, or mixture of two or three non-cross-competitive antibodies, were tested for their ability to neutralize GM-CSF. (C) The sensitivity of the above test was changed by changing the number of cells and the concentration of GM-CSF as shown. Inhibition achieved with a single antibody or three non-cross-competitive antibodies is shown for each experimental condition.

FIG. 3 shows the Fc-dependent elimination of the GM-CSF immune complex in vivo. (A) Sandwich ELISA for detecting GM-CSF in the presence of specific antibodies. A fixed amount of GM-CSF was added to mouse serum with three monoclonal antibodies (GCA21, GCA7, GCB59) added individually or in combination. Quantification of GM-CSF was performed by sandwich ELISA with antibodies specific for site I for capture and site II for detection. A serial dilution in neutral (left) or alkaline buffer (right) was added and the GM-CSF concentration was determined relative to the GM-CSF standard. The dashed line represents the concentration of GM-CSF measured in the absence of antibody. (B) Female Balb / c mice (5 per group) with 100 μg of monoclonal antibody (GCA21 (1 mAb) or GCA21 + GCA7 + GCB59 (3 mAb) in IgG or IgG-LALA) or 2 mg total from PAP patients IgG was injected and 16 hours later 2 μg of GM-CSF was injected. Day 1 and day 5 GM-CSF concentrations in untreated serum (left) or alkaline treated serum (right) are shown. (C) Proliferation of TF-1 cells in response to different dilutions of mouse serum infused with GM-CSF and the indicated antibody 24 hours prior. (D) Anti-IgG Fc of GM-CSF immune complex formed by one or three antibodies (in IgG or IgG-LALA) against Fc RIIaTZM-bl cells or Fc RIIb expressing TZM-bl cells. Binding as measured by flow cytometry with a specific antibody.

FIG. 4 shows a schematic diagram of three bispecific constructive Bs1, Bs2 and Bs3 and three triple specificity construct Ts1, Ts2 and Ts3. Positions A, B and / or C (if applicable) are shown for each of the construct types. The single chain variable domain (ScVd) of the heavy and light chains (both) of the different GM-CSF antibodies is used as a scaffold for the N-terminus and / of the heavy or light chain of one GM-CSF antibody. Alternatively, it is added to the C-terminal. The black ellipse represents the VH domain and the dark gray ellipse represents the VL domain. VHs and VLs of different ScVd are connected via a specific linker. Other linkers have been used to connect ScVd between each other to full-length antibodies used as scaffolds. The light gray ellipse represents the IgG1 CH domain and the IgG1 CL domain.

FIG. 5 shows that TsGC1 binds to GM-CSF with a very high affinity and a very slow dissociation rate (A). (BC) TsGC1 can utilize all three different specificities for binding to GM-CSF. GCA21, GCA7 and GCB59 were immobilized on an SRP chip and continuously exposed to (1) GM-CSF, (2) GCA7, GCB59 and GCA21 respectively, and (3) finally TsGC1. (E) TsGC1 can form a high molecular weight complex with GM-CSF. TsGC1 was immobilized on an SPR chip and continuously exposed to GM-CSF and soluble TsGC1 three times. The same experiment performed with GCA7 as a control is shown.

FIG. 6 shows a very strong neutralization of GM-CSF by TsgC2d and BsGC3a when compared to a single antibody or a combination of antibodies forming TsgC2d and BsGC3a.

FIG. 7 shows flow cytometry using an anti-IgG Fc-specific antibody against TZM-bl cells expressing FcRIIa or FcRIIb of the immune complex formed by GM-CSF and TsGC2d or BsGC3a. Shows the bond as measured. Binding is compared to the binding of a single antibody, as well as a combination of two or three antibodies forming BsGC3a and TsGC2d, respectively.

〔Example〕
An exemplary embodiment of the invention is shown in the following examples. The following examples are provided for purposes of illustration only and to assist those skilled in the art when using the present invention. The examples are by no means otherwise intended to limit the scope of the invention.

