EA040943B1 - ANTIBODIES TO CD154 AND METHODS FOR THEIR APPLICATION - Google Patents

Description

Cross references to related applications

The application claims benefit from U.S. Provisional Application Ser. No. 62/367,660, filed July 28, 2016, and U.S. Provisional Application Ser. No. 62/201,150, filed August 5, 2015, the entire contents of which are hereby incorporated by reference in their entirety.

Scope of the invention

The present invention relates to antibodies that specifically bind CD154, polynucleotides encoding these antibodies or fragments, as well as methods for obtaining and using these products.

Prerequisites for the creation of the invention

CD154, also referred to as CD40 ligand (CD40L), dr39, TNF-related activating protein (TRAP), antigen 5c8 or T-BAM, is a trimeric transmembrane protein from the tumor necrosis factors (TNF) superfamily. CD154 is expressed in an activation-dependent and time-limited manner on the surface of CD4 ⁺ T cells. CD154 is also expressed after activation in a subset of CD8+ T cells, basophils, mast cells, eosinophils, natural killer cells, B cells, macrophages, dendritic cells, and platelets. CD154 is also present in the blood in a soluble form.

CD154 binds to CD40 on antigen presenting cells (APCs), leading to different responses, depending on the type of target cell. The CD40-CD154 interaction is essential for normal T- and B-cell interactions, including increased costimulation, T-cell priming, cytokine production, antibody class switching, affinity maturation, and antibody and autoantibody production.

Disruption of the CD40/CD154 pathway via CD154 blockade has been shown to be beneficial in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD), type I diabetes (T1D) and allograft rejection. In humans, mutations in either CD40 or CD154 result in a hyper-IgM syndrome characterized by a deficiency of IgG or IgA isotypes (Aruffo et al., Cell 72:291, 1993).

Антитела к CD154 были описаны, например, в международных патентных публикациях № WO1993/08207, WO1994/10308, WO1996/40918, WO1993/009812, WO1999/051258, WO1995/006480, WO1995/006481, WO1995/006666, WO2001/002057, W01997 /017446, WO1999/012566, WO2001/068860, WO2005/003175, WO2006/033702, WO2006/030220, WO2008/118356, WO2012/052205, Wo2012/138768, Wo2013/055 and Wo20168.

It has been shown that antibodies to CD154 are effective in the treatment of autoimmune diseases in humans. However, thromboembolism observed during treatment, due to platelet activation, hinders long-term clinical studies. It has been shown that the cause of platelet activation by anti-CD154 antibody 5c8 is interaction with FcyRIIa on platelets (Xie et al., J Immunol 192:4083-4092, 2014).

Thus, there is a need for additional anti-CD154 antibodies with improved safety and efficacy profiles.

Statement of the Invention

The invention provides an antagonistic antibody or antigen-binding site thereof that specifically binds human CD154 with SEQ ID NO: 1, containing a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 17 (SYGIS), HCDR2 with SEQ ID NO: 23 (WISPIFGNTNYAQKFQG ) and HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), optionally HCDR1 residue S1 mutated to A, C, D, E, G, I, K, L, M, N, Q, R, T, or V;

residue I4 HCDR1 mutated to M, L, or V;

residue S5 HCDR1 mutated in A;

residue S3 HCDR2 mutated in A, T or V;

the P4 HCDR2 residue is mutated to V, T, L Q, or E;

the N8 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

the T9 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

the N10 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

residue S1 HCDR3 mutated to A or M;

the R2 residue of HCDR3 is mutated to A, S, Q, or K; and the L7 residue of HCDR3 is mutated in M.

The invention also provides an isolated antagonist antibody specifically binding to CD154 of SEQ ID NO: 1, wherein the antibody comprises the defined heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences described herein.

The invention also provides an isolated antagonist antibody specifically binding to CD154 of SEQ ID NO: 1, the antibody comprising the specific amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 described herein.

The invention also provides an isolated antagonist antibody specifically binding to CD154 of SEQ ID NO: 1, comprising

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 17, 23, 30, 37, 44 and 52, respectively;

VH with SEQ ID NO: 59 and VL with SEQ ID NO: 66; or a heavy chain of SEQ ID NO: 80 and a light chain of SEQ ID NO: 81.

The invention also provides an isolated antagonistic antibody or antigen-binding site thereof specifically binding CD154 with SEQ ID NO: 1, wherein CD154 is a homotrimer and the antibody binds the first CD154 monomer in the homotrimer within amino acid residues 182-207 of CD154 and the second CD154 monomer in the homotrimer within amino acid residues 176-253 of CD154, while the numbering of the residues corresponds to SEQ ID NO: 1.

The invention also provides an immunoconjugate containing an antibody or antigen-binding site of an antibody of the invention associated with a therapeutic agent or imaging agent.

The invention also provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.

The invention also provides a polynucleotide encoding the VH of an antibody of the invention, the VL of an antibody of the invention, or the VH and VL of an antibody of the invention.

The invention also provides a vector containing the polynucleotide of the invention.

The invention also provides a host cell containing the vector of the invention.

The invention also provides a method for producing an antibody, comprising culturing a host cell of the invention under antibody expression conditions and isolating the antibody produced by the host cell.

The invention also provides a method of treating an autoimmune disease or an immune-mediated inflammatory disease, comprising administering a therapeutically effective amount of an isolated antibody of the invention or a pharmaceutical composition of the invention to a patient in need of treatment for a time period sufficient to treat the disease.

The invention also provides an anti-idiotypic antibody that binds to an antibody of the invention.

The invention also provides a kit containing an antibody of the invention.

Brief description of the drawings

In FIG. 1 shows the effect of antibody Fc on platelet activation by immune complexes (IC) CD154:antibody. The IC of soluble human CD154 (shCD154, labeled SCD40L in the figure) and the 5c8 anti-CD154 (IgG1 isotype) antibody (IC 5c8IgG1) activated platelets, while the IC of shCD154 and 5c8 cloned on muted IgG1 scaffolds IgG1sigma, IgG1sigma-YTE, IgG2sigma or IgG2sigmaYTE (5c8IgG1z, 5c8IgG1z-YTE, 5c8IgG2z or 5c8IgG2z-YTE, respectively) had no effect. Platelet activation was assessed as the percentage of platelets expressing PAC-1 (PAC-1 antibody specifically recognizes the conformationally active form of αΠΒβ3 integrin) and CD62p (expression of P-selectin on the surface) of their total number. ADP (adenosine triphosphate): positive control. PBS (phosphate buffered saline): negative control. Platelet activation was assessed in five donors. Results are presented as the mean of each experiment ± CO.

In FIG. 2 shows that immune complexes (IC) of shCD154 (sCD40L in the figure) and anti-CD154 IgG2sigma/K antibodies C4LB5, C4LB89, C4LB189, C4LB191, C4LB199 and C4LB150, which have a muted effector function, do not activate platelets. Platelet activation was assessed as % of PAC-1 and CD62p expressing platelets of their total number. IC shCD154 and 5c8IgG1 (5c8IgG1 IC) activated platelets. ADP: positive control. Platelet activation was assessed in five donors. Results are presented as the mean of each experiment ± CO.

In FIG. 3 shows that the immune complexes (IC) of shCD154 (SCD40L) and anti-CD154 antibodies C4LB89 (IgG2sigma/K), C4LB231 (IgG1sigma/K), and C4LB232 (IgG1sigma/K) do not activate platelets. Platelet activation was assessed as % of PAC-1 and CD62p expressing platelets of their total number. 5c8-IgG1 IC-activated platelets whose activation was blocked by anti-FcyIIa antibody indicate that CD154/5c8-IgG1 IC platelet activation is mediated by FcyRIIa on platelets. ADP: positive control. PBS: negative control. Platelet activation was assessed in five donors. Results are presented as the mean of each experiment ± CO.

In FIG. 4 shows the interaction surface between CD154 and C4LB89. Aromatic residues F55, Y101 and Y102 in HCDR2 and HCDR3 are involved in most interactions (residue numbering corresponds to SEQ ID NO: 59). The light chain C4LB89 is not involved in binding.

In FIG. 5 is a 2D image of epitopic and paratopic residues identified by the crystal structure of marmoset CD154 complexed with C4LB89. Epitopic residues are circled in an ellipse and paratopic residues in HCDR1, HCDR2 and HCDR3 of the heavy chain are shown (indicated in the figure as CDR1, CDR2 and CDR3). The antibody binds simultaneously to two CD154 monomers, A and B. The numbering of epitope residues corresponds to human CD154 (SEQ ID NO: 1), and

- 2 040943 numbering of paratopic residues corresponds to the variable region of the heavy chain C4LB89 (SEQ ID

NO: 59).

In FIG. 6A shows an alignment of residues 1-180 of human CD154 (SEQ ID NO: 1, top row) and marmoset CD154 (SQ ID NO: 2, bottom row), demonstrating that the C4LB89 epitope residues of human and marmoset CD1514 are conserved. Epitopic residues on CD154 monomer 1 are underlined, and epitopic residues on monomer 2 are underlined with a double line.

In FIG. 6B shows an alignment of residues 181-261 of human CD154 (SEQ ID NO: 1, top row) and marmoset CD154 (SQ ID NO: 2, bottom row), demonstrating that the C4LB89 epitope residues for human and marmoset CD1514 are conserved. Epitopic residues on CD154 monomer 1 are underlined, and epitopic residues on monomer 2 are underlined with a double line.

In FIG. 7 shows that the immune complexes (IC) of shCD154 (SCD40L in the figure) and the anti-CD154 IgG1/κ antibody C4LB237 do not activate platelets, while the shCD154 IC and another IgG1 antibody 5c8 activate platelets. The antibodies themselves had no effect on platelet activation, measured as the percentage of PAC-1 and CD62p-expressing platelets of the total number. ADP: positive control. PBS: negative control. Platelet activation was assessed in five donors. Results are presented as the mean of each experiment ± CO. n=4 in each group.

In FIG. 8A shows that shCD154 (sCD40L in figure) and Fc-mutated C4LB119 immune complexes (ICs) activate platelets in an FcyRIIa-independent manner, while shCD154 ICs and antibodies with VH/VL domains C4LB119 expressed on IgG1 (C4LB287) activated platelets FcγRIIa dependent way. ADP: positive control. PBS: negative control.

In FIG. 8B shows that shCD154 (sCD40L in figure) and Fc-mutated C4LB94 immune complexes (ICs) activate platelets in an FcyRIIa-independent manner, while shCD154 ICs and antibodies with VH/VL domains C4LB94 expressed on IgG1 (C4LB289) activate FcyRIIa platelets. dependent way. ADP: positive control. PBS: negative control.

In FIG. 8C shows that the immune complexes (ICs) of shCD154 (sCD40L in figure) and Fc-muted C4LB83 moderately activate platelets, while shCD154 ICs and C4LB83 VH/VL domain antibodies expressed on IgG1 (C4LB288) activated platelets in an FcyRIIa dependent manner. . ADP: positive control. PBS: negative control.

Detailed description of the invention

All publications included in this specification, including, without limitation, patents and patent applications incorporated by reference, are part of this document as if they were set forth directly in this document.

It should be understood that the terms used in this document are used only for the purpose of describing specific embodiments and are not limiting. All technical and scientific terms used in this document, unless otherwise indicated, have the generally accepted meaning understood by a person of ordinary skill in the field to which the invention pertains.

As used in the present description and in the claims, the singular forms include references to plural objects, unless otherwise follows clearly from the context.

Examples of methods and materials are described herein, although any methods and materials similar or equivalent to those described herein may be used to test the present invention in practice. In describing and presenting the claims of the present invention, the following terms will be used.

The terms specific binding or specifically binds or binds refer to the binding of an antibody to an antigen or epitope within an antigen with greater affinity than to other antigens. Typically, an antibody binds to an antigen or epitope within the antigen with a dissociation constant (K _D ) of about 1x10'8 M or less, such as about 1x10'9 M or less, about ^1x10'10 M or less, about 1x10'11 M or less or about 1x10' ¹² M or less, typically with a K _D that is at least one hundred times less than the K _D value of binding to a non-specific antigen (eg bovine serum albumin (BSA), casein). The dissociation constant can be measured by standard procedures. However, antibodies that specifically bind to an antigen or epitope within an antigen may be cross-reactive with other related antigens, for example with the same antigen of other species (homologues) such as humans or monkeys, such as Masaca fascicularis (cynomolgus macaque, macaques), Pan troglodytes (chimpanzee) or Callithrix jacchus (common marmoset, marmoset). Whereas a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two different antigens or two different epitopes.

The terms neutralizing or neutralizing or neutralizing antibody or antibody antagonist or antagonist or antagonist refer to an antibody or antigen-binding site thereof that partially or completely inhibits the biological activity of human CD154. Antagonist antibodies can be detected using the CD154 biological activity assays described herein. An antagonist antibody that specifically binds human CD154 can inhibit the biological activity of human CD154 by approximately 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or

100%.

CD154 means human CD154 protein (hCD154) (eg human CD40L). The amino acid sequence of the full-length human CD154 protein is shown in SEQ ID NO: 1. Human CD154 is found on the cell membrane as a type II membrane protein and is also present in soluble form in plasma. The membrane bound form of CD154 contains residues 1261 of SEQ ID NO: 1, with the transmembrane domain located between residues 23-46 and the extracellular domain spanning residues 47-261. The soluble form of human CD154 (shCD154) is formed by proteolytic processing of the membrane bound form and contains residues 113-261 of SEQ ID NO: 1) (the amino acid sequence of shCD154 is shown in SEQ ID NO: 4). Both membrane-bound and soluble CD154 form biologically active trimers. The designation CD154 encompasses various forms of CD154, including monomeric, dimeric, trimeric, membrane-bound, and soluble forms, as well as naturally occurring variants of human CD154. The soluble human CD154 trimer (shCD154 trimer) consists of three polypeptide chains, each having the amino acid sequence of SEQ ID NO: 4.

In the context of this document, the term antibodies is intended in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies; and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site of the desired specificity. Whole antibody molecules consist of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, as well as their multimers (eg IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (consisting of the CH1, CH2 and CH3 domains). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can also be subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs), between which are frame regions (FRs). Each VH and VL consists of three CDR segments and four FR segments, arranged in the direction from the amine end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Complementarity determining regions (CDRs) represent antigen-binding sites in an antibody. CDRs can be defined using different terms: (i) complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) hypervariable regions, HVR or HV, three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refer to antibody variable domain regions that are structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol 196:901-17, 1987). The international database ImMunoGeneTics (IMGT) (http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDR, HV, and IMGT delimitations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. The designations CDR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include CDRs defined by any of the methods described above, according to Kabat, Chothia or IMGT, unless otherwise indicated in this description.

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes, IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further classified into IgA1, IgA ₂ , IgG1, IgG ₂ , IgG ₃ and IgG4 isotypes. The light chains of antibodies of any vertebrate species can be assigned, depending on the amino acid sequences of their constant domains, to one of two distinct types, namely kappa (k) and lambda (λ).

The term antibody fragments, or antigen-binding portion of an antibody, refers to the portion of an immunoglobulin molecule that retains an antigen-binding region of the heavy chain and/or light chain, such as heavy chain complementarity determining regions (HCDRs) 1, 2 and 3, light chain complementarity determining regions (LCDRs) 1, 2 and 3, heavy chain variable region (VH) or light chain variable region (VL). Antibody fragments include the well-known Fab, F(ab') ₂ , Fd and Fv fragments, as well as domain antibodies (dAbs) containing a single VH domain. The VH and VL domains can be linked together via a synthetic linker to form various types of single chain antibody configurations, in which the VH/VL domains are paired intramolecularly or intermolecularly in cases where the VH and VL domains are expressed by separate single chain antibody constructs to form a monovalent antigen binding site such as single chain Fv (scFv) or diabody; they are described, for example, in an international patent publication

- 4 040943 No. WO1998/44001, International Patent Publication No. WO1988/01649; International Patent Publication No. WO1994/13804; International Patent Publication No. WO1992/01047.

A monoclonal antibody refers to a population of antibodies with the same amino acid composition in each heavy and each light chain, except for possible well-known changes, such as the removal of the C-terminal lysine from the heavy chain of the antibody. Monoclonal antibodies generally bind to one antigenic epitope, with the exception of bispecific monoclonal antibodies, which bind to two different antigenic epitopes. Within an antibody population, monoclonal antibodies may have heterogeneous glycosylation. The monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.

The term isolated antibody means an antibody or antibody fragment that is substantially free of other antibodies having distinct antigen specificity (e.g., an isolated antibody that specifically binds CD154 is substantially free of antibodies that specifically bind antigens other than CD154. The term isolated antibody includes antibodies isolated to be of higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% pure, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The term Chothia residues means VL and VH residues of antibodies numbered according to Al-Lazikani (Al-Lazikani et al., J Mol Biol 273:927-48, 1997).

The term humanized antibodies refers to antibodies in which the antigen-binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions, whereby the framework region may not be an exact copy of the expressed human immunoglobulin or germline gene sequences.

The term human antibodies refers to antibodies having heavy and light chain variable regions in which both the framework regions and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region or part of a contrast region, the constant region is also derived from sequences of human origin.

A human antibody contains heavy or light chain variable regions that are derived from sequences of human origin if the antibody variable regions are derived from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems are phage-displayed human immunoglobulin gene libraries and non-human transgenic animals such as mice or rats harboring human immunoglobulin loci as described herein. The human antibody may contain amino acid differences from the human germline, or rearranged immunoglobulin sequences due, for example, to naturally occurring somatic mutations or intentional substitutions at the framework or antigen binding site, or both. Typically, human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by the human germline gene or rearranged immunoglobulin gene. In some cases, a human antibody may contain consensus framework sequences derived from human framework sequence assays, such as those described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3s included in human immunoglobulin gene libraries displayed on phage, for example, as described in Shi et al., J Mol Biol 397:385-96, 2010 and International Patent Publication No. WO2009/085462.

The isolated humanized antibodies are synthetic. Human antibodies, although derived from human immunoglobulin sequences, may be generated using systems such as phage display, including synthetic CDRs and/or synthetic scaffolds, or may be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies not expressed by the human germline in vivo.

Antibodies in which the antigen-binding sites are derived from a species other than a human do not fit the definition of a human antibody.

The term recombinant refers to antibodies and other proteins that are produced, expressed, created or isolated by recombinant methods.

As used herein, the term epitope refers to the portion of an antigen to which an antibody specifically binds. Typically, epitopes are composed of reactive (such as polar, nonpolar, or hydrophobic) surface groupings of components such as amino acid or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be formed from contiguous and/or non-contiguous amino acids forming a conformational spatial unit. In the case of a non-contiguous epitope, amino acids from different regions of the linear sequence of the antigen come close to each other

- 5 040943 in 3-dimensional space due to the folding of the protein molecule.

The term paratope refers to the part of an antibody to which an antigen specifically binds. The paratope may be linear or discrete, formed by spatial relationships between antibody amino acids that are not in a continuous sequence, as opposed to the interaction of linear amino acid sequences. The terms light chain paratope and heavy chain paratope, or amino acid residues of the light chain paratope and amino acid residues of the heavy chain paratope, refer to the residues of the light and heavy chains of an antibody in contact with the antigen, respectively, or in general, the residues of the paratope of an antibody refer to those amino acids of the antibody that come into contact with the antigen. .

The term bispecific refers to an antibody that specifically binds two different antigens or two different epitopes within the same antigen. A bispecific antibody may be cross-reactive with other related antigens or may bind an epitope that is common to two or more different antigens.

The term multispecific refers to an antibody that specifically binds at least two different antigens or at least two different epitopes within the same antigen. A multispecific antibody can bind, for example, to two, three, four or five different antigens or different epitopes within a single antigen.

The expression in combination with means that drugs or therapeutic agents are administered to a subject, such as a human, together in admixture, simultaneously as separate agents or sequentially as separate agents in any order.

The term biological activity of CD154 refers to any activity resulting from the binding of CD154 to its CD40 receptor. The biological activity of CD154 may be, for example, CD154-mediated activation of CD40+ B cells or dendritic cells (DC), or activation of signaling pathways downstream of CD40. The biological activity of CD154 can be measured using well known methods or the methods described herein, for example, by measuring CD154 mediated proliferation or B cell activation by assessing ICAM-1 upregulation or increased B cell cytokine production, CD154 mediated DC activation by assessing increased expression of CD80 and/or CD86 on the surface or secretion of cytokines by DC cells, or activation of the CD40 signaling pathway by assessing reporter gene assays, e.g. measuring secretion of secreted embryonic alkaline phosphatase (SEAP) by cells expressing SEAP under the control of an inducible NF-kB promoter.

The term vector refers to a polynucleotide capable of duplication within a biological system or which can be moved between such systems. Vector polynucleotides typically contain elements such as origins of replication, a polyadenylation signal, or selection markers that function to facilitate duplication or maintenance of such polynucleotides in a biological system. Examples of such biological systems may include cell, virus, animal, plant, and reengineered biological systems using biological components capable of vector doubling. The vector-containing polynucleotide may be a DNA or RNA molecule or a hybrid thereof.

The term expression vector means a vector that can be used in a biological system or a reshaped biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in an expression vector.

The term polynucleotide means a molecule containing a chain of nucleotides covalently linked through a sugar phosphate backbone or other equivalent covalent chemical structure. Double-stranded and single-stranded DNA and RNA are typical examples of polynucleotides.

The term polypeptide or protein refers to a molecule that contains at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides containing less than 50 amino acid residues may be referred to as peptides.