(Example 1: Isolation and property determination of GM-CSF-specific antibody)
Peripheral blood samples were collected from 5 patients with alveolar proteinosis (PAP). IgG memory B cells have been cryopreserved by a combination of magnetic cell isolation and fluorescence activated cell isolation (particularly using anti-FITC microbeads (Miltenyi Biotec) and later staining PBMCs with CD22-FITC (BD Phamingen)). Isolated from new PBMC or new PBMC. Then, as explained by Traggiai E. et al. (2004) Nat Med. 10 (8): 871-5 and WO 2004/076677, IgG memory B cells were subjected to 384 wells in the presence of feeder cells. In microplates, immortalized under clonal conditions with EBV (Epstein-Barr virus) and CpG. Culture supernatants were screened by ELISA for the presence of GM-CSF-specific IgG antibodies. Four immortalized B cell clones producing the GM-CSF monoclonal antibody were identified. cDNA was synthesized from positive cultures, antibody V genes (variable regions of heavy and light chains) were sequenced and analyzed using the IMGT database (http://www.imgt.org/). The V (D) J gene utilization rates of the four PAP autoantibodies are shown in Table 5.

All antibodies were recombinantly produced as IgG by transient transfection of HEK 293 Freestyle Cells (Invitrogen) with polyethyleneimine (PEI). Antibodies were then tested for binding to human GM-CSF by ELISA. Binding properties and V gene utilization are shown in Table 5. Multiple antibodies showed high affinity comparable to antibody MOR103 and namilumab, a monoclonal antibody that neutralizes GM-CSF in clinical development and serves as a reference antibody herein. EC50 values (ng / ml) were determined by ELISA and calculated on a sample-by-sample basis by non-linear regression analysis using GraphPad Prism 5 software. The EC50 value was in the range of 61.4 to 307.6 ng / ml. Interestingly, the antibody did not cross-react with both mouse and rat GM-CSF.

The reaction rate of binding was determined by surface plasmon resonance (SPR). Briefly, for SPR, protein A (450 nM) was stabilized in 10 mM acetate buffer and placed on a ProteOn sensor chip (Biorad) pre-activated by EDC / NHS via an amine bond. Immobilized (unreacted groups were blocked by infusion of ethanolamine HCl (1M)). HEPES buffered saline (HBS) (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% detergent Tween-20) was used as the running buffer. All injections were performed at a flow rate of 100 μl / min. The monoclonal antibody was diluted in HBS (200 nM) and injected onto a capture chip coated with protein A and injected with different concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM) of human GM-CSF. (HBS was injected into one channel of the chip and used as a reference for analysis). The injection time and dissociation time were 120 seconds and 600 seconds, respectively. Each mAb binding reaction with GM-CSF was evaluated using a ProteON XPR36 instrument (Biorad) and the data was processed by ProteOn Manager Software. Ka, Kd and KD were calculated by applying them to the Langmuir fit model. The KD determined was in the range of 0.18 to 0.69 nM, consistent with high affinity binding. However, the reaction rate values were very heterogeneous. For example, the antibodies GCA7 and GCB59 had similar KD values (0.38 and 0.68 nM) but showed different kinetics (GCA7 had a loose binding / dissociation rate and GCB59 had a high binding / It was characterized by dissociation rate) (Table 6).

(Example 2: Four PAP autoantibodies recognize different sites on GM-CSF and form high molecular weight immune complexes)
In order to evaluate the simultaneous binding of different mAbs to GM-CSF, SPR cross-competition experiments are performed by continuously injecting different mAbs (200 nM each) after GM-CSF capture (50 nM). This was carried out as described above (Example 1). The injection time and dissociation time were 60 seconds and 20 seconds, respectively. This revealed that GCA7, GCA21, GCB59 and GCE536 do not cross-compete between them for binding to GM-CSF (FIG. 1A). Interestingly, SPR experiments show that three non-cross-competitive antibodies can simultaneously bind to a single molecule of GM-CSF (Fig. 1B). In addition, when GM-CSF is incubated with an excess of three antibodies, the formation of high molecular weight immune complexes can be detected by size exclusion chromatography (SEC-HPLC) (FIG. 1C). In SEC-HPLC experiments, three non-cross-competitive mAbs were diluted in PBS, either alone or as a combination of three antibodies, and mixed with GM-CSF (1: 1 or 10: 1 molar ratio). 1 hour, RT). Samples were analyzed by an Agilent 1100 HPLC machine (using PBS (flow rate: 1 ml / min) as mobile phase) using a TSK-GEL G3000SW column (Tosoh, bed volume: 13 ml, void volume: 4.6 ml). A universal solvent 2 μm filter (Agilent) was placed between the injector and the column. Detection was performed by a Variable Wavelength Detector (VWD, Agilent) with an ultraviolet absorption of 220 nm.

(Example 3: Strong in vitro neutralization of GM-CSF requires a combination of three antibodies that bind to non-overlapping sites)
The neutralizing activity of autoantibodies was evaluated by measuring their ability to inhibit the proliferation of TF-1 cells in response to recombinant GM-CSF. For this purpose, TF-1 cells (CLS, Cell Lines Service) were subjected to 10% fetal bovine serum (Hyclone), 1% penicillin / streptavidin, 1% non-essential amino acids, 1% sodium pyruvate. , 0.1% 2-mercaptoethanol (all obtained from GIBCO) supplemented with RPMI 1640 medium. Cells were cultured in a humidified 37 ° C. incubator with 5% CO2. GM-CSF neutralization assay, serial dilution of mAbs (total IgG or affinity purified antibody) in growth medium without either GM-CSF or IL-3, GM- at a concentration of 100 pg / ml. This was done by adding CSF and preincubating at 37 ° C. for 1 hour in 96-well flat-bottomed cell culture plates (Costar). The cells were washed 5 times, diluted in growth medium without either GM-CSF or IL-3 and seeded with 10000 cells per well (final concentration of GM-CSF equals 50 pg / ml). ). In other tests, GM-CSF was used at final concentrations of 500 and 5000 pg / ml and 1000 cells were seeded per well. Antibodies-free cells with or without GM-CSF were used as controls to determine the maximum and minimum levels of cell proliferation. Plates were incubated for 72 hours in a humidified 37 ° C. incubator with 5% CO2 and cell proliferation was carried out for 6 hours with 0.2 μCi / well of [3H] -thymidine (PerkinElmer). Measured after incubation. GM-CSF neutralization was calculated as a percentage of TF-1 growth using the formula below.
[1- (Average CCPM of control cells cultured without signal well CCPM-GM-CSF) / (Average CCPM of control cells cultured with GM-CSF) / (Average CCPM of control cells cultured without GM-CSF) ] X 100 (CCPM = corrected count / minute)
IC90 (μg / ml) was calculated for each sample by non-linear regression analysis using GraphPad Prism 5 software. In some experiments, mouse sera were titrated in TF-1 growth medium and preincubated at 37 ° C. for 30 minutes. TF-1 cells were washed and seeded (1000 cells per well). GM-CSF (60,000-0.3 ng / ml) for titration was used as a growth control. The CCPM of each signal well was plotted against serum titer measurements.

Surprisingly, using this bioassay, GCA21, GCA7 and GCB59 were unable to neutralize GM-CSF even when tested at a concentration of 1 mg / ml (no data shown) (FIG. 2B). ). The only exception was GCE536, which had an IC90 value of 2.43 μg / ml and neutralized GM-CSF, with therapeutic antibodies Namilumab and MOR103 exhibiting IC90 values of 0.80 and 0.16, respectively. Interestingly, when combined with each other, the two non-cross-competitive antibodies showed enhanced neutralizing activity for both dose response and inhibition rate, with the combination of GCA21 and GCB59 being the most effective (Fig. 2B). Remarkably, the combination of the three non-cross-competitive antibodies (GCA21, GCA7 and GCB59) has an IC90 value of 0.08 μg / mL (expressed as the total concentration of the three mAbs) (IC90 value of therapeutic antibody MOR103 and namilumab). (Lower) led to complete inhibition of proliferation (Fig. 2B).

As expected from the law of mass action, it was found that varying cell number and GM-CSF concentration dramatically affected the sensitivity of the assay. In particular, a decrease in TF-1 cells and a decrease in the concentration of GM-CSF resulted in a more sensitive test, showing accelerated neutralization with a single antibody and multiple antibodies (Fig. 2C). In contrast, when a large number of TF-1 cells and high doses of GM-CSF were used, even the most potent neutralizing antibodies, MOR103 and namilumab, contained GM-CSF in the presence of 400-fold excess molar concentrations. I couldn't reconcile. Remarkably, under all conditions, the combination of the three non-cross-competitive antibodies was able to completely neutralize GM-CSF.

(Example 4: Fc-dependent clearance of GM-CSF immune complex in vivo)
In vivo action of a single autoantibody vs. multiple autoantibodies to demonstrate that GM-CSF can form a complex with three antibodies (causing effective in vitro neutralization of cytokine bioactivity). Tested. To this end, a group of 6-8 week old female BALB / c mice was intravenously injected with 100 μg of purified mAb or 2 mg of total IgG purified from PA96 patients. After 16 hours, 2 μg of human GM-CSF was injected. Serum samples were collected on days 1 and 5. GM-CSF was quantified by sandwich ELISA. Briefly, 10 μg / ml antibody bound to site II of GM-CSF was used to coat a 96-well Maximorp plate (Nunc) (and then blocked by PBS + 10% FBS (Gibco)). .. All sera and GM-CSF were titrated and placed in parallel under different conditions (either untreated and after alkali treatment to separate immune complexes; FIG. 3A) (one plate). 25% (vol / vol) alkaline dissociation buffer (2.5% Triton X100, 2M ethanolamine, 0.15M NaCl, pH 11.6) was added to all samples in the other plates. All samples were tested (25% (vol / vol) PBS + 10% FBS added). The plate was left at RT overnight. Detection of captured GM-CSF is bound to site I of GM-CSF (1 hour, RT) and then 0.5 μg / ml streptavidin-AP (Jackson ImmunoResearch) (1 hour, RT). This was done with 1 μg / ml biotinylated antibody. The plate was then washed, substrate (p-NPP, Sigma) was added and the plate was read at 405 nm.