In this document, conventional one- and three-letter codes for the designation of amino acids are used, as shown in table. 1.

- 6 040943

Table 1

Amino acid Three letter code Single letter code Alanine A1a A Arginine Agde R Asparagine As η N aspartate asp D Cysteine Cys C Glutamate gin E Glutamine Glu Q Glycine gly G Histidine His H Isoleucine lie I Lysine Lys TO Methionine Met M Phenylalanine Phe F Proline Pro P Serene Ser S Threonine Thr T tryptophan trp W Tyrosine Tyr Y Valine Vai V

Compositions of the present invention

The present invention provides an antagonist antibody that specifically binds CD154 with high affinity and effectively neutralizes CD154 biological activity. with the FcYRIIa receptor on platelets leads to platelet activation and aggregation and subsequent thromboembolism, it has been found that platelet activation also depends on the epitope on CD154 to which the antibody binds. In the present invention, it has been found that antibodies of the invention that bind a specific epitope on CD154 and are capable of recruiting FcyRIIa do not mediate platelet activation. In addition, the antibodies of the invention optionally undergo an Fc change to prevent triggering additional undesirable immunostimulatory functions. Therefore, the antibodies of the invention may have more favorable safety profiles in clinical use compared to existing antibodies that specifically bind CD154.

CD154 is a target in autoimmune reactions, transplant rejection reactions and other immune-related diseases in mice, non-human apes (NHP) and humans. In a number of phase II clinical trials, antibodies specifically binding to CD154 have been shown to effectively block CD154 activity in vivo and alleviate the course of the disease. CD154 antagonists differ from all other therapeutic agents in their effect on the immune response; they are the only therapeutic agents capable of inducing functional immunological tolerance, which has been demonstrated in both mice and monkeys. In mice, virtually all autoimmune diseases can be effectively ameliorated by CD154 antagonists (Noelle et al., Ann N Y Acad Sci 815: 384-391, 1997; Mackey et al., J Leukoc Biol 63: 418-428, 1998; Noelle, Agents Actions Suppl 49: 17-22, 1998; Quezada et al., Annu Rev Immunol 22: 307-328, 2004), with long-term remission observed.

The invention provides an isolated antagonistic antibody, or antigen-binding site thereof, that specifically binds CD154 with SEQ ID NO: 1.

The invention also provides an isolated antagonistic antibody or antigen-binding site thereof specifically binding CD154, wherein CD154 is a homotrimer and the antibody binds the first CD154 monomer in the homotrimer within amino acid residues 182-207 of CD154 and the second CD154 monomer in the homotrimer within amino acid residues 176- 253 CD154, with residue numbering corresponding to SEQ ID NO: 1.

An example of such an antibody is the C4LB89 antibody. Since the variable regions of antibodies C4LB235 and C4LB236 differ from those of C4LB89 by one amino acid residue in LCDR2 when compared, and since C4LB231 and C4LB232 have identical VH/VL sequences to C4LB89, these antibodies are also expected to bind the same CD154 epitope as C4LB89. Antibodies that bind CD154 within residues 182-207 and 176-253 are unable to activate platelets despite being able to interact with FcyR receptors, including FcyRIIa. Therefore, these antibodies may have an improved safety profile compared to other antagonistic antibodies that specifically bind CD154.

- 7 040943

The invention also provides an antagonist antibody or antigen-binding site thereof specifically binding human CD154 of SEQ ID NO: 1, containing a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 17 (SYGIS), HCDR2 of SEQ ID NO: 23 ( WISPIFGNTNYAQKFQG) and HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), optionally HCDR1 residue S1 mutated to A, C, D, E, G, I, K, L, M, N, Q, R, T, or V;

residue I4 HCDR1 mutated to M, L, or V;

residue S5 HCDR1 mutated in A;

residue S3 HCDR2 mutated in A, T or V;

the P4 HCDR2 residue is mutated to V, T, L Q, or E;

the N8 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

the T9 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

the N10 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

residue S1 HCDR3 mutated to A or M;

the R2 residue of HCDR3 is mutated to A, S, Q, or K; and the L7 residue of HCDR3 is mutated in M.

In some embodiments, an antibody of the present invention comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 37 (RASQSISSYLN), LCDR2 of SEQ ID NO: 44 (YANSLQS), and LCDR3 of SEQ ID NO: 52 (QQSDSIPWT), wherein optionally, the Q4 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, V, W, or Y;

the S5 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

the S7 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

residue S8 LCDR1 is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

residue A2 LCDR2 mutated to S;

the N3 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

the S4 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

the L5 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y;

residue Q6 LCDR2 is mutated to E, D, or N;

the S7 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

residue S3 LCDR3 mutated in A;

residue D4 LCDR3 mutated to N;

the S5 LCDR3 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y; and LCDR3 residue I6 is mutated to A, C, D, E, G, K, L, M, N, Q, R, S, T, or V.

The crystal structure of the complex of antibody C4LB89 and CD154 showed that the antibody binds to CD154 only at VH residues. Additional analyzes showed that some of the substitutions shown in table. 19 and tab. 20 above in the CDR of an antibody should not affect the overall structure of the complex and hence the characteristics of the antibody.

In some embodiments, an antibody of the present invention comprises light chain complementarity determining regions (LCDRs) 1, LCDR2, and LCDR3 with

SEQ ID NOs: 36, 43 and 51, respectively;

SEQ ID NOs: 37, 44 and 52, respectively;

SEQ ID NOs: 38, 45 and 53, respectively;

SEQ ID NOs: 39, 46 and 54, respectively;

SEQ ID NOs: 40, 47 and 55, respectively;

SEQ ID NOs: 41, 47 and 56, respectively;

SEQ ID NOs: 42, 48 and 57, respectively;

SEQ ID NOs: 37, 49 and 52, respectively; or

SEQ ID NOs: 37, 50 and 52, respectively.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 37, 44, and 52, respectively.

In some embodiments, an antibody of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 17, 23, 30, 37, 44, and 52, respectively.

In some embodiments, an antibody of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 17, 23, 30, 37, 49, and 52, respectively.

In some embodiments, an antibody of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 17, 23, 30, 37, 50, and 52, respectively.

In some embodiments, the implementation of the immune complex of the antibody of the invention and soluble human CD154 (shCD154) does not activate platelets, and platelet activation is measured by the expression of P-selectin on the surface of platelets.

Platelet activation is a well-known process that converts a smooth, unattached platelet into a sticky, spiked particle that releases and expresses bioactive substances and acquires the ability to bind the plasma protein fibrinogen. Activation can also occur as a result of physical stimulation by high fluid shear, such as that occurring at the site of critical arterial narrowing (Quinn et al., 2005, Platelet Function: assessment, diagnosis, and treatment, Humana Press, pp. 3 -20). Platelet activation leads to the activation of intracellular signaling pathways that cause an increase in the expression of P-selectin on the surface of platelets and an increase in the affinity of fibrinogen binding to αΠbβ3 integrin receptors. Therefore, platelet activation can be measured by measuring the increase in P-selectin expression on the surface, or by binding a probing ligand, eg PAC-1, to αΠΕβ3 integrin on platelets, eg by flow cytometry. Antibodies of the invention do not activate human platelets unless the antibody in combination with shCD154 increases surface P-selectin expression or increases the binding of a probing ligand (e.g., PAC-1) to αΠΕβ3 integrin in a statistically significant manner compared to shCD154-induced P-selectin expression on the surface. surface or by binding a probing ligand (eg, PAC-1) to the α^β3 integrin.

In some embodiments, an antibody of the invention has at least one of the following properties:

binds to CD154 with a dissociation constant (K _D ) of about 5 x 10 ^-9 M or less when KD is measured using the ProteOn XPR36 system at 25° C. in Dulbecco's PBS containing 0.03% polysorbate P20 and 100 µg/mL bovine serum albumin;

inhibits CD154-mediated proliferation of human B cells with an IC ₅₀ value of about 2.7 x 10 ^-9 M or less; or inhibits CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2.1 x 10 ^-8 M or less.

In some embodiments, an antibody of the invention binds CD154 with a dissociation constant (KD) of about 5 x 10 ^-9 M or less, about 1 x 10 ^-9 M or less, about 5 x 10 ^-10 M or less, about 1 x 10 ^-10 M or less, about 5 x 10'11 M or less, or about 1x10'11 M or less.

In some embodiments, an antibody specifically binding to CD154 cross-reacts with CD154 Macaca fascicularis (cynomolgus monkey) or CD154 Callithrix jacchus (marmoset).

The affinity of an antibody for human, cynomolgus or marmoset CD154 can be determined experimentally using any suitable method. Such methods may include the use of ProteOn XPR36, Biacore 3000 or KinExA equipment, enzyme-linked immunosorbent assay (ELISA), or competitive binding assays known to those skilled in the art. The measured affinity value for the interaction of a particular antibody with CD154 may vary when measured under different conditions (eg, osmolarity, pH). Thus, measurements of affinity and other binding parameters (eg, KD, K _on , K _off ) are typically performed under standardized conditions and using a standardized buffer solution, such as the buffer solution described herein. Those skilled in the art will appreciate that the intrinsic error of affinity measurements, such as those using Biacore 3000 or ProteOn (measured as standard deviation, CO), can typically be in the range of 5-33% for measurements, falling within typical detection limits. . Therefore, the term about characterizes the typical standard deviation in the analysis. For example, a typical CO for a KD value of 1 x 10 ^-9 M is up to ±0.33 x 10' ⁹ M.

In some embodiments, an antibody of the invention inhibits CD154-mediated human B cell proliferation with an IC ₅₀ value of about 2.7 x 10 ^-9 M or less.

In a B cell proliferation assay, 1 x 10 ⁵ B cells from human tonsils can be cultured with 100 ng/ml recombinant human IL-21, 0.5 μg/ml trimeric recombinant soluble human CD154 expressed as a leucine zipper fusion protein, and antibodies to CD154 in the range of 0.000064-25 µg/ml in a final volume of 200 µl/well. After 2 days of incubation, methyl (-3H)-thymidine (0.5 μCi/well) can be added to cultures and the effect of antibodies on human B cell proliferation can be determined after overnight incubation.

In some embodiments, an antibody of the invention inhibits CD154-induced expression of secreted embryonic alkaline phosphatase (SEAP) by an NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2 , 1x10 ^-8 M or less.

In some embodiments, an antibody of the invention inhibits CD154-induced expression of secreted embryonic alkaline phosphatase (SEAP) by an NF-kB inducible minimal interferon-β promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2.1 x 10 ^-8 M up to 5.4x10 ^-10 M.

For example, HEK-Blue™ CD40L cells (InvivoGen, San Diego, CA, USA) can be used. Human CD154 can be provided as a trimeric soluble CD154-leucine zipper fusion protein. Using well known methods, the secreted alkaline phosphatase signal can be detected and the IC ₅₀ value for inhibition calculated.

In some embodiments, in a CD154 homotrimer, an antibody of the invention binds both a first CD154 monomer and a second CD154 monomer.

In some embodiments, an antibody of the invention binds at least one, two, three, four, five, six, seven, or eight CD154 residues in the first CD154 monomer within amino acid residues 182-207 of CD154 of SEQ ID NO: 1.

In some embodiments, an antibody of the invention binds at least one, two, three, four, five, six, seven, or eight CD154 residues in the second CD154 monomer within amino acid residues 176-253 of CD154 of SEQ ID NO: 1.

In some embodiments, an antibody of the invention binds residues E182, S185, Q186, A187, P188, S214, A215, and R207 on the first CD154 monomer, with residue numbering according to SEQ ID NO: 1.

In some embodiments, an antibody of the invention binds residues T176, F177, C178, Q220, S248, H249, G250, and F253 on a second CD154 monomer, with residue numbering according to SEQ ID NO: 1.

Within means that the antibody only binds residues within the ranges of amino acids 182-207, 176-354, or 182-207 and 176-354.

An example of such an antibody is the C4LB89 antibody. Since the variable regions of antibodies C4LB235 and C4LB236 differ from those of C4LB89 by one amino acid residue in LCDR2 when compared, and since C4LB231 and C4LB232 have identical VH/VL sequences to C4LB89, these antibodies are also expected to bind the same CD154 epitope as C4LB89.

In some embodiments, an antibody of the invention binds human CD154 using paratopic residues found in the VH of the antibody.

A paratopic residue is a residue in the VH or VL of an antibody within 4 A of the CD154 residues. Paratopic residues can be identified by the crystal structures of the antibody-CD154 complex.

An example of an antibody that binds CD154 only with VH paratopic residues, without VL contact with antigen, is an antibody containing the VH and VL of the C4LB89 antibody. Since the variable regions of C4LB235 and C4LB236 differ from those of C4LB89 by one amino acid residue in LCDR2, and since C4LB231 and C4LB232 have identical VH/VL sequences to C4LB89, these antibodies are also expected to bind CD154 using only VH residues. Antibodies that bind CD154 within residues 182-207 or 176-354 of SEQ ID NO: 1 or residues E182, S185, Q186, A187, P188, S214, A215 and R207 in the first monomer of CD154 and residues T176, F177, C178, Q220 , S248, H249, G250, and F253 in the second CD154 monomer of the CD154 homotrimer are unable to activate platelets, although they are able to interact with FcyRs, including FcyRIIa. Therefore, these antibodies may have an improved safety profile compared to other antibodies that specifically bind CD154.

In some embodiments, an antibody of the present invention comprises a heavy chain variable region (VH) of SEQ ID NO: 59, optionally the VH comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a heavy chain variable region (VH) of SEQ ID NO: 59.

An antibody containing HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, or containing HCDR VH of SEQ ID NO: 59 or VH of SEQ ID NO: 59 binds CD154 with the VH antibody alone. Therefore, a VH of SEQ ID NO: 59 or a VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 17, 23, and 30, respectively, can be combined with a light chain variable region (VL) sequence, and binding of the resulting antibody to CD154 can be tested using the assays described herein to produce an antibody that specifically binds CD154.

For example, a VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 17, 23, and 30, respectively, or a VH of SEQ ID NO: 59 can be used to screen for VL domains capable of forming a dual-domain specific antigen-binding fragment capable of binding to CD154. Such screening can be performed using phage display screening methods, for example using the hierarchical dual combinatorial approach described in PCT Publication No. WO1992/01047. In this approach, an individual colony containing either an H chain clone or an L chain clone, such as VH with SEQ ID NO: 59, is used to infect the entire library of clones encoding the other chain (L or H), and the resulting double-stranded specific antigen-binding domain is selected in according to the phage display techniques described herein and tested for binding and antagonistic activity against CD154.

Alternatively, a VH of SEQ ID NO: 59 or a VH containing HCDR1, HCDR2 and HCDR3 of SEQ ID

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NO: 17, 23 and 30, respectively, can be combined with the VL domains of existing anti-CD154 antibodies or anti-CD154 antibodies described herein, and the resulting antibody is tested for binding and antagonistic activity against CD154.

In some embodiments, an antibody of the invention comprises a light chain variable region (VL) of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72, or 73, with optional VL containing one, two, three, four, five , six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 59 and a VL of SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, or 73.

In some embodiments, an antibody of the invention comprises a light chain variable region (VL) of SEQ ID NO: 66, 72, or 73, optionally the VL comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven , twelve, thirteen, fourteen or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 72.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 73.

In some embodiments, an antibody of the invention contains at least a substitution in the Fc region, and the antibody does not activate human platelets.

In some embodiments, an antibody of the invention has reduced binding to FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, or FcyRIIIb.

Reduced binding means reduced binding of the antibodies of the invention having at least one substitution in the Fc region to the FcyR receptor compared to the binding of the parent antibody lacking the substitution to the same FcyR receptor. The reduced binding may be at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10000-fold, or at least about 20000-fold. fold reduced binding. In practice, antibodies showing reduced binding to a particular FcyR are those having a statistically non-significant effector function mediated by a particular FcyR.

In some embodiments, an antibody of the invention contains at least one substitution in the Fc region.

In some embodiments, at least one substitution in the Fc region is an L234A, L235A, G237A, P238S, M252Y, S254T, T256E, H268A, A330S, or P331S substitution, with the residue numbering corresponding to the EU index.

In some embodiments, an antibody of the invention contains substitutions L234A, L235A, G237A, P238S, H268A, A330S, or P331S in the Fc region, with residue numbering corresponding to the EU index.

In some embodiments, at least one substitution in the Fc region is a V234A, G237A, P238S, M252Y, S254T, T256E, H268A, V309L, A330S, or P331S substitution, with the residue numbering corresponding to the EU index.

In some embodiments, an antibody of the invention contains substitutions V234A, G237A, P238S, H268A, V309L, A330S, and P331S in the Fc region, with residue numbering corresponding to the EU index.

The invention also provides an antagonistic antibody or antigen-binding site thereof specifically binding CD154 of SEQ ID NO: 1, comprising a heavy chain of SEQ ID NO: 80 and a light chain of SEQ ID NO: 81.

The invention also provides an antagonistic antibody or antigen-binding site thereof specifically binding CD154 of SEQ ID NO: 1, comprising a heavy chain of SEQ ID NO: 82 and a light chain of SEQ ID NO: 81.

The invention also provides an antagonistic antibody or antigen-binding site thereof specifically binding CD154 of SEQ ID NO: 1, comprising a heavy chain of SEQ ID NO: 83 and a light chain of SEQ ID NO: 81.

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SEQ ID NO: 80 (C4LB89 VH on IgGlsigma: C4LB231 HC)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTN YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRYYGDLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDT LMISRTPEVTCVWDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

SEQ ID NO: 81 (light chain C4LB89 and C4LB231)

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYANSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDSIPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKDSTYSLSSTLTLSKADYVTKSFACE KHQNSFACE

SEQ ID NO: 82 (C4LB89 VH on IgGlsigmaYTE)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTN YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRYYGDLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDT LYITREPEVTCVWDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

SEQ ID NO: 83 (C4LB89 VH on IgG2sigma)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTN

PKDTLMIS

RT PEVT CVWDVS AEDPEVQ FNWYVDGVEVHNAKTKPREE Q FNS T FRWS VL TVLHQDWLNGKE

YKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

TO

The immune effector properties of the antibodies of the invention can also be enhanced or attenuated by Fc modifications using techniques well known to those skilled in the art. For example, Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. , may be provided and/or controlled by modifiable residues in Fc responsible for these actions. For example, Fc substitutions V234A/G237A/P238S, V234A/G237A/H268Q, H268A/V309L/A330S/P331 or V2 34A/G237A/P238S/H268A/V309L/A330S/P331S (International Patent Publication No. WO11/066501), or L2634A /L235A/G237A/P238S/H268A/A330S/P331S can be incorporated into the antibodies of the invention.

The binding of antibodies of the invention to FcyRI, FcYRIIa, FcYRIIb, FcyRIIIa and FcYRIIIb can be assessed using recombinant soluble forms or cell-associated forms of the Fcy receptors. For example, direct or indirect measurements, such as competitive binding, can be used to evaluate the relative affinities and avidities of the antibodies of the invention for various FcyRs. In one assay example, the binding of a test antibody to soluble FcyR captured on a plate is assessed using competitive binding between 1 μg/ml of biotinylated human IgG1 and serial dilutions of the test antibody as a precomplex with antigen.

In some embodiments, an antibody of the invention has reduced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC).

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The terms antibody-dependent cellular cytotoxicity, antibody-dependent cell-mediated cytotoxicity, or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with effector cells with lytic activity, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors. (FcyR) expressed on effector cells. For example, NK cells express FcyRIIIa while monocytes express FcyRI, FcyRII and FcyRIIIa. To assess the ADCC activity of the antibodies of the invention, the antibody can be added to target cells in combination with effector cells of the immune system, which can be activated by antigen-antibody complexes, resulting in cytolysis of the target cell. Cytolysis is essentially detected by the release of labels (eg, radioactive substrates, fluorescent dyes, or natural intracellular proteins) from lysed cells. Examples of effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Examples of target cells are D1.1 Jurkat cells (ATCC® CRL-10915™) or T cells expressing CD154.

Antibody-dependent cellular phagocytosis (ADCP) refers to a mechanism for killing antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells. ADCP can be assessed using monocyte-derived macrophages as effector cells and CD154-expressing D1.1 Jurkat cells engineered to express GFP or another labeling molecule as target cells. The ratio of effector cells:target cells can be, for example, 4:1. Effector cells can be incubated with target cells for 4 hours with or without test anti-CD154 antibody. After incubation, cells can be separated using accutase. Identification of macrophages can be done with anti-CD11b and anti-CD14 antibodies associated with a fluorescent label, and the percentage of phagocytosis can be determined based on the % fluorescent GFP in CD11+CD14+ macrophages using standard methods.

Complement-dependent cytotoxicity, or CDC, refers to a cell death induction mechanism in which the Fc effector domain of a target-bound antibody binds and activates the C1q complement component, which in turn activates the complement cascade leading to the death of the target cell. Complement activation can also lead to the deposition of complement components on the surface of target cells, which facilitates ADCC by binding complement receptors (eg, CR3) on leukocytes. The CDC for cells expressing CD154 can be measured, for example, by seeding Jurkat cells in a suitable medium, adding anti-CD154 antibodies to the mixture, followed by the addition of pooled human serum. After the incubation period, the percentage (%) of lysed cells can be determined as % cells stained with propidium iodide in a fluorescence activated cell sorting (FACS) cytometry analysis using standard methods.

Decreased ADCC, reduced CDC, and reduced ADCP is antibody-induced ADCC, CDC, and/or ADCP that is not statistically significant in standard assays for measuring ADCC, CDC, and/or ADCP, such as in the assays described herein or in assays described in US Pat. No. 8,871,204.