In the absence of antibody, the injected GM-CSF rapidly disappeared from the serum and could not be detected after 24 hours (Fig. 3B). In contrast, when a single antibody (GCA21 or MOR103) was used, high levels of GM-CSF were recovered on day 1 and were still present on day 5. Notably, GM-CSF detection required alkaline dissociation in the case of MOR103 and not in the case of GCA21, consistent with the different dissociation rates of the two antibodies (Table 6). In particular contrast, when mice received three non-cross-competitive antibodies (GCA21, GCA7 and GCB59) or PAP IgG, low or undetectable amounts of cytokines were present 1 and 5 days after alkaline dissociation. GM-CSF was rapidly eliminated as it was only detected in serum.

To address the expected role of Fc receptors in the elimination of GM-CSF, the same antibodies were tested in variant type (not binding to C1q or Fc-γ receptors, so-called LALA). Similar to wild-type antibodies, a single LALA antibody led to increased levels of GM-CSF in serum. However, in contrast to what was observed for the three wild-type antibodies, the three LALA antibodies were unable to eliminate GM-CSF and were quantitatively recovered in serum even on the 5th day after alkaline dissociation (Figure). 3B).

To determine if antibody-bound GM-CSF is available to the organism, mouse sera were tested for their ability to support TF-1 proliferation (FIG. 3C). The sera of mice receiving GCA21 or MOR103 were consistent with a sufficient GM-CSF dissociation rate to induce vigorous proliferation of TF-1 cells and associate with cytokine receptors. In contrast, mouse sera receiving the three wild-type antibodies or PAP IgG were unable to stimulate proliferation and were consistent with in vivo elimination of immune complexes. In addition, the sera of mice receiving the three LALA antibodies contain high levels of GM-CSF, but are not irritating, and this finding is an irreversible isolation of GM-CSF in a stable immune complex. Is consistent with.

To further address the role of the Fcγ-receptor, the immune complex formed between GM-CSF and wild-type or LALA antibodies binds to TZM-bl cells expressing different Fcγ-receptors. Tested for their abilities. To this end, 10% of four TZM-bl cell lines (NIH AIDS Research & Reference Reagent Program) transfected with specific Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb or FcγIIIa), respectively. Fetal bovine serum (Hyclone), 0.025 M Hepes, 10 μg / ml gentamicin and 20 μg / ml blasticidin were maintained in DMEM supplemented. Cells were cultured in a humidified 37 ° C. incubator with 5% CO2. Expression of specific FcγRs stained TZM-bl cells with FITC-conjugated anti-CD64 (anti-FcγRI), anti-CD32 (anti-FcγRIIa and anti-FcγRIIb) and anti-CD16 (anti-FcγRIIIa) antibodies (all obtained from BD Pharmingen). Evaluated by doing. Untransfected TZM-bl cells and transfected TZM-bl cells were washed with staining buffer (PBS with 10% fetal bovine serum and 2 mM EDTA) and 50,000 cells / cell. Seeded in 96-well plates at well density. A single anti-GM-CSF mAb (GCA21) or a combination of three non-crossing competing mAbs (GCA21, GCA7 and and GCB59) at a concentration of 2.5 μg / μl, 0.05 μg / ml GM-CSF or staining. It was mixed with buffer (PBS with 10% fetal bovine serum and 2 mM EDTA). LALA types of all antibodies, and mAbs with different specificities were included as controls. Samples were incubated for 30 minutes at 37 ° C. to form immune complexes and then cooled to 4 ° C. for 30 minutes prior to adding them to TZM-bl cells. Cells were washed twice and stained with anti-human IgG Fcγ fragment specific F (ab') 2 fragment (Jackson Immuno Research. Samples were BD FACSCanto). Analyzed by (BD Biosciences) and median fluorescence intensity was analyzed and compared among multiple samples.