Antibodies of the invention with the desired affinity and neutralization profile can be selected from libraries of variants or fragments using panning selection from human CD154 or marmoset CD154, and optionally using additional antibody affinity maturation. In an example of a panning operation, phage libraries can be panned with marmoset CD154. Alternatively, antibodies of the invention can be generated by immunizing mice with human CD154 and/or marmoset CD154 and screening the hybridomas for binding to human CD154, followed by evaluation of antibody antagonist properties using the methods described herein.

In some embodiments, an antibody of the invention competes for binding to CD154 with an antibody containing VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66.

Competition for specific binding to CD154 between antibodies of the invention containing certain VH and VL sequences can be analyzed in vitro using well known methods. For example, binding of NHS MSD Sulfo-Tag™ ester labeled antibodies to CD154 in the presence of unlabeled antibody can be analyzed by solid phase ELISA or Bioacore assays, or flow cytometry can be used to demonstrate competition with antibodies of the present invention. An antibody competes for binding to CD154 with a reference antibody (e.g., an antibody containing VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66) if the antibody inhibits binding of the reference antibody to CD154 by 80% or more, e.g., 85% or more, 90% or more, or 95% or more.

In some embodiments, the VH of SEQ ID NO: 59 can be combined with the VL of any of the anti-CD154 antibodies described in International Patent Publication Nos. WO1993/08207, WO1994/10308,

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WO1996/40918, WO1993/009812, WO1999/051258, WO1995/006480, WO1995/006481, WO1995/006666,

WO2001/002057, WO1997/017446, WO1999/012566, WO2001/068860, WO2005/003175, WO2006/033702,

WO2006/030220, WO2008/118356, WO2012/052205, WO2012/138768, WO2012/138768, WO2013/055745 and

WO2013/056068 to produce an anti-CD154 antagonist antibody. The binding and antagonistic activity of the resulting antibodies can be tested using the assays and protocols described herein.

The invention also provides an antagonist antibody that specifically binds CD154 with SEQ ID NO: 1, containing HCDR1, HCDR2 and HCDR3 with

SEQ ID NOs: 16, 22 and 29, respectively;

SEQ ID NOs: 17, 23 and 30, respectively;

SEQ ID NOs: 16, 24 and 31, respectively;

SEQ ID NOs: 18, 25 and 32, respectively;

SEQ ID NOs: 19, 26 and 33, respectively;

SEQ ID NOs: 20, 27 and 34, respectively; or

SEQ ID NOs: 21, 28 and 35, respectively.

The invention also provides an antagonist antibody that specifically binds CD154 with SEQ ID NO: 1, containing HCDR1, HCDR2 and HCDR3 with

SEQ ID NOs: 16, 22 and 29, respectively;

SEQ ID NOs: 17, 23 and 30, respectively;

SEQ ID NOs: 16, 24 and 31, respectively;

SEQ ID NOs: 18, 25 and 32, respectively;

SEQ ID NOs: 19, 26 and 33, respectively;

SEQ ID NOs: 20, 27 and 34, respectively; or

SEQ ID NOs: 21, 28, and 35, respectively, and LCDR1, LCDR2, and LCDR3 with

SEQ ID NOs: 36, 43 and 51, respectively;

SEQ ID NOs: 37, 44 and 52, respectively;

SEQ ID NOs: 38, 45 and 53, respectively;

SEQ ID NOs: 39, 46 and 54, respectively;

SEQ ID NOs: 40, 47 and 55, respectively;

SEQ ID NOs: 41, 47 and 56, respectively;

SEQ ID NOs: 42, 48 and 57, respectively;

SEQ ID NOs: 37, 49 and 52, respectively; or

SEQ ID NOs: 37, 50 and 52, respectively.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 16, 22, and 29, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 36, 43, and 51, respectively; or VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 44, and 52, respectively; or VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 16, 24, and 31, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 38, 45, and 53, respectively; or VH with SEQ ID NO: 60 and VL with SEQ ID NO: 67.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 18, 25, and 32, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 39, 46, and 54, respectively; or VH of SEQ ID NO: 61 and VL of SEQ ID NO: 68.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 19, 26, and 33, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 40, 47, and 55, respectively; or VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 20, 27, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 41, 47, and 56, respectively; or VH of SEQ ID NO: 63 and VL of SEQ ID NO: 70.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 21, 28, and 35, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 42, 48, and 57, respectively; or VH of SEQ ID NO: 64 and VL of SEQ ID NO: 71.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 49, and 52, respectively; or VH of SEQ ID NO: 59 and VL of SEQ ID NO: 72.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 50, and 52, respectively; or VH of SEQ ID NO: 59 and VL of SEQ ID NO: 73.

In some embodiments, an antibody of the invention comprises VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 58, 59, 60, 61, 62, 63, or 64.

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In some embodiments, an antibody of the invention comprises VH and VL, wherein VL comprises the amino acid sequence of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72, or 73.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NOs: 58, 59, 60,

61, 62, 63 or 64, and VL with SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72 or 73.

In some embodiments, an antibody of the invention comprises the amino acid sequences HCDR1, HCDR2, and HCDR3 VH of SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, or 73, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 VL of SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72 or 73, with CDRs determined by Kabat, Chothia and/or IMGT.

Variants of the antibodies of the invention containing the amino acid sequence VH or VL shown in table. 8, tab. 9 and tab. 14 are within the scope of the invention. For example, variants may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions in VH and/or VL that do not adversely affect properties. antibodies. In some embodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the VH or VL amino acid sequence of the invention.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % identity=number of identical positions/total number of positions χ 100), taking into account the number of gaps and the length of each gap entered for optimal alignment of two sequences.

Percent identity between two amino acid sequences can be determined using the algorithm described in E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is built into the ALIGN program (version 2.0) using weight table of PAM120 residues with a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity of two amino acid sequences can be determined using the algorithm described in Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970) ) embedded in the GAP program from the GCG software package (available at http://_www_gcg_com) using either a Blossum 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight gaps 1, 2, 3, 4, 5 or 6.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 58, 59, 60, 61, 62, 63, or 64, wherein the antibody exhibits one or more of the following properties:

the immune complex of the antibody and shCD154 does not activate platelets, and platelet activation is measured by the expression of P-selectin on the surface of platelets;

binds to CD154 with a dissociation constant (K _D ) of about 5χ10 ^-9 M or less when K _D is measured using the ProteOn XPR36 system using the experimental design described in Example 1 to measure affinity;

inhibits CD154-mediated proliferation of human B cells with an IC ₅₀ value of about 2.7χ10 ^-9 M or less; or inhibits CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2.1 χ10 ^-8 M or less.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, or 73, wherein the antibody exhibits one or more of the following properties:

the immune complex of the antibody and shCD154 does not activate platelets, and platelet activation is measured by the expression of P-selectin on the surface of platelets;

binds to CD154 with a dissociation constant (K _D ) of about 5χ10 ^-9 M or less when K _D is measured using the ProteOn XPR36 system using the experimental design described in Example 1 to measure affinity;

inhibits CD154-mediated proliferation of human B cells with an IC ₅₀ value of about 2.7χ10 ⁹ M or less; or inhibits CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2.1 χ10 ^-8 M or less.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 58, 59, 60, 61, 62, 63 or 64, and a VL that is at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% identical to VL with SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72 or 73, and the antibody exhibits one or more of the following properties:

the immune complex of the antibody and shCD154 does not activate platelets, and platelet activation is measured by the expression of P-selectin on the surface of platelets;

binds to CD154 with a dissociation constant (KD) of about 5x10' ⁹ M or less when KD is measured using the ProteOn XPR36 system using the experimental design described in Example 1 to measure affinity;

inhibits CD154-mediated proliferation of human B cells with an IC ₅₀ value of about 2.7x10' ⁹ M or less; or inhibits CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40, with an IC ₅₀ value of about 2.1x10'8 M or less.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 58.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 59.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 60.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 61.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 62.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 63.

In some embodiments, an antibody of the invention contains a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH from SEQ ID NO: 64.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 65.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 66.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 67.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 68.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 69.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 70.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 71.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 72.

In some embodiments, an antibody of the invention contains a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL with SEQ ID NO: 73.

In some embodiments, an antibody of the present invention comprises a VH of SEQ ID NO: 58 and a VL of SEQ ID NO: 65, wherein the VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven , eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 59 and a VL of SEQ ID NO: 66, wherein the VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 61 and a VL of SEQ ID NO: 68, wherein the VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 63 and a VL of

SEQ ID NO: 70, wherein VH, VL, or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions .

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 64 and a VL of SEQ ID NO: 71, wherein the VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 72, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises a VH of SEQ ID NO: 59 and a VL of SEQ ID NO: 73, wherein the VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

In some embodiments, an antibody of the invention comprises the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in Table 1. 8, tab. 9 and tab. 14, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of antagonist antibodies that specifically bind CD 154.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 22, and 29, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 17, 23, and 30, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 16, 24, and 31, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 18, 25, and 32, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 19, 26, and 33, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 20, 27, and 34, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the invention comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 21, 28, and 35, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 36, 43, and 51, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 37, 44, and 52, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 38, 45, and 53, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 39, 46, and 54, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 40, 47, and 55, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 41, 47, and 56, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 42, 48, and 57, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 37, 49, and 52, respectively, and conservative modifications thereof.

In some embodiments, an antibody of the present invention comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 37, 50, and 52, respectively, and conservative modifications thereof.

Antibodies of the invention containing certain sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 and conservative modifications thereof exhibit one or more of the following properties:

the immune complex of the antibody and shCD154 does not activate platelets, and platelet activation is measured by the expression of P-selectin on the surface of platelets;

bind to CD154 with a dissociation constant (K _D ) of about 5x10'9 M or less when K _D is measured using the ProteOn XPR36 system using the experimental design described in Example 1 to measure affinity;

inhibit CD154-mediated human B cell proliferation with an IC ₅₀ value of about

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2.7χ10' ⁹ M or less; or inhibit CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by an NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40 with an IC ₅₀ value of about

2.1 x 10' ⁸ M or less.

The term conservative modification means amino acid modifications that do not significantly alter or affect the binding characteristics of an antibody containing such amino acid sequences. Conservative modifications include substitutions, additions, and deletions of amino acids. Conservative substitutions are those in which an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids with acidic side chains (eg, aspartic acid, glutamic acid), basic side chains (eg, lysine, arginine, histidine), non-polar side chains (eg, alanine, valine, leucine , isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g. phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (eg, glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (eg, asparagine, glutamine), beta-branched side chains (eg, threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Moreover, any natural residue in the polypeptide can be replaced by alanine, according to the method previously described as alanine-scanning mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). Amino acid substitutions in the antibodies of the invention can be performed by well known methods, for example by PCR mutagenesis (US Patent No. 4,683,195). Alternatively, variant libraries can be generated by known methods, for example by using random (NNK) or non-random codons, such as DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) . The characteristics of the resulting antibody variants can be examined using the assays described herein.

Although the embodiments given in the examples contain pairs of variable regions (one for the heavy chain and one for the light chain), one skilled in the art will appreciate that alternative embodiments may contain single heavy or light chain variable regions. A single variable region can be used to screen for variable domains capable of forming a dual-domain specific antigen-binding fragment capable of, for example, specifically binding to CD154. Such screening can be done using phage display techniques, for example using the hierarchical dual combinatorial approach described in International Patent Publication No. WO1992/01047 as described herein.

Antibodies of the invention can be obtained using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975, can be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other animal host such as a hamster, rat, or monkey is immunized with CD154 from a human, marmoset, or fragments of CD154, such as a soluble form of CD154, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)) . Colonies arising from single cells of the immortalized hybridoma are selected based on the production of antibodies with desired qualities such as binding specificity, cross-reactivity or lack of it, and affinity for the antigen.

Various animal hosts can be used to produce the antibodies of the invention. For example, Balb/c mice can be used to create antibodies. Antibodies derived from Balb/c mice and other non-human animals can be humanized using various techniques to generate sequences that are more human-like. Examples of humanization techniques involving selection of human acceptor scaffolds are known and include CDR grafting (US Patent No. 5,225,539), Specificity Determining Region (SDR) grafting (US Patent No. 6,818,749), Remodeling (Padlan, Mol Immunol 28:489-499, 1991) , remodeling of specificity-determining residues (US Patent Publication No. 20100261620), human adaptation (or adaptation of human scaffolds) (US Patent Publication No. US2009/0118127), superhumanization (US Patent No. 7,709,226) and guided selection (Osbourn et al. (2005 ) Methods 36:61-68, 2005; US patent No. 5,565,332). In these methods, parental antibody CDRs are transferred to human scaffolds, which can be selected based on their shared homology with parental scaffolds, based on scaffold CDR length information, homology or canonical structure, or a combination thereof.

Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by incorporating altered scaffold support residues to maintain binding affinity (backmutations) using techniques such as those described in International Patent Publication No. WO90/007861 and in international patent publication no. WO92/22653, or by introducing a variation in any of the CDRs, for example to improve the affinity of the antibody.

Transgenic mice carrying human immunoglobulin (Ig) loci in their genome can be used to generate human antibodies to the target protein and are described, for example, in International Patent Publication No. WO90/04036; US Pat. No. 6,150,584; International Patent Publication No. WO99/45962, International Patent Publication No. WO02/066630, International Patent Publication No. WO02/43478, Lonberg et al. (1994) Nature 368:856-9; Green et al. (1994) Nature Genet. 7:13-21; Green & Jacobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93; Bruggemann et al. (1991) Euro. J. Immunol. 21:1323-1326; Fishwild et al. (1996) Nat. Biotechnol. 14:845-851; Mendez et al. (1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol. Methods 231:11-23; Yang et al. (1999) Cancer Res. 59:1236-1243; Bruggemann and Taussig (1997) Curr. Opin. Biotechnol. 8:455-458; International Patent Publication No. WO02/043478). Endogenous immunoglobulin loci in such mice can be destroyed or removed, and at least one complete or partial human immunoglobulin locus can be inserted into the mouse genome using homologous or non-homologous recombination using transchromosomes or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbor Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. can be used to generate human antibodies directed against a selected antigen using the technology described above. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com).

Human antibodies can be selected from a phage display library where the phage is engineered to express human immunoglobulins or portions thereof such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., J. Mol Biol 296:57 -86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., Nature Biotechnology 14:309-314, 1996; Sheets et al., PITAS (USA) 95:6157-6162 , 1998; Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991). Antibodies of the invention can be isolated, for example, from a phage display library expressing antibody light and heavy chain variable regions as fusion proteins with bacteriophage coat protein pIX as described in Shi et al., J Mol Biol 397:385-96, 2010 and International Patent Publication No. WO09/085462). Libraries can be screened for phage binding to human, marmoset and/or cynomolgus CD154, and the resulting positive clones can be further characterized, Fab can be isolated from clone lysates, and full-length IgG can then be expressed. Such use of phage display techniques for the isolation of human antibodies is, for example, described in: US Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 issued by Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 issued to Dower et al.; US Pat. Nos. 5,969,108 and 6,172,197 issued to McCafferty et al.; and US Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 issued by Griffiths et al.

The production of immunogenic antigens and the production of monoclonal antibodies can be accomplished by any suitable technique, such as production of a recombinant protein. Immunogenic antigens can be administered to animals in the form of purified protein or protein mixtures, including whole cells or cell or tissue extracts, or the antigen can be formed de novo in the body of the animal from nucleic acids encoding said antigen or portion thereof.

The antibodies of the invention may be human or humanized.

In some embodiments, an antibody of the invention comprises a VH framework derived from the human VH1_1-69, VH4_4-39, VH1_1-02, or VH4_4-59 germline germline gene.

In some embodiments, an antibody of the invention comprises a VL framework derived from the human VKIV_B3, VKI_O12, or VL3_3R germline gene.

The antibodies of the invention may be of the IgA, IgD, IgE, IgG or IgM type. The antibodies of the invention may be of the IgG1, IgG2, IgG3, IgG4 type.

Antibodies of the invention can additionally be engineered to produce a modified antibody with similar or altered properties compared to the parent antibody. Antibodies of the invention can be engineered VH, VL, VH and VL regions, constant regions, VH framework, VL framework, or any or all of the six CDRs.

The antibodies of the invention can be constructed by CDR transplantation. One or more CDR sequences of antibodies of the invention can be grafted onto another framework sequence. CDR grafting can be performed using the methods described herein. In some embodiments, the antibodies of the invention comprise a VH containing HDCR1 with SEQ ID NO: 16, 17, 18, 19, 20 or 21, HCDR2 with SEQ ID NO: 22, 23, 24, 25, 26, 27 or 28, HCDR3 with SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35 and VL containing LCDR1 of SEQ ID NO: 36, 37, 38, 39, 40, 41 or 42, LCDR2 of SEQ ID NO: 43, 44 , 45, 6, 47, 48, 49 or 50, and/or LCDR3 with SEQ ID NO: 51, 52, 53, 54, 55, 56 or

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57, wherein the VH framework is not derived from VH1_1-69, VH4_4-39, VH1_1-02, or VH4_4-59, and the VL framework is not derived from VKIV_B3, VKI_O12, or VL3_3R. Framework sequences for use can be obtained from public DNA databases or published literature including germline antibody gene sequences. For example, germline DNA and protein encoded sequences for human light and heavy chain variable region genes can be found in the international information system IMGT®, international ImMunoGeneTics information system® http://_www-imgt_org.

Framework sequences that can be used to replace existing framework sequences in the antibodies of the invention are those that have the highest percentage identity with C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191, C4LB199, C4LB231, C4LB232, C4LB35 and C4LB256. The framework sequences of parental and engineered antibodies can be subjected to further modification, such as by backmutations, to restore and/or improve the binding of the resulting antibody to the antigen, as described, for example, in US patent No. 6,180,370. The framework sequences of parental and engineered antibodies can be further modified by mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes and therefore reduce the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication No. 20030153043.

CDR residues of antibodies of the invention can be mutated to improve one or more of the binding properties of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding or other functional property of interest can be assessed in in vitro or in vivo assays as described herein and provided in the examples . Examples of substitutions that can be implemented include the conservative modifications described above. Moreover, within a CDR, as a rule, no more than one, two, three, four, or five residues are changed.

Fc substitutions can be introduced into an antibody of the invention to modulate the half-life of the antibody. For example, one or more of the M252Y, S254T, and T256E substitutions can be introduced to increase the half-life of the resulting antibody (Dall'Acqua et al., J Biol Chem 281:23514-240, 2006).

In addition, the antibodies of the invention may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or artificial covalent modification such as the addition of polyethylene glycol moieties (pegylation) or lipidization. Such modifications may occur in vivo or in vitro. For example, the antibodies of the invention can be polyethylene glycol conjugated (PEGylated) to improve their pharmacokinetic profiles. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies to PEG has been shown to improve pharmacodynamics while not affecting function (Knigh et al., Platelets 15:409-18, 2004; Leong et al., Cytokine 16:106-19, 2001; Alfthan et al., Protein Eng. 16:761-70, 2003).

Antibodies of the invention, or fragments thereof, modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, or other desirable biological or biophysical characteristics are within the scope of the present invention. The stability of an antibody is determined by a number of factors, including: (1) the spatial folding of individual domains, which affects their natural stability; (2) surface interactions of proteins that affect the pairing of HC and LC; (3) the depth of location of polar and charged residues; (4) a network of H-bridges between polar and charged residues; and (5) surface charge and distribution of polar residues along with other intra- and intermolecular interactions (Worn et al., J Mol Biol 305:989-1010, 2001). Residues capable of destabilizing the structure can be identified based on the crystal structure of the antibody or, in certain cases, by molecular modeling, and the effect of residues on antibody stability can be tested by creating and evaluating variants containing mutations at the identified residues. One way to increase the stability of an antibody is to increase the average transition temperature (T _m ), measured using differential scanning calorimetry (DSC). In general, the T _m of a protein is directly correlated with its stability and inversely correlated with its susceptibility to tertiary disruption and denaturation in solution, as well as degradation processes that depend on the protein's susceptibility to tertiary disruption (Remmele et al., Biopharm 13: 36-46, 2000). A number of studies indicate a correlation between the degree of physical stability of structures, measured as thermal stability by DSC, and physical stability measurements made using other methods (Gupta et al., AAPS PharmSci 5E8, 2003; Zhang et al., J Pharm Sci 93:3076 -89, 2004; Maa et al., Int J Pharm 140:155-68, 1996; Bedu-Addo et al., Pharm Res 21:1353-61, 2004; Remmele et al., Pharm Res 15:200-8 , 1997). The results of structural studies suggest that T _m Fab affects the long-term physical stability of the respective monoclonal antibody (mAb).

- 20 040943

In some embodiments, an antibody of the invention is a bispecific antibody.

In some embodiments, an antibody of the invention is a multispecific antibody.

Monospecific antibodies of the invention that specifically bind CD154 can be designed as bispecific antibodies, which are also within the scope of the present invention. From the VL and/or VH regions of antibodies of the invention using published methods, single chain bispecific antibodies can be constructed as structures such as TandAb® configurations (International Patent Publication No. WO1999/57150; US Patent Publication No. 2011/0206672), or bispecific scFVs as structures such as those described in US Pat. No. 5,869,620; International Patent Publication No. WO1995/15388, International Patent Publication No. WO1997/14719 or International Patent Publication No. WO2011/036460.