Strong binding was observed only for FcγRIIa-expressing cells and FcγRIIb-expressing cells when the immune complex was formed by three wild-type antibodies (not LALA) (Fig. 3D). Given these, the above results indicate that a single antibody prolongs the half-life of GM-CSF and builds a circulating pool of cytokines available to the organism, even when strongly in vitro neutralized. Shown. In contrast, three or more antibodies lead to the formation of immune complexes that are efficiently eliminated via Fc-dependent mechanisms.

(Example 5: Genetic engineering preparation of a multispecific antibody having the highest GM-CSF neutralizing activity)
Epitope binding sites of GCA21, GCA7, GCB59 and GCE536, specific in view of the need to target multiple independent sites on GM-CSF to achieve efficient in vivo neutralization and elimination of cytokines. Bispecific and trispecific antibodies carrying linkers and fully human IgG Fc were designed and generated (Table 7). Six different constructs were used to generate multispecific antibodies (Fig. 4). In particular, three constructive Ts1, Ts2 and Ts3 were used to generate six different trispecific hexavalent antibodies, and three constructive Bs1, Bs2 and Bs3 were of 10 different bispecificities. A tetravalent antibody was used to generate (FIGS. 4 and 7). Constructive Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3 were designed in accordance with US Patent Application Publication No. 2009/0155275.

(Example 6: Evaluation of productivity and aggregation of multispecific antibody)
Anti-GM-CSF multispecific antibody was produced in 293F cells and purified by protein A. Quantification was performed by the Pierce bicinchoninic acid (BCA) protein assay according to the manufacturer's instructions (Thermo Scientific). The assay is a detergent-compatible, BCA-based formation for colorimetric detection and quantification of total proteins. Productivity varied according to different antibodies (Bs3CG1a, Bs1CG2a, Bs1CG3a, Bs3GC3b, BsGC5 were produced at concentrations greater than 30 μg / ml). Aggregation of multispecific antibodies (final concentration: 1 mg / ml) was analyzed by measuring their turbidity in OD340 with or without GM-CSF (final concentration: 0.1 mg / ml). (Table 8).

(Example 7: Multispecific antibody binds to GM-CSF with high affinity)
All multispecific antibodies were tested by ELISA for binding to GM-CSF. Binding was highly specific with high affinity for GM-CSF, as shown by EC50 values in Table 9 below. The use of different binding sites for multispecific antibodies was tested by SPR experiments using TsGC1 as a model. In this experiment, GM-CSF was bound by two of the surplus of the three antibodies forming TsGC1, followed by binding of Ts1GC1 to GM-SCF via a third specificity. It was revealed. Ts1GC1 has a very high affinity (very low KD as shown in FIG. 5A). In various SPR experiments, GM-CSF was associated with two of the three antibodies that make up Ts1GC1 so that only one of the three GM-CSF epitopes remains unbound by Ts1GC1. It formed a complex (FIGS. 5B-D). Furthermore, when immobilized on the SPR chip, TsGC1 is a high molecular weight complex when the chip is continuously exposed to soluble GM-CSF and Ts1GC1 multiple times, unlike conventional antibodies (eg GCA7). Was able to be formed (Fig. 5E).

(Example 8: Neutralization of very strong GM-CSF with multispecific antibody)
GM-CSF neutralization was tested using an in vitro bioassay based on TF-1 cells as described above (Example 3). Here, two different GM-CSF concentrations (50 and 500 pg / ml) and two different cell numbers / wells (1000 and 10000) were tested. The IC90 values are recorded in Table 10. Surprisingly, all multispecific antibodies completely inhibited TF-1 proliferation under all conditions tested at very low concentrations (<1 ng / ml for most multispecific antibodies). Notably, using less rigorous conditions, MOR103 and namilumab required more than 100-fold and 690-fold concentrations, respectively, compared to the best multispecific antibody (TsGC2a). Under strict conditions, most multispecific antibodies were able to neutralize GM-CSF at a concentration of 10 ng / ml, and MOR103 and namilumab required concentrations above 1 mg / ml. Two multispecific antibodies Ts3GC2 and Bs1GC3a were selected for their overall properties and compared to the single antibody or combination of antibodies for which they were obtained.

(Example 9: The immune complex of GM-CSF having multiple specificity binds to Fcγ receptors IIa and IIb)
To seek binding of the Fcγ receptor, the immune complex formed between GM-CSF and the multispecific antibody is expressing TZM-bl cells expressing the FcγRIIa receptor and the FcγRIIb receptor. Their ability to bind to TZM-bl cells was tested. Multispecific antibody was mixed with 0.05 μg / ml GM-CSF or staining buffer. Samples were incubated at 37 ° C. for 30 minutes to allow the formation of immune complexes and then cooled to 4 ° C. for 30 minutes prior to adding them to TZM-bl cells. Cells were washed twice and stained with 2 anti-human IgG Fcγ fragment-specific F (ab') fragments. Samples were analyzed by BD FACSCanto (BD Biosciences) and median fluorescence intensity was analyzed and compared among multiple samples. Strong binding was observed with GM-CSF and different multispecific antibodies, along with immune complexes formed on FcγRIIa-expressing cells and FcγRIIb-expressing cells (Table 11). In particular, TsGC2d and Bs1GC3a showed the strongest binding to FcγRIIa and FcγRIIb in the presence of GM-CSF, and they weakly bound to the same FcR in the presence of GM-CSF (FIG. 7). Considering these, the above results suggest that GM-CSF in immune complexes with multispecific monoclonal antibodies can be eliminated from the human body via an Fc-dependent mechanism.

(A) SPR cross-competition between GCA21, GCA7 and GCB59, (B) multi-channel chips covered with GCA21, GCA7 and site IV GCB59 are soaked by GM-CSF and into excess amounts of the same antibody. It was continuously exposed. (C) Samples containing three non-cross-competitive antibodies, either alone or with GM-CSF added at equimolar concentrations (1: 1) or 10-fold excess of antibody (10: 1). SEC-HPLC profile. It shows strong in vitro neutralization of GM-CSF by a combination of three antibodies. It shows the Fc-dependent clearance of the GM-CSF immune complex in vivo. Schematic representations of the three bispecific constructive Bs1, Bs2 and Bs3 and the three triple specificity construct Ts1, Ts2 and Ts3 are shown. It is shown that TsGC1 binds to GM-CSF with a very high affinity and a very slow dissociation rate (A). (BC) TsGC1 can utilize all three different specificities for binding to GM-CSF. GCA21, GCA7 and GCB59 were immobilized on the SRP chip and exposed to (1) GM-CSF, (2) GCA7, GCB59 and GCA21 respectively, and (3) finally TsGC1. (E) TsGC1 can form a high molecular weight complex with GM-CSF. TsGC1 was immobilized on an SPR chip and was sequentially exposed to GM-CSF and soluble TsGC1 three times. The same experiment performed with GCA7 as a control is shown. It shows a very strong neutralization of GM-CSF by TsgC2d and BsGC3a when compared to a single antibody or a combination of antibodies forming TsgC2d and BsGC3a. Binding of CSF and immune complexes formed by TsGC2d or BsGC3a as measured by flow cytometry with anti-IgG Fc-specific antibodies to TZM-bl cells expressing FcRIIa or FcRIIb. Is shown.

Claims (32)