From the VL and/or VH regions of the antibodies of the invention, bispecific full length antibodies can be constructed wherein each antibody arm binds a different antigen or epitope. Such bispecific antibodies can be generated by modulating CH3 interactions between the heavy chains of two antibodies to form bispecific antibodies using techniques such as those described in US Pat. No. 7,695,936; International Patent Publication No. WO2004/111233; US Patent Publication No. 2010/0015133; US Patent Publication No. 2007/0287170; International Patent Publication No. WO2008/119353; US Patent Publication No. 2009/0182127; US Patent Publication No. 2010/0286374; US Patent Publication No. 2011/0123532; International Patent Publication No. WO2011/131746; International Patent Publication No. WO2011/143545; or US Patent Publication No. 2012/0149876.

For example, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and generating a bispecific heterodimeric antibody from the two parental monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization, according to the methods described in International Patent Publication No. WO2011/131746. In these methods, two monospecific bivalent antibodies are designed to have specific substitutions in the CH3 domain to promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to ensure that the cysteines in the hinge region undergo disulfide bond isomerization; thus forming a bispecific antibody by exchanging Fab arms. The incubation conditions can be optimally reverted to non-reducing. Examples of suitable reducing agents include 2-mercaptoethylamine (2MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably the reducing agent is selected from the group , consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. For example, an incubation of at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of 5 to 8 can be used, e.g. at pH 7.0 or at pH 7.4.

Examples of CH3 mutations that can be used in the first heavy chain and second heavy chain of the bispecific antibody include K409R and/or F405L.

Additional bispecific structures into which the VL and/or VH regions of the antibodies of the invention can be inserted can be, for example, double variable domain (DVD) immunoglobulins (International Patent Publication No. W02009/134776) or structures comprising various dimerization domains for connecting two arms of antibodies with different specificities, such as a leucine zipper or collagen dimerization domains (International Patent Publication No. WO2012/022811, US Patent No. 5932448; US Patent No. 6833441). DVDs are full-length antibodies containing a heavy chain having the structure VH1-linker-VH2-CH and a light chain having the structure VL1-linker-VL2-CL; wherein the linker is optional.

The invention also provides an antagonist antibody specifically binding to CD154 of SEQ ID NO: 1 having defined VH and VL sequences, the VH of the antibody being encoded by a first polynucleotide and the VL of the antibody being encoded by a second synthetic polynucleotide. The polynucleotide may be a complementary deoxynucleic acid (cDNA) and may have optimized codons for expression in a suitable host. Codon optimization is a well known technology.

In some embodiments, the polynucleotides encoding the VH or VL antibodies of the invention have the sequences of SEQ ID NO: 76, 77, 78, or 79.

SEQ ID NO: 76 (encoding VH in C4LB231)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGAC

CATCACCTGTCGGGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGC

- 21 040943

AAGGCCCCCCAAGCTGCTGATCTACTACGCCAACAGCCTGCAGAGCGGCGTGCCCAGCAGATTCA GCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGACTTCGC CACCTACTACTGCCAGCAGAGCGACAGCATCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAA ATGAAG

SEQ ID NO: 77 (encoding VL in C4LB231)

CAGGTCCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGT GTCCTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACGGCATCAGCTGGGTCCGACAGGCCCCA GGACAGGGCCTGGAATGGATGGGCTGGATCAGCCCCATCTTCGGCAACACCAACTACGCCCAGA AATTCCAGGGCAGAGTGACCATСАСCGCCGACGAGAGCACCAGCACCGCCTAGATGGAAGTGAG CAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCCGGTACTACGGCGACCTG GACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT

SEQ ID NO: 78 (encoding VH in C4LB191)

CAGGTGCAGCTGGTGCAGAGCGGCGCTCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGT GAAGAAACCTGGCGCCAGCATGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACTAC TACATCCACTGGGTGCGCCAGGCCCCAGGCCAGGGACTGGAATGGGTGGGACGGTTCAACCCCA ACAGCGGCGACACCAACGGCGCCCAGAAATTCCAGGGCAGAGTGACCATGACCCGGGACACCAG CATCAGCACCGCCTACATGGAACTGACCCGGCTGCGGAGCGACGACACCGCCGTGTACCACTGT GCCAGAGAGGGCGAGCTGGCCGGCATCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACAG TGTCCAGC

SEQ ID NO: 79 (encoding VL in C4LB191)

AGCTACGAGCTGACCCAGCCCCCCAGCGTGTCCGTGTCTCCTGGCCAGACCGCCAGCAT CACCTGTAGCGGCGACAAGCTGGGCGACAAATACGTGTCCTGGAACCACCAGAAGCCCGGCCAG AGCCCCGTGCTGGTGATCTACCAGGACCGGAAGAGGCCCAGCGGCATCCCCGAGAGATTCAGCG GCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCGGCACCCAGGCCATGGACGAGGCCGA CTACTACTGCCAGGCCTGGGACAGCAGCACCGTGGTGTTCGGCGGAGGCACCAAGCTGACCGTG CTG

The invention also provides an isolated polynucleotide encoding any of the antibody heavy chain variable regions, antibody light chain variable regions, antibody heavy chains, and/or antibody light chains of the invention. Some examples of polynucleotides are described herein, however, other polynucleotides which, given the degeneracy of the genetic code or the preference for codon usage in a given expression system, encode antibodies of the invention are also within the scope of the present invention. Examples of polynucleotides are, for example, those containing the sequences shown in SEQ ID NOs: 76, 77, 78 and 79. Polynucleotide sequences encoding the VH or VL (or fragment thereof) of an antibody of the invention may be operably linked to one or more regulatory elements. , such as a promoter or enhancer that allows the expression of the nucleotide sequence in the intended host cell. The polynucleotide may be cDNA.

The invention also provides a vector containing the polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon vectors, or any other vectors suitable for introducing the synthetic polynucleotide of the invention into a given organism or genetic environment by any means. For example, polynucleotides encoding the variable regions of the light and/or heavy chains of the antibodies of the invention, optionally linked to constant regions, are inserted into expression vectors. The light and/or heavy chains can be cloned in the same or different expression vectors. DNA segments encoding immunoglobulin chains may be operably linked in the expression vector(s) to control sequences for expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (eg, naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected for expression of the antibody. After introducing the vector into the appropriate host, the host is incubated under conditions acceptable for

- 22 040943 high-level expression of proteins encoded by the introduced polynucleotides.

In general, suitable expression vectors can replicate in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors contain selection markers, eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to allow detection of such cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Examples of promoters for expression in a eukaryotic cell include promoter and enhancer elements of immunoglobulin light and/or heavy chain genes; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; SV40 early and late promoters; a promoter present in the long terminal repeats of the retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. The selection of an appropriate vector and promoter is well within the ability of one of ordinary skill in the art.

Large numbers of acceptable vectors and promoters are known to those skilled in the art; many are available on the market to generate the claimed recombinant constructs. The following vectors are provided as an example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, San Diego, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).

The invention also provides a host cell containing one or more vectors of the invention. The term host cell refers to the cell into which the vector is introduced. It should be understood that the term host cell is used to refer not only to a specific claimed cell, but also to the progeny of such a cell, as well as a stable cell line created from a specific claimed cell. Since some modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but may also be encompassed by the term host cell as used herein. Such host cells can be eukaryotic, prokaryotic, plant or archaeal cells.

E. coli (Escherichia coli), bacilli such as hay bacillus (Bacillus subtilis), and other enterobacteria such as salmonella, serrations and various types of Pseudomonas are examples of prokaryotic host cells. Other microorganisms such as yeast are also used for expression. Saccharomycetes (eg S. cerevisiae) and pichias are examples of suitable yeast host cells. Examples of eukaryotic cells may be mammalian, insect, avian or other animal cells. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines, such as murine SP2/0 cell lines (ATCC, Manassas, VA, USA, CRL-1581), NS0 (European cell cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580). An example of a human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include lines derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologies, Walkersville, MD, USA), CHO-K1 (ATCC CRL-61) or DG44.

The invention also provides a method for producing an antibody of the invention, comprising culturing a host cell of the invention under antibody expression conditions and isolating the antibody produced by the host cell. Methods for producing antibodies and purifying them are well known to those skilled in the art. Once synthesized (chemically or recombinantly), whole antibodies, their dimers, individual light and/or heavy chains, or other antibody fragments such as VH and/or VL, can be purified according to standard procedures, including ammonium sulfate precipitation, the use of affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like. (Essentially, see Scopes, Protein Purification (SpringerVerlag, N.Y., 1982). A claimed antibody may be substantially pure, e.g., at least about 80 to 85% pure, at least about 85 to 90% pure. %, pure at least from about 90 to 95%, pure at least from about 98 to 99% or more, for example, free of contaminants such as cellular debris, macromolecules, etc., other than the claimed antibody.

The invention also provides a method for producing an antagonist antibody specifically binding CD154 with SEQ ID NO: 1, comprising combining in an expression vector a first polynucleotide encoding the VH antibody and a second polynucleotide encoding the VL antibody;

transformation of the host cell with this expression vector;

culturing the host cell in a culture medium under conditions in which VL and VH are expressed to form an antibody; And

- 23 040943 isolation of the antibody from the host cell or culture medium.

Polynucleotides encoding certain VH or VL sequences of the invention can be incorporated into vectors using standard molecular biology techniques. Transformation of the host cell, culture, expression and purification of the antibody are performed using well known methods.

Methods of treatment

Antagonist antibodies of the invention that specifically bind CD154, such as antibodies C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191, C4LB199, C4LB231, C4LB232, C4LB35 and C4LB236, can be used to treat and/or prevent any condition or disease in which antagonistic effects in against CD154 may be therapeutically effective and may reduce the symptoms of the disease. Examples include the treatment of allergic, autoimmune, cancerous, transplantation conditions, graft-versus-host disease (GVHD), inflammatory and other conditions, especially conditions in which tolerance induction and/or suppression of humoral immunity is therapeutically desirable. Diseases that can be treated with antibodies of the invention include immune-mediated inflammatory diseases or autoimmune diseases such as arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease, transplantation, kidney transplantation, skin transplantation, bone marrow transplantation, graft-versus-host disease (GVHD). ), immune thrombocytopenia (ITP), multiple sclerosis, thyroiditis, type I diabetes, or atherosclerosis.

In addition to simply blocking CD154-CD40 interactions, anti-CD154 therapy leads to the induction of immunological tolerance (Gordon et al., Diabetes 47: 1199-1206, 1988); Markees et al., Transplantation 64: 329-335, 1997; Jarvinen et al., Transplantation 76: 1375-1379, 2003; Quezada et al., Blood 102: 1920-1926, 2003; Frleta et al., J Immunother 26: 72-84, 2003; Elster et al., Transplantation 72: 1473-1478, 2001; Benda et al., Cell Transplantation 11: 715-720, 2002; Wekerle and Sykes, Annual review of medicine 2001. 52: 353-370 ¹⁹ ; Camirand et al., Transplantation 73: 453-461, 2002).

The invention also provides a method for treating an immune-mediated inflammatory disease or an autoimmune disease, comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need thereof for a time period sufficient to treat the immune-mediated inflammatory disease or autoimmune disease.

The invention also provides a method for treating arthritis comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need thereof for a time period sufficient to treat the arthritis.

In some embodiments, the arthritis is juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis, or gouty arthritis.

The invention also provides a method for treating lupus, comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need thereof for a time period sufficient to treat lupus.

In some embodiments, the lupus is systemic lupus erythematosus (SLE) or cutaneous lupus erythematosus (CLE).

In some embodiments, the subject has lupus nephritis.

The invention also provides a method for treating inflammatory bowel disease, comprising administering a therapeutically effective amount of an antibody of the invention to a subject in need thereof for a time period sufficient to treat the inflammatory bowel disease.

In some embodiments, the inflammatory bowel disease is Crohn's disease.

In some embodiments, the inflammatory bowel disease is ulcerative colitis.

The terms treatment or treat mean therapeutic treatment. Subjects in need of treatment include those subjects diagnosed with a disorder or a symptom of a disorder. Subjects that can be treated also include those who are susceptible to or susceptible to the disorder, or those in need of prophylaxis for the disorder. Beneficial or desirable clinical outcomes include relief of symptoms, reduction in disease severity, stabilization (i.e., no worsening) of the course of the disease, delay or retardation of disease progression, alleviation or temporary amelioration of the disease state, and remission (partial or complete) as detectable, so undetectable. Beneficial clinical outcome in a treated subject includes, for example, reduced B cell or dendritic cell proliferation, decreased levels of pro-inflammatory cytokines, adhesion molecules, proteases, immunoglobulins (in cases where the CD40-bearing cell is a B cell), combinations thereof, increased production of anti-inflammatory proteins, decreased number of autoreactive cells, increased immune tolerance, inhibition of survival of autoreactive cells, and/or reduction of one or more symptoms mediated by stimulation of CD40-expressing cells by CD154.

- 24 040943

Clinical response can be assessed using screening techniques such as magnetic resonance imaging (MRI), radiography, computed tomography (CT), flow cytometry or fluorescence activated cell sorting (FACS), histology, macropathology, and blood chemistry, including but not limited to , changes determined by the method of solid-phase ELISA, radioimmunoassay (RIA), chromatography, etc.

Examples of antibodies that can be used in the methods of the invention include the VH, VL, HCDR and/or LCDR regions shown in Table 1. 2, tab. 3, tab. 4, tab. 5, tab. 6, tab. 7, tab. 8, tab. 9, tab. 13 and tab. 14, and antibodies C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191, C4LB199, C4LB231, C4LB232, C4LB35 and C4LB236.

The methods of the invention can be used to treat a subject within any classification of animals. Examples of subjects that can be treated include mammals such as humans, rodents, dogs, cats, and farm animals.

Antibodies of the invention can also be used to obtain a drug for such treatment, and the drug is obtained for administration in dosages defined herein.

The antibodies of the invention may be administered in combination with a second therapeutic agent.

The second therapeutic agent may be any known therapeutic agent for autoimmune and inflammatory diseases, including any agent or combination of agents known to be useful or that has been or is being used to treat autoimmune and inflammatory diseases. Such therapies and therapeutic agents include surgery or surgical procedures (eg, splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukophoresis, cell, tissue, or organ transplantation, bowel procedures, organ perfusion, etc.), radiation therapy, such therapy such as steroid and non-steroid therapy, hormonal therapy, cytokine therapy, therapy with dermatological agents (eg, topical agents used to treat skin conditions such as allergies, contact dermatitis, and psoriasis), immunosuppressive therapy, and therapy with other anti-inflammatory monoclonal antibodies.

The second therapeutic agent may be a corticosteroid, an antimalarial drug, an immunosuppressant, a cytotoxic drug, or a B cell modulator.

In some embodiments, the second therapeutic agent is prednisone, prednisolone, methylprednisolone, deflascort, hydroxychloroquine, azathioprine, methotrexate, cyclophosphamide, mycophenolate mofetil (MMF), sodium mycophenolate, cyclosporine, leflunomide, tacrolimus, RITUXAN® (rituximab), or BENLYSTA® (belimumab). ).

In some embodiments, the antibodies of the invention are administered in combination with a second therapeutic agent. Examples of second therapeutic agents include corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine, cytotoxic drugs, immunosuppressive drugs, immunomodulating antibodies, methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506; ProGrafrM), mycophenolate mofetil and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualine, leflunomide and its malononitriloamide analogues; antibodies to CTLA4 and fusion Igs, antibodies to B-lymphocyte stimulators (e.g. LYMPHOSTAT-BTM) and CTLA4-Ig fusion proteins (BLySlg), antibodies to CD80, anti-T cell antibodies such as anti-CD3 antibodies (OCTZ), anti-CD4 antibodies, corticosteroids such as, for example, clobetasol, halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinol, fluocinonide, prednisone, prednisolone, methylprednisolone; non-steroidal anti-inflammatory drugs (NSAIDs), such as, for example, sulfasalazine, medicines containing mesalamine (known as 5-ASA agents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozin , piroxicam, rofecoxib, salicylates, sulindac and tolmetin; phosphodiesterase-4 inhibitors, anti-TNFa antibodies REMICADE® (infliximab), SIMPONI® (golimumab) and HUMIRA® (adalimumab), thalidomide or analogues such as lenalidomide.

The antibodies of the invention may be administered in combination with a second therapeutic agent simultaneously, sequentially or separately.

Treatment efficacy or RA may be assessed using efficacy as measured by clinical responses as defined by the American College of Rheumatology criteria, the European League Against Rheumatism criteria, or any other criteria. See, for example, Felson et al. (1995) Arthritis Rheum. 38:727-35 and van Gestel et al. (1996) Arthritis Rheum. 39:34-40.

Introduction. Pharmaceutical compositions

The invention provides pharmaceutical compositions of antagonistic antibodies specifically binding to CD154 and a pharmaceutically acceptable carrier. For therapeutic use

- 25 040943 it is possible to obtain antibodies of the invention in the form of pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. The term carrier refers to the diluent, adjuvant, excipient or carrier vehicle with which the active compound is administered. Such carriers may be liquids such as water and oils, including oils derived from petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine solution. These solutions are sterile and essentially free of particulate matter. They can be sterilized using well known standard sterilization techniques (eg filtration). The compositions may contain pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules or antibodies of the invention in such a pharmaceutical formulation can vary greatly, those. from less than about 0.5 wt.%, usually at least from about 1 to 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, 30 wt.%, 35 wt.%, 40 wt.%, 45 wt.% or 50 wt.%, and will be mainly selected based on the required dose, fluid volumes, viscosity values, etc. according to the specific route of administration chosen. Suitable carrier media and formulations including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, ^21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 6911092, see especially pp. 958-989.

The route of administration of the antibodies of the invention in the methods of the invention may be any suitable route, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transmucosal (oral, intranasal, intravaginal, rectal), or other methods known to the skilled person, as is well known in this area.

The antibodies of the invention can be administered to a subject by any suitable route, such as parenterally by intravenous (IV) infusion or bolus injection, intramuscularly, subcutaneously, or intraperitoneally. IV infusion can be carried out, for example, for 15, 30, 60, 90, 120, 180 or 240 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 h.

The dose administered to a subject with an immune-mediated inflammatory disease or an autoimmune disease such as rheumatoid arthritis is sufficient to attenuate or at least partially reverse the disease being treated (therapeutically effective amount) and may sometimes range from 0.005 mg/kg to about 100 mg/kg, for example, from about 0.05 mg/kg to about 20 mg/kg, or from about 0.1 mg/kg to about 20 mg/kg, or from about 1 mg/kg to about 20 mg/kg kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or, for example, about 1, 2, 3, 4, 5, b, 7, 8, 9 or 10 mg/kg, but may be even higher, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be administered, such as 50, 100, 200, 500, or 1000 mg, or the dose may be based on the patient's body surface area, such as 500, 400, 300, 250, 200, or 100 mg/m ² . For the treatment of an immune-mediated inflammatory disease such as rheumatoid arthritis, 1 to 8 doses (eg, 1, 2, 3, 4, 5, b, 7, or 8) can usually be administered, but 9, 10, 11, 12 can also be administered. , 13, 14, 15, 16, 17, 18, 19, 20 or more doses.

Administration of antibodies of the invention can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months , four months, five months, six months or more. Repeated courses of treatment in the form of long-term administration are also possible. Repeated administration can be carried out at the same dose or at a different dose. For example, the antibodies of the invention may be administered by infusion at 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 8 mg/kg, or 16 mg/kg one week apart for 8 weeks, followed by infusion into 8 mg/kg or 16 mg/kg every two weeks for an additional 16 weeks, followed by 8 or 16 mg/kg every four weeks.

The antibodies of the invention may be administered as maintenance therapy, eg once a week for a period of 6 months or more.

For example, the antibodies of the invention can be administered as a daily dose of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg per day on at least one of days 1, 2, 3, 4, 5, b, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39 or 40, or alternatively at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after the start of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof.

The antibodies of the invention can also be administered prophylactically to reduce the risk of developing an immune-mediated inflammatory disease or an autoimmune disease such as arthritis or rheumatoid arthritis and/or to delay the onset of an immune-mediated inflammatory disease of an autoimmune disease.

- 26 040943

Thus, a pharmaceutical composition of the invention for intramuscular injection can be prepared at a content of 1 ml of sterile buffered water and from about 1 ng to about 100 mg/kg, for example from about 50 ng to about 30 mg/kg, or more preferably from about 5 mg to about 25 mg/kg of an antibody of the invention.

For example, a pharmaceutical composition containing antibodies of the invention for administration to an 80 kg patient by intravenous infusion may be formulated to contain about 200 ml of sterile Ringer's solution and about 8 mg to about 2400 mg, from about 400 mg to about 1600 mg or about 400 mg to about 800 mg of the antibodies of the invention. Methods for preparing compositions for parenteral administration are well known and described in more detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, PA. A therapeutically effective amount of an antibody of the invention effective for treating an immune-mediated inflammatory disease or an autoimmune disease can be determined by standard assay methods. For example, in vitro assays can be used to help determine the optimal dosage range. The dose of antibodies of the invention that may be effective in the treatment of immune-mediated inflammatory diseases or autoimmune diseases such as arthritis or rheumatoid arthritis can optionally be determined by administration of the antibodies in appropriate animal models well known in the art. The choice of a specific effective dose (eg, through clinical trials) can be performed by those skilled in the art based on several factors. Such factors include: the disease to be treated or prevented, the symptoms noted, the body weight of the patient, the immunological status of the patient, and other factors known to the person skilled in the art. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease and should be determined based on the judgment of the healthcare professional and the condition of each patient. Effective doses can be extrapolated from dose-response curves obtained in in vitro test systems or animal models. Antibodies of the invention can be tested for their efficacy and effective dosage using any of the models described herein.

Antibodies of the invention may be lyophilized for storage and reconstituted in an acceptable vehicle prior to use. This technique has been shown to be effective for standard protein preparations and well known lyophilization and reconstitution techniques can be used.

Anti-idiotypic antibodies

The present invention provides an anti-idiotypic antibody that binds to an antibody of the invention.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 61 and VL of SEQ ID NO: 68.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 63 and VL of SEQ ID NO: 70.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 64 and VL of SEQ ID NO: 71.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 72.

The invention also provides an anti-idiotypic antibody specifically binding an antibody comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 73.

An anti-idiotypic (Id) antibody is an antibody that recognizes the antigenic determinants (eg, paratope or CDR) of an antibody. The Id antibody may be blocking or non-blocking antigen. An antigen-blocking Id antibody can be used to detect a free antibody in a sample (eg, the anti-CD154 antibody of the invention described herein). A non-blocking Id antibody can be used to detect all antibodies (free, partially bound to an antigen, or fully bound to an antigen) in a sample. An Id antibody can be obtained by immunizing an animal with an antibody for which an anti-Id antibody is obtained.

An anti-Id antibody can also be used as an immunogen to induce an immune response in another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody may be epitopically identical to the primary mAb that induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of the mAb, other clones expressing antibodies of identical specificity can be identified. Anti-Id antibodies can be subjected to variations (thereby variants of anti-I antibodies) and/or derivatization using any suitable technique, for example, described in other parts of this document in relation to antibodies that specifically bind antibodies to HLA-DR.

Immunoconjugates

The term immunoconjugate means an antibody of the invention conjugated to one or more heterologous molecules. In some embodiments, an antibody of the invention is conjugated to one or more cytotoxic agents. Examples of such cytotoxic agents include chemotherapeutic agents or drugs, growth inhibitory agents, toxins (eg, protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), and radioactive isotopes.

In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC) wherein an antibody of the invention is conjugated to one or more drugs, such as maytansinoid (see, for example, US Pat. Nos. 5,208,020, 5,416,06)); auristatin, such as the monomethylauristatin drug groups DE and DF (MMAE and MMAF) (see, for example, US Pat. 5.739, 116, 5.767.285, 5.770.701, 5.770.710, 5.773.001 and 5.877.296 Hinman et al. (1993) Cancer Res 53:3336-3342 and Lode et al. (1998) Cancer Res 58:2925); an anthracycline such as daunomycin or doxorubicin (see e.g. Kratz et al., (2006) Current Med. Chem 13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem Letters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagy et al., (2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529 -1532 (2002); King et al., (2002) J Med Chem 45:4336-4343 and US Pat. No. 6,630,579), methotrexate, vindesine, taxane such as docetaxel, paclitaxel, larotaxel, tezetaxel and orthotaxel.

In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated with an enzymatically active toxin or fragment thereof, such as diphtheria toxin A chain, non-binding diphtheria toxin active moieties, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain , Modexin A-chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), omordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecenes.

In some embodiments, an antibody of the invention is conjugated to a radioactive atom to form a radioconjugate. To obtain radioconjugates, there are various radioactive isotopes. Examples include At211, 1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu radioactive isotopes. When used to detect a radioconjugate, it may contain a radioactive atom for scintigraphy, such as tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody of the invention and a cytotoxic agent can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC ), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis-(p-azidobenzoyl)hexanediamine) , bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene-2,6 diisocyanate) and compounds with two active fluorine groups (such as 1,5-difluoro-2,4 dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et al., (1987) Science 238:1098. Carbon-14 labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating a radionuclide to an antibody. See, for example, WO94/11026. The linker may be a cleavable linker that facilitates the release of the cytotoxic drug into the cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide containing linker can be used (Chari et al., (1992) Cancer Res 52: 127-131; US Pat. No. 5,208,020).

Immunoconjugates or ADCs can be prepared with cross-linker reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo -KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfone) benzoate), which are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, State Illinois, USA).

The invention also provides an immunoconjugate containing an antibody that specifically binds CD154 with SEQ ID NO: 1 of the invention, associated with a therapeutic agent or imaging agent.

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Additional embodiments of the invention

Below are some additional embodiments of the invention in accordance with the descriptions provided in other parts of this document. The elements from the embodiments of the invention set forth above, described as being related to the invention disclosed herein, also apply to each and every one of these additionally numbered embodiments.

1) An isolated antagonist antibody or antigen-binding site thereof specifically binding human CD154 with SEQ ID NO: 1, containing a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 17 (SYGIS), HCDR2 with SEQ ID NO: 23 (WISPIFGNTNYAQKFQG ) and HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), optionally

a) HCDR1 residue S1 is mutated to A, C, D, E, G, I, K, L, M, N, Q, R, T, or V;

b) HCDR1 residue I4 is mutated to M, L or V;

c) the S5 HCDR1 residue is mutated in A;

d) the S3 residue of HCDR2 is mutated to A, T, or V;

e) the P4 HCDR2 residue is mutated to V, T, L Q or E;

f) the N8 residue of HCDR2 is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

g) the T9 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

h) the N10 HCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

l) HCDR3 residue S1 is mutated to A or M;

j) the R2 residue of HCDR3 is mutated to A, S, Q, or K; and k) the L7 residue of HCDR3 is mutated into M.

2) An antibody according to claim 1 containing a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 37 (RASQSISSYLN), LCDR2 with SEQ ID NO: 44 (YANSLQS) and LCDR3 with SEQ ID NO: 52 (QQSDSIPWT), and optionally

a) the Q4 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, V, W, or Y;

b) the S5 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

c) the S7 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

d) the S8 LCDR1 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

e) LCDR2 residue A2 is mutated to S;

f) the LCDR2 N3 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

g) the S4 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

h) LCDR2 L5 residue is mutated to A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y;

i) the Q6 LCDR2 residue is mutated to E, D or N;

j) the S7 LCDR2 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

k) the S3 LCDR3 residue is mutated in A;

l) the D4 LCDR3 residue is mutated to N;

m) the S5 LCDR3 residue is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y; And

n) LCDR3 residue 16 is mutated to A, C, D, E, G, K, L, M, N, Q, R, S, T, V.

3) Antibody according to embodiment 1 or 2, containing HCDR1 with SEQ ID NO: 17 (SYGIS), HCDR2 with SEQ ID NO: 23 (WISPIFGNTNYAQKFQG), HCDR3 with SEQ ID NO: 30 (SRYYGDLDY), LCDR1 with SEQ ID NO: 37 (RASQSISSYLN), LCDR2 of SEQ ID NO: 44 (YANSLQS), and LCDR3 of SEQ ID NO: 52 (QQSDSIPWT).

4) An antibody according to any one of embodiments 1-3, comprising

a) VH with SEQ ID NO: 59 and VL with SEQ ID NO: 66; or

b) a heavy chain of SEQ ID NO: 80 and a light chain of SEQ ID NO: 81.

5) An isolated antagonist antibody specifically binding soluble human CD154 (shCD154) of SEQ ID NO: 4.

6) An antibody according to any one of embodiments 1-5, containing at least one substitution in the Fc region.

7) An antibody according to any one of embodiments 1-6 that does not activate human platelets.

8) An antibody according to any one of embodiments 1-7, wherein the antibody binds to shCD154 with a KD dissociation constant of about 5x10'9M or less, about 1x10'9M or less, about ^5x10'10M or less, about 1x10' 10 M or less, about 5x10'11 M or less, or about 1x10'11 M or less, wherein K _D is measured using a ProteOn XPR36 system using the experimental design described in Example 1 to measure affinities.

9) An antibody according to any one of embodiments 1-8, wherein the antibody inhibits shCD154 mediated proliferation of human B cells with an IC ₅₀ value of about 7.0 x 10'11 M to about 6 x 10' ¹⁰ M.

10) An antibody according to any one of embodiments 1-9, wherein the antibody inhibits shCD154 mediated expression of secreted embryonic alkaline phosphatase (SEAP) by an NF-κΒ inducible interferon-β (IFN-β) minimal promoter in cells

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HEK293 stably expressing SEAP and human CD40 with an IC ₅₀ value of about 2.1 x 10' ⁸ M or less.

11) An antibody according to any one of embodiments 1-10, wherein the antibody competes for binding to shCD154 with an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence of SEQ ID NO: 59 , and VL contains the sequence SEQ ID NO: 66.

12) An antibody according to any one of embodiments 1-11, wherein the antibody comprises the amino acid sequences of heavy chain complementarity determining regions (HCDR) 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) VH of SEQ ID NOs: 58, 59 , 60, 61, 62, 63, or 64, optionally having one, two, or three conservative amino acid substitutions in HCDR1, HCDR2, and/or HCDR3, with HCDR defined by the Kabat, Chothia, or IMGT system.

13) An antibody according to any one of embodiments 1-12, wherein the antibody comprises the amino acid sequences of light chain complementarity determining regions (LCDR) 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) VL of SEQ ID NOs: 65, 66 , 67, 68, 69, 70, 71, 72, or 73, optionally having one, two, or three conservative amino acid substitutions in LCDR1, LCDR2, and/or LCDR3, where LCDRs are defined by the Kabat, Chothia, or IMGT system.

14) An antibody according to any one of embodiments 1-13 containing the amino acid sequences HCDR1, HCDR2 and HCDR3 with

a) SEQ ID NO b) SEQ ID NO c) SEQ ID NO d) SEQ ID NO e) SEQ ID NO

16, 22

17, 23

16, 24

18, 25

19, 26 and 29, respectively, and 30, respectively, and 31, respectively, and 32, respectively, and 33, respectively, and and and and and their conservative modifications; their conservative modifications; their conservative modifications; their conservative modifications; their conservative modifications;

f) SEQ ID NOs: 20, 27 and 34, respectively, and conservative modifications thereof, or

g) SEQ ID NOs: 21, 28 and 35, respectively, and their conservative modifications.

15) An antibody according to any one of embodiments 1-10, comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 with

a) SEQ ID NO b) SEQ ID NO c) SEQ ID NO d) SEQ ID NO e) SEQ ID NO

36, 43

37, 44

38, 45

39, 46

40, 47 and and and and and

51, respectively,

52, respectively,

53, respectively,

54, respectively,

55, respectively, and and and and and their conservative modifications; their conservative modifications; their conservative modifications; their conservative modifications; their conservative modifications;

f) SEQ ID NOs: 41, 47 and 56, respectively, and conservative modifications thereof;

g) SEQ ID NOs: 42, 48 and 57, respectively, and conservative modifications thereof;

h) SEQ ID NOs: 37, 49 and 52, respectively, and conservative modifications thereof, or

i) SEQ ID NOs: 37, 50 and 52, respectively, and conservative modifications thereof.

16) An antibody according to any one of embodiments 1-15 containing HCDR1, HCDR2 and HCDR3 with

a) SEQ ID NOs: 16, 22 and 29, respectively;

b) SEQ ID NOs: 17, 23 and 30, respectively;

c) SEQ ID NOs: 16, 24 and 31, respectively;

d) SEQ ID NOs: 18, 25 and 32, respectively;

e) SEQ ID NOs: 19, 26 and 33, respectively;

f) SEQ ID NOs: 20, 27 and 34, respectively, or

g) SEQ ID NOs: 21, 28 and 35, respectively.

17) An antibody according to any one of embodiments 1-16, comprising the amino acid sequences LCDR1, LCDR2, and LCDR3 with

a) SEQ ID NOs: 36, 43 and 51, respectively;

b) SEQ ID NOs: 37, 44 and 52, respectively;

c) SEQ ID NOs: 38, 45 and 53, respectively;

d) SEQ ID NOs: 39, 46 and 54, respectively;

e) SEQ ID NOs: 40, 47 and 55, respectively;

f) SEQ ID NOs: 41, 47 and 56, respectively;

g) SEQ ID NOs: 42, 48 and 57, respectively;

h) SEQ ID NOs: 37, 49 and 52, respectively, or

i) SEQ ID NOs: 37, 50 and 52, respectively.

18) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 16, 22, and 29, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 36, 43, and 51, respectively .

19) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 44, and 52, respectively .

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20) Antibody according to any one of embodiments 1-17, containing HCDR1, HCDR2 and

HCDR3 with SEQ ID NOs: 16, 24 and 31, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 38, 45 and 53, respectively.

21) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 18, 25, and 32, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 39, 46, and 54, respectively .

22) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 19, 26, and 33, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 40, 47, and 55, respectively .

23) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 20, 27, and 34, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 41, 47, and 56, respectively .

24) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 21, 28, and 35, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 42, 48, and 57, respectively .

25) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 49, and 52, respectively .

26) An antibody according to any one of embodiments 1-17, comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 17, 23, and 30, respectively, and LCDR1, LCDR2, and LCDR3 of SEQ ID NOS: 37, 50, and 52, respectively .

27) An antibody according to any one of embodiments 1-26, containing a VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VH with SEQ ID NO: 58, 59, 60, 61, 62, 63 or 64.

28) An antibody according to any one of embodiments 1-27, comprising a VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to VL with SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72 or 73.

29) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

30) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

31) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67, wherein VH, VL or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

32) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 61 and VL of SEQ ID NO: 68, wherein VH, VL or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

33) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

34) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 63 and VL of SEQ ID NO: 70, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

35) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 64 and VL of SEQ ID NO: 71, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

36) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 72, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions.

37) An antibody according to any one of embodiments 1-28, comprising VH of SEQ ID NO: 59 and VL of SEQ ID NO: 73, wherein VH, VL, or both VH and VL optionally contain one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen

- 31 040943 or fifteen amino acid substitutions.

38) An antibody according to any one of embodiments 1-37, which contains at least one substitution in the Fc region.

39) An antibody according to any one of embodiments 1-38, wherein at least one Fc region substitution is L234A, L235A, G237A, P238S, H268A, A330S, and P331S, with residue numbering corresponding to the EU index.

40) An antibody according to any one of embodiments 1-38, wherein at least one substitution in the Fc region is V234A, G237A, P238S, H268A, V309L, A330S, and P331S, with residue numbering corresponding to the EU index.

41) An antibody according to any one of embodiments 1-40, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 isotype.

42) An antibody according to any one of embodiments 1-41, wherein all paratopic residues of the antibody are in the VH.

43) An antibody according to any one of embodiments 1-42, wherein the antibody simultaneously binds the first CD154 monomer and the second CD154 monomer of the soluble human CD154 trimer.

44) The antibody of embodiment 43, wherein the antibody binds at least one, two, three, four, five, six, seven, or eight CD154 residues in the first CD154 monomer within amino acids 182-207 of CD154 in SEQ ID NO: 1, where the numbering of residues corresponds to SEQ ID NO: 1.

45) The antibody of embodiment 43 or 44, wherein the antibody binds at least one, two, three, four, five, six, seven, or eight CD154 residues in the second CD154 monomer within amino acids 176-253 of CD154 in SEQ ID NO: 1, where the numbering of residues corresponds to SEQ ID NO: 1.

46) An antibody according to any one of embodiments 43-45, wherein the antibody binds residues E182, S185, Q186, A187, P188, S214, A215, and R207 in the first CD154 monomer, with residue numbering according to SEQ ID NO: 1.

47) An antibody according to any one of embodiments 43-46, wherein the antibody binds residues T176, F177, C178, Q220, S248, H249, G250, and F353 in the second CD154 monomer, with residue numbering according to SEQ ID NO: 1.

48) A bispecific molecule comprising an antibody or antigen-binding site thereof, according to any one of embodiments 1-47, associated with a second antigen-binding molecule having a different binding specificity.

49) An antibody according to any one of embodiments 1-47, or a bispecific molecule according to 48, which

a) in the immune complex with shCD154 does not activate human platelets;

b) binds to shCD154 with a dissociation constant K _D of about 5x10'9 M or less;

c) inhibits shCD154 mediated human B cell proliferation; or

d) inhibits the biological activity of CD154 in the NF-kB-SEAP reporter gene assay.

50) An immunoconjugate comprising an antibody or antigen-binding site thereof according to any one of embodiments 1-47 associated with a therapeutic agent or an imaging agent.

51) A pharmaceutical composition comprising the antibody of Embodiment 1-47 and a pharmaceutically acceptable carrier.

52) A polynucleotide encoding an antibody VH, an antibody VL, or an antibody VH and an antibody VL according to any one of embodiments 1-47.

53) A vector containing the polynucleotide of embodiment 52.

54) Host cell containing the vector of embodiment 53.

55) A method for producing an antibody, comprising culturing the host cell of embodiment 54 under antibody expression conditions, and isolating the antibody produced by the host cell.

56) An antibody according to any one of embodiments 1-47 or a pharmaceutical composition according to embodiment 51 for use in the treatment of an autoimmune disease or an immune-mediated inflammatory disease.

57) An antibody according to any one of embodiments 1-47 for use in the treatment of diseases such as arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease, transplantation, kidney transplantation, skin transplantation, bone marrow transplantation, graft versus host disease (GVHD ), immune thrombocytopenia (ITP), multiple sclerosis, thyroiditis, type I diabetes, or atherosclerosis.

58) An antibody according to any one of embodiments 1-47 for use in the treatment of rheumatoid arthritis.

59) An antibody according to any one of embodiments 1-47 for use in the treatment of

- 32 040943 chunks.

60) An antibody according to any one of embodiments 1-47 for use in transplantation treatment.

61) An antibody according to any one of embodiments 1-47 for use in the treatment of inflammatory bowel disease.

62) An antibody according to any one of embodiments 1-47 for use in the treatment of any one of embodiments 56-61 in combination with a second therapeutic agent.

63) The antibody of embodiment 62, wherein the second therapeutic agent is non-steroidal anti-inflammatory drugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine, corticosteroids, cytotoxic drugs, immunosuppressive drugs, and/or antibodies.

The invention also provides an isolated antibody or antigen-binding site thereof specifically binding human CD154 with SEQ ID NO: 1, containing a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 17 (SYGIS), HCDR2 with SEQ ID NO: 23 ( WISPIFGNTNYAQKFQG), HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), Light Chain Complementarity Determining Region (LCDR) 1 of SEQ ID NO: 37 (RASQSISSYLN), LCDR2 of SEQ ID NO: 44 (YANSLQS), and LCDR3 of SEQ ID NO: 52(QQSDSIPWT).

The invention also provides an isolated antagonist antibody specifically binding soluble human CD154 (shCD154) with SEQ ID NO: 4, containing HCDR1 with SEQ ID NO: 17 (SYGIS), HCDR2 with SEQ ID NO: 23 (WISPIFGNTNYAQKFQG), HCDR3 with SEQ ID NO: 30 (SRYYGDLDY), LCDR1 with SEQ ID NO: 37 (RASQSISSYLN), LCDR2 with SEQ ID NO: 44 (YANSLQS), and LCDR3 with SEQ ID NO: 52 (QQSDSIPWT).

In some embodiments, the antibody comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66.

In some embodiments, the antibody binds to shCD154 with a KD dissociation constant of about 5x10'9 M or less, about 1x10'9 M or less, about 5x10'1° M or less, about ^1x10'10 M or less, about 5x10'11 M or less, or about 1x10'11 M or less, with KD measured using a ProteOn XPR36 system using the experimental design described in Example 1 to measure affinity.

In some embodiments, the antibody inhibits shCD154 mediated human B cell proliferation with an IC ₅₀ value of about 7.0 x 10' ¹¹ M to about 6 x 10' ¹⁰ M.

In some embodiments, the antibody inhibits shCD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by an NF-kB inducible minimal interferon-β (IFN-β) promoter in HEK293 cells stably expressing SEAP and human CD40 with an IC ₅₀ value of about 2.1x10 '8 M or less.

In some embodiments, the antibody is of the IgG1 isotype, optionally containing heavy chain substitutions L234A, L235A, G237A, P238S, H268A, A330S, and P331S compared to wild-type IgG1.

In some embodiments, the antibody comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66, and is of the IgG1 isotype, optionally containing heavy chain substitutions L234A, L235A, G237A, P238S, H268A, A330S, and P331S compared to IgG1 wild type.

In some embodiments, the antibody comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66 and is an IgG1/K isotype containing heavy chain substitutions L234A, L235A, G237A, P238S, H268A, A330S, and P331S compared to IgG1 wild type.

In some embodiments, the antibody is of the IgG2 isotype, optionally containing heavy chain substitutions V234A, G237A, P238S, H268A, V309L, A330S, and P331S compared to wild-type IgG2.

In some embodiments, the antibody comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66 and is an IgG2/K isotype containing heavy chain substitutions V234A, G237A, P238S, H268A, V309L, A330S, and P331S compared to IgG2 wild type.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 80 and a light chain (LC) of SEQ ID NO: 81.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 80 and a light chain (LC) of SEQ ID NO: 81.

In some embodiments, the antibody is a bispecific antibody.

The antibody is suitable for use in therapy, for example in the treatment of an autoimmune disease.

The antibody is suitable for use in therapy, for example in the treatment of an immune-mediated inflammatory disease.

The antibody is suitable for use in therapy, for example in the treatment of arthritis.

The antibody is suitable for use in therapy, for example in the treatment of systemic lupus erythematosus

- 33 040943 ki.

The antibody is suitable for use in therapy, for example in the treatment of inflammatory bowel disease.

The antibody is suitable for use in therapy, such as transplant therapy.