It contains (a) at least two different epitope binding sites; and (b) Fc moieties.
Each of the at least two different epitope binding sites specifically binds to an individual epitope of the cytokine, whereby the plurality of individual epitopes of the cytokine to which the at least two different epitope binding sites bind. , Multiple epitopes that do not overlap,
An isolated multispecific anti-cytogenic antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof is bispecific, trispecific, quadrispecific or quintugenous, and preferably the antibody or antigen-binding fragment thereof is bispecific, trispecific or It is quadruspecific, more preferably the antibody or antigen-binding fragment thereof is bispecific or trispecific, and more preferably the antibody or antigen-binding fragment thereof is trispecific. The antibody according to claim 1 or an antigen-binding fragment thereof. Claim 1 or 2, wherein the antibody molecule comprises exactly two copies of each of the different epitope binding sites that specifically bind to at least two different non-overlapping sites in the cytokine. The antibody or antigen-binding fragment thereof. The anti-cytokine antibody is selected from the group consisting of anti-colony stimulating factor antibody and anti-interferon antibody, and preferably the above anti-cytokine antibody is selected from the group consisting of anti-GM-CSF antibody and anti-interferon beta antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. The antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-cytokine antibody is an anti-GM-CSF antibody. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody is a bispecific tetravalent antibody or a trispecific hexavalent antibody. The above antibody or its antigen-binding fragment
(C) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising at least one linker. A claim, wherein the linker comprises an amino acid sequence according to SEQ ID NO: 143 or 144 or an amino acid sequence according to a functional sequence variant thereof, or comprises the amino acid sequence or a functional sequence variant thereof. 7. The antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof is IgG type, preferably IgG1 type, and more preferably contains a heavy chain constant region of IgG1 CH1-CH2-CH3 type and a light chain constant region of IgG1 CK type. A heavy chain constant region containing an amino acid sequence or a plurality of functional sequence variants thereof, more preferably according to SEQ ID NO: 140, or a heavy chain definition consisting of the amino acid sequence or a plurality of functional sequence varians thereof. An IgG CK-type light chain constant consisting of a normal region and a light chain constant region containing an amino acid sequence according to SEQ ID NO: 141 or a plurality of functional sequence variants thereof, or an amino acid sequence or a plurality of functional sequence variants thereof. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which comprises a normal region. The antibody according to any one of claims 1 to 9, wherein the antibody or an antigen-binding fragment thereof is a construct type selected from the group consisting of Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3. Antigen binding fragment. 10. The antibody or antigen-binding fragment thereof follows constructive Ts3, preferably the antibody or antigen-binding fragment thereof is a trispecific antibody according to constructive T3. The antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. The above antibody or its antigen-binding fragment
(I) Strict conditions with IC ₉₀ of 150 ng / ml or less;
(Ii) Lower strict conditions with IC ₉₀ of 20 ng / ml or less;
Neutralize the cytokines under more stringent conditions with an IC ₉₀ of (iii) 160 ng / ml or less; and / or under very stringent conditions with an IC ₉₀ of (iv) 1000 ng / ml. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, characterized in that. A light chain in which the antibody or antigen-binding fragment thereof comprises at least one CDRH1, at least one CDRH2 and at least one CDRH3, and at least one CDRL1, at least one CDRL2 and at least one CDRL3. It comprises a chain and the at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NO: 3, 51, 69 or 107 or a plurality of functional sequence variants thereof, preferably at least one multiple. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the chain CDRH3 comprises an amino acid sequence according to SEQ ID NO: 3 or 69 or a plurality of functional sequence variants thereof. The above antibody or its antigen-binding fragment
(I) According to SEQ ID NOs: 1 to 5 and 7, or SEQ ID NOs: 1 to 4 and 6 to 7, respectively, CDRH1 amino acid sequence, CDRH2 amino acid sequence, CDRH3 amino acid sequence, CDRL1 amino acid sequence, CDRL2 amino acid sequence and CDRL3 amino acid sequence or their respective. Multiple functional sequence variants;
(Ii) CDRH1 amino acid sequence, CDRH2 amino acid sequence, CDRH3 amino acid sequence, CDRL1 amino acid sequence, CDRL2 amino acid sequence and CDRL3 amino acid sequence or a plurality of functional sequence variants thereof, respectively according to SEQ ID NOs: 49-53 and 55;
(Iii) CDRH1 amino acid sequence, CDRH2 amino acid sequence, CDRH3 amino acid sequence, light chain CDRL1 amino acid sequence, light chain CDRL2 amino acid sequence and light according to SEQ ID NOs: 67-71 and 73, or SEQ ID NOs: 67-70 and 72-73, respectively. Chain CDRL3 amino acid sequence or multiple functional sequence variants thereof; and / or (iv) heavy chain CDRH1 amino acid sequence, heavy chain CDRH2 according to SEQ ID NOs: 105-109 and 111, or SEQ ID NOs: 105-108 and 110-111. Claims 1-14, comprising an amino acid sequence, a heavy chain CDRH3 amino acid sequence, a light chain CDRL1 amino acid sequence, a light chain CDRL2 amino acid sequence and a light chain CDRL3 amino acid sequence or a plurality of functional sequence variants thereof. The antibody or antigen-binding fragment thereof according to any one of. The above antibody or its antigen-binding fragment