The antibody is suitable for use in therapy, for example in kidney transplant therapy. The antibody is suitable for use in therapy, for example in skin graft therapy. The antibody is suitable for use in therapy, for example in bone marrow transplantation therapy.

The antibody is suitable for use in therapy, for example in the treatment of graft versus host disease.

The antibody is suitable for use in therapy, for example in the treatment of immune thrombocytopenia.

The antibody is suitable for use in therapy, for example in the treatment of multiple sclerosis.

The antibody is suitable for use in therapy, for example in the treatment of thyroiditis.

The antibody is suitable for use in therapy, for example in the treatment of type I diabetes.

The antibody is suitable for use in therapy, for example in the treatment of atherosclerosis.

The antibody is suitable for use in therapy, for example in the treatment of rheumatoid arthritis.

The antibody is suitable for use in therapy, for example in the treatment of Crohn's disease.

The antibody is suitable for use in therapy, for example in the treatment of ulcerative colitis.

The antibody is suitable for use in therapy, for example in the treatment of inflammatory bowel disease in combination with a second therapeutic agent.

The present invention will now be described with reference to the following non-limiting specific examples.

Example 1. Materials and methods.

Getting used proteins.

Because endogenous CD154 is involved in signal transduction as a trimer, recombinant CD154 has been expressed in various ways to produce a functional recombinant trimer. Soluble human CD154 (shCD154; SEQ ID NO: 4), soluble CD154 Callithrix jacchus (common marmoset; herein referred to as marmoset) (smCD154; SEQ ID NO: 5), or soluble CD154 Macaca fascicularis (cynomolgus monkey, herein referred to as cynomolgus monkey) (scCD154; SEQ ID NO: 6) were cloned and expressed as fusions to His6 (SEQ ID NO: 10) (shCD154-his, SEQ ID NO: 7; smCD154-his, SEQ ID NO: 8; scCD154- his, SEQ ID NO: 9) or as a leucine zipper (ILZ) fusion protein (SEQ ID NO: 11) (shCD154-ILZ, SEQ ID NO: 12; smCD154-ILZ, SEQ ID NO: 13; scCD154-ILZ , SEQ ID NO: 14). Cloning, expression and purification of proteins were performed using standard methods. Fusions to His and ILZ were predominantly trimers. The smCD154 and smCD154-ILZ proteins were biotinylated using EZLink™ sulfo-NHS-LC-Biotin and labeling kit (Thermo, cat. no. 21327), biotinylation success was analyzed using the HABA-avidin assay (Thermo, cat. no. 46610) and Octet. Cells expressing human CD40 (SEQ ID NO: 15) were used in some of the assays. Some assays used D1.1 Jurkat cells (ATCC® CRL-10915™) endogenously expressing human CD154.

Human CD154; SEQ ID NO: 1

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNL HEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIA AHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQA PFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGT GFTSFGLLKL

- 34 040943

CD154 marmosets; SEQ ID NO: 2

MIETYNQPVPRSAATGPPVSMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNL HEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEEKKKENSFEMQKGDQNPQIA AHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQA PFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGASVFVNVTDPSQVSHGT GFTSFGLLKL

CD154 cynomolgus monkey; SEQ ID NO: 3

MIETYNQPSPRSAATGLPVRMKIFMYLLTIFLITQMIGSALFAVYLHRRLDKIEDERNL HEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEEKKKENSFEMQKGDQNPQIA AHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQA PFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGT GFTSFGLLKL shCD154; SEQ ID NO: 4

SVFVNVTDPSQVSHGTGFTSFGLLKL smCD154; SEQ ID NO: 5

MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYI YAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGA SVFVNVTDPSQVSHGTGFTSFGLLKL

SCGD154; SEQ ID NO: 6

shCD154-his; SEQ ID NO: 7

HLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL smCD154-his; SEQ ID NO: 8

GSHHHHHHGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGK QLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSI HLGGIFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL scCD154-his; SEQ ID NO: 9

GSHHHHHHGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGK QLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSI

- 35 040943

His6; SEQ ID NO: 10

HHHHH

ILZ; SEQ ID NO: 11

RMKQIEDKIEEILSKIYHIENEIARIKKLIGER shCD154-ILZ; SEQ ID NO: 12

GSHHHHHHGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSMQKGDQNPQI AAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQ APFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDSFQVGLSHL TGFTCDPSQVGLSHL TGFTCDSFQVGLSHL TGFTCDPSQVGLSHL SEQ ID NO: 13

GSHHHHHHGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSMQKGDQNPQI AAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQ APFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGGIFELQPGASVFVNVTDSFQVGLSHL TGFTCDSFQVLSHL TGFTCDSFQVLSHL TGFTCDSFQVLSHL SEQ ID NO: 14

human SEQ ID NO: 15

MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETEC LPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHR SCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLWQQAGTNKTDWCG PQDRLRALWIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPV QETLHGCQPVTQEDGKESRISVQERQ

Affinity measurements.

Affinity measurements using surface plasmon resonance (SPR) were performed using a ProteOn XPR36 system (BioRad). A biosensor surface was prepared by binding an anti-human IgG Fc antibody (Jackson cat. no. 109-005-098) to an alginate polymer layer-modified GLC chip surface (BioRad, cat. no. 176-5011) using the manufacturer's instructions for amine coupling. Approximately 4700 RU (response units) of the test antibodies were immobilized. Kinetic experiments were performed at 25° C. in running buffer solution (DPBS+0.03% polysorbate P20+100 μg/ml BSA). To perform kinetic experiments, 100 RU of antibodies were captured, followed by injections of analytes (shCD154-his and smCD154-his) at concentrations ranging from 0.391 nM to 100 nM (in 4-fold serial dilution). The association phase was monitored for 3 min at 50 μl/min, followed by a 15 min buffer run (dissociation phase). The chip surface was regenerated with two 18 second pulses of 100 mM H ₃ PO ₄ (Sigma, cat. no. 7961) at 100 μl/min.

The obtained data were processed using the ProteOn Manager software. First, the data was corrected against the background using intermediate plots. Subsequently, a double standard subtraction was performed on the data using buffer injection instead of analyte injections. Kinetic data analysis was performed using a 1:1 Langmuir binding model.

CD154-induced activation of Ramos cells.

The ability of anti-CD154 antibodies to inhibit Ramos cell activation was assessed using CD54 as a cell activation marker. Ramos cells (Burkitt's lymphoma cells, ATCC® CRL-1596™), cultured according to the supplier's protocol, were plated in a 96-well V-bottom plate at a density of 2.0 x 10 ⁵ cells/well in complete growth medium at 100 μl/well . Test antibodies at concentrations of 0.2, 2 or 20 μg/ml were pre-incubated with 40 ng/ml smCD154his for 1 hour at room temperature (RT) and subsequently added to the cells. The tablet was covered and incubated overnight (37°C, 5% CO ₂ ). The next day, the assay plate was centrifuged and the depleted growth medium was removed. The resulting cell pellet was washed with cold PBS/2% FBS and subsequently the cells were stained with PE labeled anti-CD54 antibody (ICAM-1) or an appropriate isotype control for 1 h at 4°C. Cells were washed with cold PBS/2% FBS, resuspended in 100 μl/well of cold PBS/2% FBS and the fluorescent signal (yellow channel) measured on a flow cytometer. Antibodies were considered antagonists when the following criteria were met: % activity against antibody 5C8 > 5% activity against 5C8, where % activity is the normalized percentage of inhibition against 5C8 at the highest

- 36 040943 tested concentration.

Analysis of the NF-kB-SEAP reporter gene.

The ability of anti-CD154 antibodies to inhibit CD154-induced signaling pathways downstream of CD40 was assessed using HEK-Blue™ CD40L cells (Invivogen) engineered to express human CD40 and transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of inducible NF- kB promoter (minimal IFN-β promoter). Cells were stimulated with human or cynomolgus monkey CD154 or Jurkat cells. HEK-Blue™ CD40L cells were cultured according to the supplier's protocol and all activity assays were performed in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1X Glutamax. Cells were seeded in 96-well tissue culture plates at a cell density of 2.5 or 5x10 ⁴ cells per well in a volume of 100 μl and incubated overnight (37° C., 5% CO ₂ ). The next day, 4X solutions of shCD154-His or shCD154-ILZ or D1.1 Jurkat cells were pre-incubated with 4X solutions of anti-CD154 antibodies (at appropriate concentrations) in a 1:1 ratio to obtain 2X solutions of mixtures of CD154:antibody precomplexes. The CD154:antibody mixtures were incubated at RT for 1 h and the D1.1 Jurkat:mAb mixture was incubated at 37°C and 5% CO2 for 1 h. Blue™ CD40L, added 100 µl/well of 2X precomplex solutions; the final assay volume was 200 µl/well with final CD154 concentrations of 80 ng/ml shCD154-His or 40 ng/ml shCD154-ILZ, or 2.5-6.0x104 D1.1 Jurkat cells. After a 16-24 h treatment period (37° C., 5% CO2), supernatants were analyzed for phosphatase activity (SEAP) by measuring absorbance (650 nm) for supernatants injected at 40 µl/well and incubated with 160 µl/well QUANTI- Blue™ (Invivogen) at 37°C for 30-60.

Analysis of Jurkat cell-mediated activation of dendritic cells.

The ability of anti-CD154 antibodies to inhibit Jurkat-mediated dendritic cell (DC) activation was assessed by measuring the reduction in the production of various DC cytokines. Human monocytes (Biologic Specialties) were cultured with 50 ng/ml IL-4 and GM-CSF for six days. On day 3, the cells were replaced with fresh medium (with IL-4 and GM-CSF). Immature DC cells (iDC) (CD1a ⁺ CD14 ^low CD83 ^- ) were used in cell assays on day 6. 2.5x105 D1.1 Jurkat cells (irradiated at 1000 rad) were incubated with 0.000064-25 µg/ml µg/ml anti-CD154 antibody for 15-20 min and subsequently co-cultured with 2.5x10 ⁴ iDC in a final volume of 200 µl /well in a 96-well round bottom plate. After 48 hours of incubation, supernatants were collected for cytokine analysis.

Analysis of Jurkat Cell-Mediated B-Cell Activation.

The ability of anti-CD154 antibodies to inhibit Jurkat-mediated B cell activation was assessed by analyzing the effect of antibodies on B cell proliferation. 1x105 D1.1 Jurkat cells (irradiated at 5000 rad) were co-cultured with 1x105 B cells from human tonsils in the presence of IL21 (100 ng/ml) and 0.0077 ng/ml - 15 μg/ml anti-CD154 antibody in a final volume of 200 μl /well in a 96-well round bottom plate. After 2 days of incubation, methyl (-3H)thymidine (0.5 μCi/well) was added to the cultures and the proliferation of human B cells was determined after overnight incubation.

Analysis of CD154 mediated B cell activation.

The ability of anti-CD154 antibodies to inhibit recombinant CD154-mediated B cell activation was evaluated in human or cynomolgus monkey B cells. 1x105 B cells from human tonsils or cynomolgus spleen cells were cultured with 100 ng/ml rhIL-21, 0.5 µg/ml shCD154ILZ and 0.0077 ng/ml - 15 µg/ml anti-CD154 antibodies in a final volume of 200 µl/ well in a 96-well round bottom plate. After 2 days of incubation, methyl (-3H)-thymidine (0.5 μCi/well) was added to the cultures and the proliferation of human B cells was determined after overnight incubation.

Example 2 Isolation of Anti-CD154 Antibodies from Phage Display Libraries.

CD154 binding Fabs were selected from de novo generated pIX phage display libraries as described in Shi et al., J Mol Biol 397:385-96, 2010; International Patent Publication No. WO2009/085462; US Patent Publication No. US2010/0021477). Briefly, libraries were generated by diversifying human scaffolds in which the IGHV1-69*01, IGHV3-23*01, and IGHV5-51*01 germline VH genes were recombined with the human IGHJ-4 minigene via the H3 loop, and the human VL germline kappa genes were recombined. lines O12 (IGKV1-39*01), L6 (IGKV3-11*01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1 minigene to assemble the complete VH and VL domains. For diversification, the positions of the variable regions of the light and heavy chains near the H1, H2, L1, L2, and L3 loops were chosen, corresponding to the positions for which frequent contact with protein and peptide antigens was detected. Sequence diversification at selected positions was limited to residues appearing at each of the positions in the IGHV or IGLV germline gene families for the respective IGHV or IGLV genes. Diversification of the H3 loop was created using synthetic short or medium loops of 7-14 amino acids in length. The distribution of amino acids in H3 was performed similarly to the observed amino acid variation in human antibodies. Configuration

- 37 040943 libraries are described in detail in Shi et al., J Mol Biol 397:385-96, 2010. The scaffolds used to create the libraries were named according to their origin from the human VH and VL germline gene. Three heavy chain libraries were combined with four germline light chains or germline light chain libraries to create 12 unique VH:VL combinations for panning experiments on smCD154 or cells expressing full-length cynomolgus CD154.

Libraries were panned against either full-length cynomolgus CD154 (SEQ ID NO: 3) stably expressed in CHO cells or biotinylated and non-biotinylated smCD154 (SEQ ID NO: 5). After several cycles of panning, ELISA was performed with polyclonal phages using smCD154 as antigens to detect specific enrichment in separate panning experiments. Phages harvested from these panning experiments, which showed enrichment for smCD154 binders, were further screened with monoclonal Fab ELISA, in which Fab proteins expressed from individual Fab clones were used as binders of non-biotinylated smCD154 binders applied directly to the plate. Fab clones for which the binding signal was four times that of the negative control Fab were selected for screening in full IgG format. Selected Fabs were cloned into the IgG2sigma/κappa framework and further characterized for binding to D1.1 Jurkat cells. IgG2sigma is a muted Fc and has substitutions V234A, G237A, P238S, H268A, V309L, A330S and P331S compared to wild-type IgG2. A description of IgG2sigma is provided in US Pat. No. 8,961,967.

Example 3 Obtaining antibodies to CD154 in rats.

Anti-CD154 antibodies were generated using transgenic rats expressing human immunoglobulin loci, OmniRat®; OMT Inc. The endogenous immunoglobulin loci in OmniRat® are replaced by human IgK and Igλ loci, and the chimeric human/rat IgH locus with V, D and J segments of human origin is linked to the rat CH locus. The IgH locus contains 22 human VHs, all of the human D and J _H segments in natural configuration are linked to the rat CH locus. The preparation and characterization of OmniRat® rats is described in Osborn, et al. J Immunol 190: 1481-1490, 2013; and International Patent Publication No. WO2014/093908.

OmniRat® rats were immunized with smCD154 using a repeat immunization at multiple sites (RIMMS) protocol. After a 45 day immunization schedule, lymph nodes were harvested from all four rats and used to generate hybridomas. Hybridoma supernatants in 96-well plates were screened by binding in solid phase ELISA to detect mAbs exhibiting binding to smCD154, from which hybridoma supernatants showing a signal greater than 3 times the mean signal of the negative control were selected.

The selected antibodies were cloned as full length IgG2sigma/λ. Antibodies showing antagonistic activity against CD154-induced activation of Ramos cells were selected for further characterization.

Example 4 Characterization of antibodies.

Several anti-CD154 antibodies derived from phage display or transgenic animals expressing human immunoglobulin loci exhibiting antagonistic activity as described in Examples 2 and 3 were sequenced and further characterized for binding to human and cynomolgus dendritic cells, for their ability to inhibit functions human and cynomolgus monkey dendritic cells and B cells, and on the effector functions of antibodies. The VH and VL regions of the antibodies were sequenced using standard methods.

In table. 2 shows the HCDR1 amino acid sequences of selected antibodies.

In table. 3 shows the HCDR2 amino acid sequences of selected antibodies.

In table. 4 shows the HCDR3 amino acid sequences of selected antibodies.

In table. 5 shows the LCDR1 amino acid sequences of selected antibodies.

In table. 6 shows the LCDR2 amino acid sequences of selected antibodies.

In table. 7 shows the LCDR3 amino acid sequences of selected antibodies.

In table. 8 shows the VH amino acid sequences of selected antibodies.

In table. 9 shows the VL amino acid sequences of selected antibodies.

- 38 040943

table 2 mAb ID HCDR1 Subsequence SEQID NO: C4LB5 SYAIS 16 C4LB89 SYGIS 17 C4LB94 SYAIS 16 C4LB150 SYSFYWG 18 C4LB189 AYYIH 19 C4LB191 DYYIH 20 C4LB199 SFIYYWG 21

Table 3

mAb ID HCDR2 Subsequence SEQID NO: C4LB5 GIIPIFGTANYAQKFQG 22 C4LB89 WIS PIFGNTNYAQKFQG 23 C4LB94 GIS PYFGNTNYAQKFQG 24 C4LB150 SLYYSGSTYYNPSLKS 25 C4LB189 RINPDSGGTDYAQRFQG 26 C4LB191 RFNPNSGDTNGAQKFQG 27 C4LB199 CIYSSGGTYYNPSLKS 28

Table 4

mAb ID HDDR3 Subsequence SEQID NO: C4LB5 GASVWDGPAEVFDY 29 C4LB89 SRYYGDLDY thirty C4LB94 DTGWVGAFYLDY 31 C4LB150 LQLGTTTDYFDH 32 C4LB189 DWNYYDGSGYFGPGYYGLDV 33 C4LB191 EGELAGIFFDY 34 C4LB199 LWLGTTTDYFDY 35

Table 5

mAb ID LCDR1 Subsequence SEQID NO: C4LB5 KSSQSVLASSNNENFLA 36 C4LB89 RASQSISSYLN 37 C4LB94 KS SQSVLYS SNNKNYLA 38 C4LB150 SGDELGDKFAC 39 C4LB189 SGDKLGDKYVC 40 C4LB191 SGDKLGDKYVS 41 C4LB199 SGDKLGDKFAC 42

Table 6

mAb ID LCDR2 Subsequence SEQID NO: C4LB5 SASTRES 43 C4LB89 YANSLQS 44 C4LB94 WASTRES 45 C4LB150 QENKRPS 46 C4LB189 QDRKRPS 47 C4LB191 QDRKRPS 47 C4LB199 QDDKRPS 48

-39040943

Table 7

mAb ID LCDR3 Subsequence SEQID NO: C4LB5 QQAYTTPFT 51 C4LB89 QQSDSIPWT 52 C4LB94 QQYYSTPLT 53 C4LB150 QAWDSDTAV 54 C4LB189 QAWDSGTW 55 C4LB191 QAWDSSTW 56 C4LB199 QAWDSNTW 57

Table 8 mAb ID Name vh vh Subsequence SEQ ID NO: C4LB5 C4LH12 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCARGASVWDGPAEVFDYWGQGTLVTVSS 58 C4LB89 C4LH165 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLE WMGWISPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCARSRYYGDLDYWGQGTLVTVSS 59 C4LB94 C4LH99 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE WMGGISPYFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCARDTGWVGAFYLDYWGQGTLVTVSS 60 C4LB150 C4LH201 QLQLQESGPGLVKPSETLSLTCTVSGGSISSYSFYWGWIRQPPGQG LEWIGSLYYSGSTYYNPSLKSRATMSWTSKTQFSLNLNSVTAADT 61 AVYYCARLQLGTTTDYFDHWGQGTLVTVSS C4LB189 C4LH240 QVQLVQ S GAEVKKРGAS VKVS CKAS GYT FAAYYIHWVRQAP GQGLE WMGRINPDSGGTDYAQRFQGRVTMTRDTSISTAYMELSRLRSDDTA VFYCARDWNYYDGSGYFGPGYYGLDVWGQGTTVTVSS 62 C4LB191 C4LH242 Q VQ LVQ S GAEVKK P GASMKVS C KAS GYT FT DYYIHWVRQAP GQGLE WVGRFNPNSGDTNGAQKFQGRVTMTRDTSISTAYMELTRLRSDDTA VYHCAREGELAGIFFDYWGQGTLVTVSS 63 C4LB199 C4LH250 QVQLQESGPGLVKPSETLSLTCTVSGDSISSFIYYWGWIRQPPGKG LDWVGCIYSSGGTYYNPSLKSRVTISVDTSKNQFSLKLPSVTAADT AVYYCARLWLGTTTDYFDYWGQGTLVTVSS 64

Table 9

mAh ID VL VL Subsequence SEQ ID NO: C4LB5 C4LL8 DIVMTQSPDSLAVSLGERATINCKSSQSVLASSNNENFLAWYQQKP GQPPKLLLYSASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQAYTTPFTFGQGTKVEIK 65 C4LB89 C4LL49 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL LIYYANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSD SIPWTFGQGTKVEIK 66 C4LB94 PH9L2 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKP GQPPKLLLYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSTPLTFGQGTKVEIK 67 C4LB150 C4LL82 SYELTQPPSVSVSPGQTASITCSGDELGDKFACWYQQKPGQSPVLV IWQENKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDS DTAVFGGGTKLTVL 68 C4LB189 C4LL116 SYELTQPPSVSVSPGQTASVTCSGDKLGDKYVCWYQRKPGQSPVLV IYQDRKRPSGIPERFSGSNSGNTATLTISGTQAIDEEADYYCQAWDS GTWFGRGTKLTVL 69 C4LB191 IAPL39 SYELTQPPSVSVSPGQTASITCSGDKLGDKYVSWNHQKPGQSPVLV IYQDRKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDS STWFGGGTKLTVL 70 C4LB199 C4LL125 SYELTQPPSVSVSPGQTVSITCSGDKLGDKFACWYQQKPGQSPVW IYQDDKRPSGIPERFSGSTSGNTATLTISGTQAMDEADYYCQAWDS NTWFGGGTKLTVL 71

- 40 040943

Antibodies inhibited both the function of endogenous CD 154 on Jurkat cells (Dl.l Jurkat cells in Table 10) and the recombinantly expressed human CD 154 trimer (expressed as shCD154-ILZ or shCD154-his) as measured in the reporter gene assay NF-kV SEAP. The antibodies inhibited signaling with IC ₅₀ values in the range of 0.08-21.15 nM. Values IC ₅₀ for selected antibodies in this analysis are shown in table. 10. The IC ₅₀ values in the table for each antibody represent the highest and lowest IC ₅₀ values obtained in separate experiments, with a single value indicating that the antibody was tested in a single experiment or there is only one valid IC ₅₀ value.

Table 10

mAb ID Analysis of the NF-kB SEAP reporter gene; _IC50 (hM) Dl.l. Jurkat* shCD154-ILZ* shCD154-his* C4LB5 1.55 0.54 1.03-1.37 C4LB89 0.08-0.32 1.91-2.44 3.93-5.69 C4LB94 0.13 4.37 7.57-21.15 C4LB150 3.57 4.95 8.87-9.81 C4LB189 2.06 1, 63 6.51-13.60 C4LB191 2.19-3.46 C4LB199 1.21 1, 09 2.00-2.70

*CD154 format used for alarm induction

The ability of antibodies to inhibit dendritic cell activation was assessed using IL-12p40 secretion as a marker of DC activation. The antibodies inhibited DC activation induced by endogenous CD 154 on Jurkat cells with IC ₅₀ values ranging from 0.02 to 0.49 nM. In table. 11 shows the IC ₅₀ values obtained from the analysis of selected antibodies. The 1C ₅₀ range in the table represents the smallest and largest 1C ₅₀ values obtained in separate experiments on 2-4 donors in 1-6 replicates.

Table 11

mAb ID Analysis of Jurkat cell-mediated activation of dendritic cells; 1С ₅₀ (nM) C4LB5 0.32-0.49 C4LB89 0.02-0.09 C4LB94 0.07-0.09 C4LB150 0.25-0.30 C4LB189 0.15-0.16 C4LB191 0.38-0.39 C4LB199 0.19-0.20

The ability of antibodies to inhibit human or cynomolgus B cell activation was measured using B cell proliferation as an indicator. The antibodies inhibited proliferation induced by both endogenous CD 154 (Dl.l Jurkat cells) and recombinant human CD 154 trimer (shCD154-ILZ) with IC ₅₀ values ranging from 0.01-5.35 nM. In table. 12 shows IC ₅₀ values for various antibodies obtained from human or cynomolgus B cell activation assays. The 1C ₅₀ range in the table represents the smallest and largest 1C ₅₀ values obtained in separate experiments on 2-4 donors in 1-6 repetitions. The only 1C ₅₀ value in the table indicates the presence of one valid 1C ₅₀ value.

Table 12

mAb ID Analysis of B-cell activation; 1С ₅₀ (nM) Dl.l Jurkat Cells/Human Cells* shCD154ILZ/human B cells* shCD154-ILZ/B cells of the cynomolgus macaque* C4LB5 0.19-0.28 2.74 5.35 C4LB89 0.01-0.02 0.20-0.48 0.13-0.44 C4LB94 0.01-0.03 0.07-0.14 0.30-0.31 C4LB150 0.27-0.31 HB 1.14-2.00 C4LB189 0.47-0.65 HB 4.35-5.16 C4LB191 0.64-1.00 HB 0.25-0.36 C4LB199 0.04-0.27 HB 1.89-2.31

HB: not performed *CD154 format used for signaling induction/source of B cells

Example 5 Design of antibodies to minimize the risk of post-translational modification.

The VL of the C4LB89 antibody contained a putative deamination site in LCDR2 (N52-S53 in the C4LL49 light chain, SEQ ID NO: 66). Individual substitutions were made in each position (N52S and

-41 040943

S53T) to VL. The mutated light chains were co-expressed with the parent C4LH165 heavy chain (SEQ ID NO: 59) to generate C4LB235 and C4LB236 antibodies as IgG2sigma/K. The amino acid sequences of LCDR2 and VL from C4LB235 and C4LB236 are shown in Table 1. 13 and tab. 14, respectively. C4LB235 contains HCDRs of SEQ ID NOs: 17, 23, and 30, LCDRs of SEQ ID NOs: 37, 49, or 52, VHs of SEQ ID NOs: 59, and VLs of SEQ ID NOs: 72. C4LB236 contains HCDRs of SEQ ID NOs: 17, 23 and 30, LCDR with SEQ ID NO: 37, 50 or 52, VH with SEQ ID NO: 59 and VL with SEQ ID NO: 73.

Both antibodies were tested for their ability to inhibit the proliferation of cynomolgus monkey B cells. C4LB235 showed similar potency to C4LB89 and C4LB236 showed reduced activity compared to C4LB89.

Example 6 Anti-CD154 antibodies with muted effector function do not induce platelet activation.

Anti-CD154 antibodies have been clinically investigated with positive results in patients with autoimmune diseases, but due to cases of thromboembolism (TE), further clinical development of these antibodies has been halted. The humanized antibody 5c8 (IgG1/K) is an anti-CD154 antibody that has resulted in TE in the clinic (Yazdany et al., Lupus 13:377-380, 2004). It has been hypothesized that thromboembolism (TE) mediated by humanized 5c8 is the result of platelet activation and aggregation due to the formation of higher order anti-CD154/CD154 antibody immune complexes (IC) that cross-link platelets by binding Fc to the platelet FcYRIIa receptor. In vitro, the Fc-mutated engineered 5c8 antibody (D265A substitution in IgG1) that does not bind the FcYRIIa receptor does not activate platelets (Xie et al., J Immunol 192:4083-4092, 2014).

Anti-CD154 antibodies having impaired binding to at least FcYRIIa and having reduced effector functions may thus be a more acceptable therapeutic agent with a reduced risk of developing TE.

To this end, anti-CD154 antibodies with silted effector function were obtained, having various substitutions in Fc, and tested in terms of their effect on platelet activation.

The VH and VL of the humanized 5c8 antibody (Karpusas et al., Structure 9: 321-329, 2001) were cloned as IgG1sigma/K, IgG1sigmaYTE/K, IgG2sigma/K, or IgG2sigmaYTE/K to evaluate the effect of Fc on platelet activation; the resulting antibodies were named 5c8IgG1sigma, 5c8IgG1sigmaYTE, 5c8IgG2sigma and 5c8IgG2sigmaYTE). IgG1sigma contains substitutions L234A, L235A, G237A, P238S, H268A, A330S and P331S compared to wild-type IgG1. IgG1sigmaYTE contains substitutions L234A, L235A, G237A, P238S, M252Y, S254T, T256E H268A, A330S and P331S. IgG2sigma contains substitutions V234A, G237A, P238S, H268A, V309L, A330S and P331S. IgG2sigmaYTE contains substitutions V234A, G237A, P238S, M252Y, S254T, T256E, H268A, V309L, A330S and P331S. The numbering of residues corresponds to the EU index. IgG2sigma backbone antibodies lack effector functions and FcYR binding as described in US Pat. No. 8,961,967. The YTE substitution (M252Y, S254T, T256E) is described in Dall'Acqua et al., J Biol Chem 281:23514-24, 2006.

The VH and VL domains of humanized 5c8 are described in SEQ ID NOs: 74 and 75, respectively.

SEQ ID NO: 74

QVQLVQSGAEWKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTN

FNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS

SEQ ID NO: 75

DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLE

SGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIK

- 42 040943

5c8IgG1sigma, 5c8IgG1sigmaYTE, 5c8IgG2sigma and 5c8IgG2sigmaYTE were studied for effects on platelet activation.

Blood from healthy human donors pre-screened for low response to shCD154 only was used. Platelet activation was studied by flow cytometry using validated platelet activation markers PAC-1 (activated GPIIb/IIIa) and CD62p (P-selectin). Briefly, whole blood (WB) was added to a modified Tyrodes-HEPES buffer solution containing 1 mM CaCl ₂ , and anti-PAC1 and anti-CD62p antibodies with or without FcYRIIa blocking antibody (clone IV.3, StemCell Technologies, no. 60012) were added to the mixture. ), after which it was incubated for 25 minutes. Preformed immune complexes of soluble CD154 (PeproTech cat. no. 310-02; SEQ ID NO: 4 or Tonbo Biosciences cat. no. 217088)/antibody with a 3:1 molar ratio of CD154 and anti-CD154 antibody were added to the mixture and incubated for an additional 20 minutes; platelets were fixed in 1% formalin followed by FACS analysis. Platelet activation for each condition was assessed as the % proportion of selected platelets (CD61-positive events) expressing PAC-1 and CD62p; for each treatment condition, 5000 CD61 (platelet) expression events were recorded and analyzed. The results of the experiment are shown in Fig. 1. CD154/5c8IgG1 IC (wild-type IgG1) activated platelets, while CD154 IC with 5c8-IgG1sigma, 5c8IgG1sigmaYTE, 5c8IgG2sigma and 5c8IgG2sigma-YTE did not activate platelets. Platelet activation by ADP was not inhibited by immune complexes (data not shown). None of the antibodies alone activated platelets (data not shown).

Anti-CD154 antibodies C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191, C4LB199 (all IgG2sigma mAbs with silenced effector function) were also tested in a platelet activation assay to confirm that the antibodies had lost FcYRIIa binding and did not activate platelets. In FIG. 2 shows experimental results demonstrating that CD154 immune complexes complexed with C4LB5, C4LB89, C4LB150, C4LB189, C4LB191, or C4LB199 did not activate platelets. IC CD154/C4LB94 induced PAC-1 on platelets. The results demonstrate that anti-CD154 antibodies with IgG1sigma, IgG1sigmaYTE, IgG2sigma or IgG2sigmaYTE isotypes generally cannot activate platelets and therefore may have an improved safety profile relative to wild-type IgG1 antibodies.

Example 7 Effect of Isotype Switching on Antibody Properties.

The variable regions of the C4LB89 antibody were cloned as IgG1sigma/K and IgG1sigmaYTE isotypes to evaluate potential differences in functionality and opportunities for development. The new antibodies were named C4LB231 (IgG1sigma) and C4LB232 (IgG1sigmaYTE).

The resulting IgG1sigma and IgG1sigmaYTE antibodies were compared with the parent antibody in terms of functionality. Antibodies C4LB231 and C4LB232 were comparable in function to the parental C4LB89. In table. 15 shows the IC50 values or range of _IC50 values for each antibody in the functional assays shown in the table. The IC ₅₀ range in the table represents the smallest and largest IC ₅₀ values obtained from experiments with 2-4 donors in 1-6 replicates. A single IC ₅₀ value in the table indicates the presence of one valid IC ₅₀ value.

Table 15

Analysis CD154 format used for signaling induction mAb C4LB231 C4LB232 NF-kB SEAP reporter gene analysis; 1С ₅₀ (nM) Dl.l Jurkat 0.27 NF-kB SEAP reporter gene analysis; 1С ₅₀ (nM) shCD154-ILZ 1.21 1.25-1.45 NF-kB SEAP reporter gene analysis; 1С ₅₀ (nM) shCD154-his 2.15 1.77-1.99 Analysis of Jurkat cell-mediated activation of dendritic cells; IL-12p40 1C ₅₀ (nM) DI.1 Jurkat 0.02-0.04 0.03-0.03 proliferation of human B cells, 1C ₅₀ (nM) DI.1 Jurkat 0.01-0.01 0.01-0.01 proliferation of human B cells, 1C ₅₀ (nM) shCD154-ILZ 0.38-0.67 0.42-0.74 cynomolgus monkey B cell proliferation, 1C ₅₀ (nM) shCD154-ILZ 0.25-0.55 0.20-0.55

C4LB231 and C4LB232 have also been tested for their effects on platelets. Neither IC shCD154:C4LB231 nor IC shCD154:C4LB232 activated platelets from baseline. IC CD154/5c8IgG1 activated platelets and activation was blocked in the presence of IV.3, and

- 43 040943 this shows that platelet activation is mediated by IC binding to FcyRIIa. In FIG. 3 shows the results of the experiment.

Example 8 Anti-CD154 antibodies bind human CD154 with high affinity.

Affinity measurements were performed using ProteOn as described in Example 1. Association rate, dissociation rate, and affinity values are shown in Table 1. 16. The parameters shown in this table are derived from a 1:1 Langmuir binding model for all samples except C4LB94 and C4LB150, which are approximated by a two-state binding model.

Table 16

Sample ka (1/ms) kd (1/s) K _D (M) C4LB5 5.70E+05 1.78E - 04 3.12E - 10 C4LB89 1.61E+06 3.80E - 04 2.35E - 10 C4LB94 1.58E+06 3.29E - 03 2.09E - 09 C4LB150 2.27E+06 6.17E - 03 2.72E - 09 C4LB189 3.55E+05 2.06E - 04 5.81E - 10 C4LB191 5.62E+0 6 1.76E - 04 3.13E - 11 C4LB199 1.60E+0 6 4.04E - 04 2.53E - 10

Example 9 Crystal structure of marmoset CD154 complexed with C4LB89.

The epitope of the C4LB89 antibody was determined by X-ray crystallography. His-tagged C4LB89 Fab fragment and His-tagged soluble marmoset CD40L form (smCD154-his) were expressed in HEK293 GnTI cells and purified using affinity chromatography and size exclusion chromatography. The smCD154:C4LB89 complex was incubated overnight at 4°C, concentrated and separated by size exclusion chromatography. The complex was crystallized from a solution containing 16% PEG 3350, 0.2 M ammonium citrate, 0.1 M MES, pH 6.5 by vapor diffusion. The crystals belonged to the cubic space group P2i3 with a unit cell size of 162.1 A. The structure of the complex was determined by the molecular substitution method using C4LB89 Fab and CD40L crystal structures as search models (PDB entry identifier 1ALY).

The smCD154:C4LB89 complex is a symmetrical trimer located on the 3rd order crystallographic symmetry axis. C4LB89 binds mCD154 at the interface between the two subunits at an epitope distal to the cell surface. The epitope contains 16 residues, 8 from each of the two CD154 subunits. The epitopic residues are E182, S185, Q186, A187, P188, S214, A215 and R207 in the first subunit of CD154 and T176, F177, C178, Q220, S248, H249, G250 and F253 in the second subunit of CD154. Epitope residue numbering corresponds to full-length human CD154 of SEQ ID NO: 1. A paratope is defined as antibody residues within 4 A of CD154 residues. The C4LB89 paratope includes 9 residues from the C4LB89 heavy chain: S31 and Y32 from HCDR1, S52, I54, F55 and N57 from HCDR2 and R100, Y101 and Y102 from HCDR3. The numbering of the paratope residues corresponds to VH C4LB89 with SEQ ID NO: 59. The light chain is not involved in contacts with mCD154. Given the number of contacts, F55 in HCDR2 is a key element for antigen recognition. F55 makes contact with CD154 residues T176, F177, C178, Q220, S248, H249, G250 and F253. HCDR3 residues Y101 and Y102 are also involved in binding. In FIG. 4 shows contact residues of HCDR2 and HCDR3 and no LC binding to CD154. In FIG. 5 shows in drawing the remains of an epitope and a paratope.

The soluble CD154 proteins of humans and marmosets differ by only 8 amino acid residues. All human mCD154 C4LB89 epitope residues are conserved. Therefore, the epitope is expected to be conservative when comparing human and marmoset CD154. The alignment of full-length human and marmoset CD154 proteins is shown in FIG. 6.

Example 10 Platelet activation by anti-CD154 antibodies is epitope dependent.

The C4LB89 variable regions (VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66) were cloned as IgG1/K to give the C4LB237 antibody. C4LB237 was confirmed to retain binding to human CD154 (Table 17) as measured using ProteOn as described in Example 1. The affinity of C4LB237 for human CD154 was 23.6 ± 5.4 pM. It turned out that switching the isotype of VH/VL derived from C4LB89 from IgG2sigma to IgG1 changed the binding affinity of the resulting antibodies.

As expected, C4LB237 bound human FcyRIIa and FcyRIIIa with KDs of 0.994 μM and 0.146 μM, respectively. C4LB237 showed activity similar to C4LB231 and higher than C4LB89 in NF-KB SEAP reporter gene assays when shCD154-his was used to induce signaling (Table 18).

C4LB237 was tested for an effect on platelet activation. IC shCD154:C4LB237 did not activate platelets above baseline as shown in FIG. 7. This result indicates that in addition to Fc, the ability of the antibody, or lack of it, to activate platelets is affected by the epitope of the antibody.

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Table 17

Sample ka (1/Ms) 10 ⁶ kd (1/s) 10~ ⁰⁵ K _D (pM) C4LB231 (n=8) 2.53±0.15 7.81±0.69 31.0±3.3 C4LB232 (n=8) 2.54±0.15 8.89±0.91 35.2±5.2 C4LB237 (n=4) 2.59±0.20 6.05±0.98 23.6±5.4

Table 18

mAb 1С ₅₀ (nM) 95% CI 1C ₅₀ (nM) C4LB231 2.32 2.11-2.55 C4LB237 2.56 2.43-2.70 C4LB89 6.22 5.27-7.34

Example 11 Introduction of neutral mutations in C4LB89.

Crystal structure analyzes of C4LB89 complexed with CD 154 revealed positions in the CDR of C4LB89 that could be mutated without affecting the overall structure of the complex and would therefore not be expected to affect the properties of the C4LB89 antibody. These neutral mutations in the light chain CDRs are listed in Table 1. 19, and neutral mutations in the CDR of the heavy chain - in table. 20. The numbering of residues that can be mutated is shown both on individual CDRs and on VL or VH. For example, the Q4 residue in LCDR1 with SEQ ID NO: 37 (RASQSISSYLN) can be mutated into A, C, D, E, F, G, Η, I, K, L, Μ, N, R, S, Τ, V, W or Y, and expect that the characteristics of the antibody will not change substantially. The corresponding residue in VL of SEQ ID NO: 66 is Q27.

Mutations shown in Table. 19 or tab. 20 are made in C4LB89 singly or in combination using standard methods. The resulting VH/VL pairs were expressed, and mutated antibodies were isolated and characterized using the methods described herein.

Table 19

LCDR C4LB89 Remainder LCDR C4LB89 VL C4LB89 (SEQ ID NO: 66) Possible replacements LCDR1 SEQ ID NO:37 (RASQSISSYLN) Q4 Q27 A, C, D, E, F, G, Η, I, K, L, M, N, R, S, T, V, W, Y S5 S28 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y S7 S30 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y S8 S31 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y CDR2 with SEQ ID NO:44 (YANSLQS) A2 A51 S N3 N52 A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, Y S4 S53 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y L5 L54 A, C, D, E, F, G, Η, I, K, Μ, N, Q, R, S, T, V, W, Y Q6 Q55 E, D, N S7 S56 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y LCDR3 with SEQ ID NO: 52 (QQSDSIPWT) S3 S91 A D4 D92 N S5 S93 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y 16 194 A, C, D, E, G, K, L, M, N, Q, R, S, T, V

-45040943

Table 20

HCDR C4LB89 Remainder HCDR C4LB89 VH C4LB89 (SEQ ID NO: 59) Remainder position Possible replacements HCDR1 SEQ ID NO: 17 (SYGIS) SI S31 A, C, D, E, G, I, K, L, M, N, Q, R, T, V 14 134 M, L, V S5 S35 A HCDR2 with SEQ ID NO: 23 (WISPIFGNTNYAQKF QG) S3 S52 A, T, V 15 154 V, T, L Q, E N8 N57 A, C, D, E, F, G, Η, I, K, L, M, Q, R, S, T, V, W, Y T9 T58 A, C, D, E, F, G, Η, I, K, L, M, N, Q, R, S, V, W, Y N10 N59 A, C, D, E, F, G, Η, I, K, L, M, Q, R, S, T, V, W, Y HDDR3 with SEQ ID NO: 30 (SRYYGDLDY), SI S99 A, M R2 R100 A, S, Q, K R7 L105 M

Example 12 Evaluation of Platelet Activation and Higher Order Immune Complex Formation.

When evaluating the crystal structure data of the Fab region and shCD154 region complex for earlier experiments with C4LB231 and 5C8IgG1 using gel filtration high performance liquid chromatography (GP-HPLC) and dynamic light scattering (DLS), as well as studying data on platelet activation, a hypothesis was put forward that small differences in the binding of anti-CD154 antibodies to the shCD154 trimer may facilitate the formation of a higher order immune complex: 1) Fab C4LB231 binds between 2 subunits of the sCD154 trimer, and Fab 5C8 binds 1 subunit of SCD154, 2) Fab C4LB231 is more rigid in this conformation, than Fab 5c8 3) and the binding angle of anti-CD154 antibody to SCD154.

To further evaluate the role of the antibody epitope mediating platelet activation and higher order immune complex formation with shCD154, VH and VL of various anti-CD154 antibodies were cloned and expressed as Fc-muted IgG2sigma and IgG1sigma isotypes or as IgG1. Created antibodies are presented in table. 21. Platelet activation assays were performed as described in Example 6. For evaluations in higher order immune complex formation, gel filtration high performance liquid chromatography (GP-HPLC) and dynamic light scattering (DLS) methods were used to obtain complexes of antibodies with shCD154 in molar ratios of 1: 1 (also referred to as 10:10) and 10:1 in order to detect the presence of concentration-dependent differences in the formation of immune complexes.