(I) A VH amino acid sequence according to SEQ ID NO: 37 or a plurality of functional sequence variants thereof, and a VL amino acid sequence according to SEQ ID NO: 38 or a plurality of functional sequence variants thereof;
(Ii) A VH amino acid sequence or a plurality of functional sequence variants thereof according to SEQ ID NO: 63, and a VL amino acid sequence or a plurality of functional sequence variants thereof according to SEQ ID NO: 64;
(Iii) VH amino acid sequence according to SEQ ID NO: 95 or a plurality of functional sequence variants thereof, and VL amino acid sequence according to SEQ ID NO: 96 or a plurality of functional sequence variants thereof; and / or (iv) to SEQ ID NO: 130. Any of claims 1-15, comprising a VH amino acid sequence according to it or a plurality of functional sequence variants thereof, and a VL amino acid sequence according to SEQ ID NO: 131 or a plurality of functional sequence variants thereof. The antibody or antigen-binding fragment thereof according to. The heavy chain of the antibody or antigen-binding fragment thereof is selected from a plurality of amino acid sequences according to SEQ ID NOs: 4, 52, 70, 108 or a plurality of functional sequence variants thereof, CDRL1 amino acid sequence, SEQ ID NOs: 5, 6 , 53, 54, 71, 72, 109, 110, or multiple CDRL2 amino acid sequences selected from multiple functional sequence variants thereof, and / or multiple according to SEQ ID NOs: 7, 55, 73, 111. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which comprises a CDRL3 amino acid sequence selected from the amino acid sequence of the above or a plurality of functional sequence variants thereof. The heavy chain of the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence selected from a plurality of amino acid sequences according to SEQ ID NOs: 38, 64, 96, 131 or a plurality of functional sequence variants thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence according to SEQ ID NO: 38 or 96 or a plurality of functional sequence variants thereof, and more preferably, the antibody or antigen-binding fragment thereof contains SEQ ID NO: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, characterized in that it comprises a VL amino acid sequence according to 96 or a plurality of functional sequence variants thereof. (I) The antibody or antigen-binding fragment thereof is a construct type selected from the group consisting of construct types Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3; and (ii) SEQ ID NOs: 1 to 7 and 67 to Position the CDRH1 amino acid sequence, the CDRH2 amino acid sequence, the CDRH3 amino acid sequence, the CDRL1 amino acid sequence, the CDRL2 amino acid sequence and the CDRL3 amino acid sequence, or a plurality of functional sequence variants thereof selected from the group consisting of a plurality of amino acid sequences according to 73. Included in either A and / or B; preferably CDRH1 amino acid sequence, CDRH2 amino acid sequence, CDRH3 amino acid sequence, CDRL1 amino acid according to SEQ ID NOs: 67-71 and 73, or SEQ ID NOs: 67-70 and 72-73. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, characterized in that the sequence, the CDRL2 amino acid sequence and the CDRL3 amino acid sequence, or a plurality of functional sequence variants thereof are contained at position A. .. The antibody or antigen binding fragment thereof, gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1b, gBs2GC1c, gBs2GC1d, gBs1GC2a, gBs3GC2b, gBs1GC3a, gBs3GC3b, gBs3GC4, and that according to GBs3GC5, preferably the antibody or The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antigen-binding fragment conforms to gTs3GC2d or gBs1GC3a. The antibody or antigen binding fragment thereof, Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, it is Bs3GC3b, Bs3GC4 and Bs3GC5, this 2 Ts3GC2d or Bs1GC3a The antibody or antigen-binding fragment thereof according to claim 20, characterized in that. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a human monoclonal antibody, a purified antibody or a single-chain antibody. .. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, for use as a medicine. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, for use in the prevention, treatment or weakening of inflammatory and / or autoimmune diseases. A nucleic acid molecule comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24. The polynucleotide sequences are SEQ ID NOs: 8-36, 39-48, 56-62, 65-66, 74-94, 97-104, 112-129, 132-139, 152, 154, 156, 158, 160, It contains a nucleic acid sequence according to any one of 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190 or one of its functional sequence variants. The nucleic acid molecule according to claim 25, which comprises the nucleic acid sequence. The polynucleotide sequence is 1,65-66, 97-104, 132-139, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 26. The nucleic acid molecule of claim 26, comprising or consisting of a nucleic acid sequence according to any one of 178, 180, 182, 184, 186, 188, 190 or a functional sequence variant thereof. A vector comprising the nucleic acid molecule of any of claims 25-27. A cell that expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 24; or comprises the vector according to claim 28. An isolated immunogenic polypeptide or purified immunogen comprising at least two epitopes that specifically bind to the antibody or antigen-binding fragment thereof according to any one of claims 1-24. Primary polypeptide. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, the nucleic acid according to any one of claims 25 to 27, the vector according to claim 28, the cell according to claim 29, or the cell according to claim 29. A pharmaceutical composition comprising the immunogenic polypeptide according to 30, as well as a pharmaceutically acceptable excipient, diluent or carrier. (I) Prevention, treatment or weakening of inflammatory and / or autoimmune diseases; or (ii) for use in the diagnosis of inflammatory and / or autoimmune diseases, according to any of claims 1-24. Antibodies or antigen-binding fragments thereof, nucleic acids according to claims 25 to 27, vectors according to claim 28, cells according to claim 29, or immunogenic polypeptides according to claim 30, or claims. 31. The pharmaceutical composition.