Table 21

Name of the antibody Isotype VH SEQ ID NO: VL SEQ ID NO: 5C8IgG2sigma (C4LB71) IgG2n 74 75 5C8IgGl (MSCB8) IgGl C4LB89 IgG2a 59 66 C4LB231 IgGla C4LB237 IgGl C4LB119 IgG2a 84 85 C4LB290 IgGla C4LB287 IgGl C4LB83 IgG2n 86 87 C4LB288 IgGl C4LB94 IgG2a 60 67 C4LB234 IgGln C4LB289 IgGl IgG2n: IgG2sigma IgGla: IgGlsigma

- 46 040943

SEQ ID NO: 84 VH C4LB119

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYISWVRQAPGQGLEWMGAIDPYFGYAN

YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTGLNYGGFDYWGQGTLVTVSS

SEQ ID NO: 85 VL C4LB119

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP

ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK

SEQ ID NO: 86 VH C4LB83

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWIIPIFGNTN

YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREKDFRGYTKLDYWGQGTLVTVSS SEQ ID NO: 87 VL C4LB83

DIQMTQSPSSLSASVGDRVTITCRASQSINNWLNWYQQKPGKAPKLLIYAASSLQSGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSFPYTFGQGTKVEIK

For C4LB83 and C4LB119 binding characteristics and functionality were evaluated using the protocols described in example 1, and the data are shown in table. 22.

Table 22

Antibody ka (1/ms) kd (1/s) K _D (M) B Cell Activation Assay, 1C ₅₀ (nM) * C4LB83 2.10E+06 4.70E - 03 2.23E - 09 0.64-1.09 C4LB119 1.84E+06 8.80E - 03 4.79E - 09 0.81-2.03 *shCD154-ILZ induced human B cell activation. The range shows the smallest and largest values of 1C ₅₀ obtained in separate experiments on 2 donors

Methods GF-HPLC.

Immune complexes of the antibodies indicated in table. 21 and Alexafluo-448 labeled shCD154 were prepared in 1:1 and 10:1 molar ratios of antibody and shCD154 in 110 µl of 1x PBS and incubated at 37°C for 30 min. For each run, 100 μl of each sample containing 5 μg of AF488 labeled shCD154 and 15 μg (1:1 ratio) or 150 μg (10:1 ratio) of anti-CD154 mAb was injected into the column (Agilent, 1100/1200 system). Molecular weight was determined based on the retention time of the standards (Bio-Rad). The relationship between molecular weight and protein retention time satisfies the following equation:

log (M) - b - c T, where M is the molecular weight, T is the retention time, and b and c are constants. A linear curve of log(M) versus T was obtained from the GP-HPLC chromatogram of the standards with a least squares fit with R ² =0.9928.

DLS.

DLS measurement is based on the principles of light scattering and molecular or Brownian motion. Brownian motion, the characteristic motion of molecules in solution, causes light to scatter both in and out of phase, resulting in amplifying and attenuating interference. The result is that the scattered light intensity fluctuates with time. In DLS or QELS (Quasi-Electric Light Scattering) analysis, this time-dependent fluctuation in scattered light intensity is captured by a fast photon counter. The fluctuations are directly proportional to the diffusion rate. Solving the correlation data using the Stokes-Einstein equation gives the hydrodynamic radius. Unlike SEC-MALS or SEC methods, where the degree of sample discrimination allows distinguishing between monomer and dimer, DLS only distinguishes substances that differ by a size factor of ~4, and therefore discrimination will start from the monomer and tetramer; although monomer and dimer are indistinguishable, a weighted average of the mixture will be determined.

Stokes - Einstein equation: ^R h=kT/ 6πηϋ;

where D=diffusion coefficient, k=Boltzmann constant,

T=temperature,

P=viscosity.

Individual antibodies at a concentration of 10 μm or immune complexes of the antibodies shown in table. 21 and shCD154 was prepared at 10:1 or 10:10 molar concentrations of antibody and shCD154 (10 μM antibody per 1 μM or 10 μM shCD154) in PBS. All samples were prepared in glass vials with vial inserts (250 μl deactivated glass with polymer foot, Agilent cat. no. 5181-8872) and the samples were added in the following order: PBS, mAb, shCD154. Samples were mixed by gentle shaking at RT, shaking (Nutating Mixer (VWR) ~ 30 rpm for approximately 23 hours. If necessary, antibodies were first concentrated by standard methods.

- 47 040943

The particle size and distribution of the substance in all samples was determined on a flatbed scanner DynaPro Plate Reader DLS (Wyatt Technologies Corporation) at 23°C. DLS validity was first validated with BSA (data not shown). Measurements were performed by injecting 30 μl of sample into each of 3 wells (triple measurements). DLS measurements were carried out on a DynaPro flatbed scanner. Twenty 5 second readings were taken for each sample and the instrument performed automatic laser power adjustments. The parameters used in data analysis were the viscosity at 23°C for PBS, which was 1.019 cP, and the value of the refractive index at 589 nm and 23°C for PBS, which was 1.333. The program used the globular protein model. The signals were combined into peaks where peak 1 was 0.1-10 nm, peak 2 was 10-100 nm, peak 3 was 100-1000 nm and peak 4 was 1000-5000 nm. Recorded visible precipitation in the sample (if any). Data analysis was performed using Dynamics software (Wyatt Technology Inc.). Build graphs of the percentage of the mass relative to the radius of the particles (Rh). Peak radius, polydispersity, weight percentage and intensity percentage were calculated and recorded.

Results. platelet activation.

The ability of shCD154 immune complexes and antibodies expressed as Fc-muted IgG2sigma or IgG1sigma or wild-type IgG1 to activate platelets was assessed. The 5c8 antibody cloned on different IgG scaffolds was used as a control. In FIG. 3 shows that the 5c8IgG1:shCD154 immune complexes activated platelets in an FcyRIIa-dependent manner, since the anti-FcyRIIa antibody inhibited 5c8IgG1-mediated platelet activation. The 5c8 VH/VL regions cloned in IgG1sigma or IgG2sigma effector Fc muted form lost their ability to activate platelets (FIG. 1). These results are consistent with those described earlier. Surprisingly, however, the experiments performed in Example 10 showed that immune complexes with the wild-type IgG1 antibody (C4LBB237:shCD154) did not activate platelets (FIG. 7), indicating the need for further research on the possible dependence of platelet activation on the epitope.

The VH/VL regions of 4 individual antibodies were cloned as wild-type IgG2sigma, IgG1sigma or IgG1 to evaluate the effect of the epitope and/or Fc on platelet activation as well as higher order immune complex formation. In contrast to data previously described in the literature, it was found that platelet activation was in some cases mediated by the antibody epitope independently of FcyRIIa-mediated cross-linking.

In FIG. 8A, FIG. 8B and FIG. 8C is a summary of the assays. Fc-mutated immune complexes of antibodies C4LB119 (FIG. 8A) and C4LB94 (FIG. 8B) activated platelets in an FcyRIIa-independent manner, since pre-blocking with anti-FcyRIIa did not inhibit platelet activation. IC C4LB83, also an Fc-muted antibody, moderately activated platelets (Fig. 8C). IC antibodies with identical VH/VL domains cloned on wild-type IgG1 activated platelets in an FcyRIIa-mediated manner in each case (C4LB278 in Fig. 8A, C4LB289 in Fig. 8B and C4LB288 in Fig. 8C). Thus, several antibodies have been identified that mediate platelet activation despite the presence of muted Fc. One VH/VL domain pair was found not to mediate platelet activation on either muted (C4LB231) or wild-type IgG1 (C4LB237). These data demonstrate that the antibody epitope plays a role in mediating platelet aggregation.

Formation of the immune complex of a higher order.

GP-HPLC and DLS methods were used to further evaluate the presence of higher order immune complexes and the approximate size of these immune complexes for various anti-CD154 antibodies shown in Table 1. 21, and shCD154. Since shCD154 in solution is a trimer, the expected stoichiometric ratio of antibody to shCD154 trimer in solution is 3:1. , smaller immune complexes should have longer retention times, except for very large immune complexes that are unable to elute from the column and show low % recovery. In the case of DLS, the larger the radius (Rh) value, the larger the immune complex. DLS plate techniques do not distinguish between IgG monomers, dimers, trimers, and tetramers. Thus, the resulting Rh values will be a weighted average of the monomer-tetramer, therefore Rh values closer to 6.5 may represent mAb solutions containing higher order particles, typically a dimer. If mAb binding to shCD154 is stoichiometric, two species of particles may be present - 3:1 complex (~500 kDa) and unbound antibody (~150 kDa), which correspond to Rh values of ~8.8 nm and 5-6.5 nm for the complex 3:1 and free mAb, respectively. No technique can accurately determine the molecular weight of immune complexes, but it is possible to obtain comparative sizes. In table. 23 shows the relationship between retention times and hydrodynamic radii with approximate molecular weights.

- 48 040943

Table 23

immune complex Approximate molecular weight (kDa) GF-HPLC Retention time (min) DLS hydrodynamic radius (nm) mAb 150 ~ 9 min 5 to 6.5 mAb:shCD154 (trimer) 550 ~ 7 min ~ 8.8 Immune complexes of a higher order ~ 1000 < 7 min ~ 12

DLS.

mAb sizes in the absence of CD154 and antibody:shCD154 immune complex were evaluated under conditions where antibody:shCD154 complexes were formed in excess antibody (10:1 molar ratio) and at an equivalent concentration (1:1 molar ratio). Under conditions of excess antibody, the antibody typically saturates regions of CD154 to form an immune complex, and there is an excess of unbound antibody. At an equivalent concentration, there is no free antibody or free shCD154. The Rh values obtained for each mAb in the absence of shCD154 and in the presence of shCD154 at molar ratios of 10:1 and 10:10 are shown in Table 1. 24.

A typical mAb with a nominal MM of 150 kDa gives an Rh value of 5.0-6.5 nm. IgG dimers, often seen in size exclusion chromatography, cannot be distinguished by plate DLS techniques. The resulting Rh values will be a weighted average of the monomer-tetramer, therefore Rh values closer to 6.5 may represent mAb solutions containing a higher order particle, typically a dimer.

For mAbs in the absence of CD154, expected R _h values of 5.5-6.3 nm were observed for all mAbs except for C4LB287 and C4LB234, for which R _h values were 6.9 and 7.1 nm, respectively, suggesting that these antibodies have intrinsic tendencies to aggregate.

Rh values for antibody:shCDl54 complexes formed at a ratio of 10:1 were substantially less than about 8.8 nm, indicating formation of a 3:1 stoichiometric complex with no higher order immune complex formation except for 5c8IgG2sigma (C4LB71) and C4LB234 ( the R _h values were 8.9 and 9.3 nm, respectively), indicating the formation of higher order immune complexes.

Increasing the concentration of shCD154 to 10 μM led to the appearance of antibody:shCD154 immune complexes with increased Rh compared to complexes with 1 μM CD154 (antibody to CD154 ratio was 10:1). Some antibody:shCD154 complexes showed heterogeneity with the presence of high molecular weight secondary particles ranging from 7-25% of the total weight. For C4LB71, 5C8IgG1, C4LB89, C4LB119, C4LB94, C4LB234, and C4LB289 ICs, the Rh values were 22.1, 615, 56.3, 45.0, 18.3, 18.7, and 19.6 nm, respectively. In addition, C4LB89, C4LB119, C4LB83, C4LB228 also formed 900-4000 nm particles, and C4LB89 and C4LB119 formed precipitates indicating the formation of very large immune complexes. Particles with Rh around 12 nm typically correspond to a mass of ~1000 kDa, hence these mAbs form very large immune complexes with CD154 under these conditions.

Table 24

Antibody Isotype Hydrodynamic radius (Rh), nm 100* 10:1* 10:10* 5c8IgG2a (C4LB71) IgG2a 5.8 θ.9 22.1 5c8IgGl IgGl 5.7 8.3 19.6 C4LB89 IgG2a 5.9 5.3, (4082) 615ppt C4LB231 IgGla 5.9 8.2 10.5 C4LB237 IgGl 6, 1 7.4 9.8 C4LB119 IgG2a 6.3 8.0 56.3ppt C4LB290 IgGla 6, 1 8.3 13.3 C4LB287 IgGl 6, 9 8.2 12.9 C4LB83 IgG2a 6 6, 1 14.5, (3632) C4LB288 IgGl 6, 1 6, 6 10.9 C4LB94 IgG2a 6, 1 6, 9 45 C4LB234 IgGla 7.1 9.3, (83) 5.3, (18.3) C4LB289 IgGl 5.6, 7.8, (17, 1350) 3.7, (18.7) Ratio of antibody and shCD154 Rh secondary particles are in parentheses if wt % was > 25% ppt: a precipitate precipitated out of the solution IgG2a: IgG2sigma IgGla: IgGlsigma

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GF-HPLC.

In table. 25 shows the retention time, recovery rate, and estimated molecular weight (MW) of anti-CD154 antibodies alone or in immune complex with shCD154 as determined by GP-HPLC analysis. A typical antibody had an MM of about 150 kDa, and the shCD154 trimer had an MM of about 50 kDa. Therefore, the mAb:shCD154 trimer complex with a 3:1 stoichiometric ratio has an expected MW of about 500 kDa.

Table 25

mAb ID Type Ratio* Retention time (min) Square peak % extraction MM (kDa) 5c8IgG2a IgG2a 1: 1 6.24 6887 85.2 1596.8 (C4LB71) 10: 1 6.28 7348 90.9 1538.2 5c8IgGl IgGl 1: 1 7.3 1096 55.7 592.8 10: 1 6, 92 3280 69.2 845.6 C4LB89 IgG2a 1: 1 6, 19 356 16.9** 1673.2 10: 1 6.35 3662 45.3** 1440.8 C4LB231 IgGla 1: 1 6.97 6766 83.8 807 10 : 1 6.97 7181 88.9 807 C4LB237 IgGl 1: 1 6.99 6506 80.5 792.1 10: 1 6.99 7977 98.7 792.1 C4LB119 IgG2a 1: 1 9.59 307 5.6** 69.7 10: 1 8.02 4667 8.3** 302.4 C4LB290 IgGla 1: 1 7.01 4957 61.4 777.4 10: 1 7.01 6983 86.4 777.4 C4LB287 IgGl 1: 1 7 5937 73.5 784.7 10: 1 7.02 8011 99.2 770.2 C4LB83 IgG2a 1: 1 7.86 5193 64.3 351.2 10: 1 7.84 3355 86.9 357.8 C4LB288 IgGl 1: 1 7.62 4169 70.6 439.5 10: 1 7.37 5582 95.5 555.2 C4LB94 IgG2a 1: 1 6, 01 3233 40.0** 1979.9 10: 1 6, 02 7748 95.9 1961.5 C4LB234 IgGla 1: 1 6.26 5534 86.6 1567.3 10: 1 6.53 7922 98.1 1217.6 C4LB289 IgGl 1: 1 6.25 5800 83.5 1582 10: 1 6.53 7958 98.5 1217.6

*(rnAb: CD154) “Recovery < 50%

Antibodies with identical VH/VL are divided into groups separated by blank lines

IgG2a: IgG2sigma

IgGla: IgGlsigma

All antibodies formed an immune complex with shCD154. C4LB231, C4LB237, C4LB290, C4LB287 (all IgGlsigma) formed an immune complex with SCD154 and eluted at 7.0 min. However, C4LB290 and C4LB287 had a lower % recovery under 1:1 conditions compared to 10:1 conditions, while C4LB231 and C4LB237 had a higher % recovery (>80%). C4LB289 (IgGl), C4LB234 (IgGlsigma), 5c8IgGlsigma (C4LB71) and C4LBB94 (IgG2sigma) eluted earlier, at 6.2-6.5 min, indicating that the aforementioned mAbs were forming larger complexes. 5c8IgGl had a broader peak at the expected retention time for the mAb: shCD154 3:1 trimer, however, the broad peak and lower recovery (56% under 1:1 conditions) indicates the possible formation of higher order immune complexes that could not enter to interact with the column or not to enter it. C4LB89 and C4LB119 (both IgG2sigma) formed an immune complex with shCD154 and eluted with a broad peak and very low recovery (6-17% under 1:1 conditions), probably due to the formation of large off-column complexes. As a rule, antibodies against IgG2sigma isotypes formed larger immune complexes compared to antibodies against IgGlsigma or IgGl.

In table. 26 shows a summary of antibody characterization data. In general, platelet activation data, GP-HPLC and DLS data, taken together, indicate that platelet activation is not

Claims (22)

due to active Fc. The data indicate that Fc-mutated antibodies such as IgGlsigma and IgG2sigma are able to form larger immune complexes, larger than the expected 3:1 complex of mAb and shCD154 trimer, and that some Fc-mutated antibodies are capable of platelet activation. These data support the conclusion that both VH/VL domains (eg, the epitope to which the antibody binds) and higher order immune complex formation are involved in platelet activation. Table 26 Antibody Isotype Platelet activation** Platelet activation independent of FcyRIIa IC, RT by GF- HPLC (min) IC, Rh 10:1* (nm) by DLS 10:10* 10:1* 1:1* 5c8IgG2a (C4LB71) IgG2a No 6.3 6.2 θ.9 22.1 5c8IgGla IgGla No 5c8IgGl IgGl Yes No 6, 9 7.3 8.3 19.6 C4LB89 IgG2a No — 6, 4# 6.2# 5.3, 4082 ^Λ 615 r C4LB231 IgGla No 7.0 7.0 8.2 10.5 C4LB237 IgGl No 7.0 7.0 7.4 9.8 C4LB119 IgG2a Yes Yes 8.0# 9.6# 8.0 56.3 r C4LB290 IgGla 7.0 7.0 8.3 13.3 C4LB287 IgGl Yes No 7.0 7.0 8.2 12.9 C4LB83 IgG2a Negligible Yes 7.8 7.9 6, 1 14.5, 3632 ^l C4LB288 IgGl Yes No 7.4 7.6 6, 6 10.9 C4LB94 IgG2a Yes Yes 6.0 6, 0# 6, 9 45 C4LB234 IgGln 6.5 6.3 9.3 5.3, 18.3 ^l C4LB289 IgGl Yes No 6.5 6.3 7.8 3.7, 18.7 Antibody to shCD154 Ratio **Assessed by either PAC-1 or CD62p expression #% GP-HPLC recovery < 50% ^A Rh secondary particles reported if % wt. > 25% p: a precipitate precipitated from the solution IgG2a: IgG2sigma IgGla: IgGlsigma CLAIM 1. An isolated antagonist antibody or antigen-binding fragment thereof that specifically binds to human CD154 with SEQ ID NO: 1 and contains a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 37 (RASQSISSYLN), LCDR 2 with SEQ ID NO :44 (YANSLQS) and LCDR 3 with SEQ ID NO:52 (QQSDSIPWT), heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO:17 (SYGIS), HCDR2 with SEQ ID NO:23 (WISPIFGNTNYAQKFQG) and HCDR3 with SEQ ID NO:30 (SRYYGDLDY) 2. An isolated antagonist antibody or antigen-binding fragment thereof that specifically binds to human CD154 with SEQ ID NO:1 and contains HCDR 1 with SEQ ID NO:17 (SYGIS), HCDR2 with SEQ ID NO:23 (WISPIFGNTNYAQKFQG), HCDR3 with SEQ ID NO:30 (SRYYGDLDY), LCDR1 with SEQ ID NO:37 (RASQSISSYLN), LCDR2 with SEQ ID NO:49 (YASSLQS) and LCDR3 with SEQ ID NO:52 (QQSDSIPWT). 3. An isolated antagonist antibody or antigen-binding fragment thereof that specifically binds to human CD154 with SEQ ID NO:1 and contains HCDR1 with SEQ ID NO:17 (SYGIS), HCDR2 with SEQ ID NO:23 (WISPIFGNTNYAQKFQG), HCDR3 with SEQ ID NO:30 (SRYYGDLDY), LCDR1 with SEQ ID NO:37 (RASQSISSYLN), LCDR2 with SEQ ID NO:50 (YANTLQS), and LCDR3 with SEQ ID NO:52 (QQSDSIPWT). 4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody has at least one of the following properties: a) the immune complex of the antibody and soluble human CD154 (shCD154) does not activate thrombo- - 51 040943 cytes if platelet activation is measured by the expression of P-selectin on the surface of platelets; b) binds to CD154 with a dissociation constant (KD) of about 5x10 ^-9 M or less when KD is measured using the ProteOn XPR36 system at 25°C in Dulbecco's phosphate buffered saline containing 0.03% polysorbate P20 and 100 mcg/ml bovine serum albumin; c) inhibits CD154 mediated proliferation of human B cells with an IC ₅₀ value of about 2.7 x 10 ^-9 M or less; or d) inhibits CD154-mediated expression of secreted embryonic alkaline phosphatase (SEAP) by NF-kB inducible minimal interferon-β (IFNβ) promoter in HEK293 cells stably expressing SEAP and human CD40 with an IC ₅₀ value of about 2.1 x 10 ^-8 M or less. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein CD154 is a homotrimer and the antibody binds the first CD154 monomer in the homotrimer within amino acid residues 182-207 of CD154 and the second CD154 monomer in the homotrimer within amino acid residues 176-253 CD154, where the numbering of residues corresponds to SEQ ID NO:1. 6. The antibody or antigen-binding fragment thereof according to claim 5, where the antibody binds residues E182, S185, Q186, A187, P188, S214, A215 and R207 in the first CD154 monomer, where the numbering of the residues corresponds to SEQ ID NO: 1. 7. The antibody or antigen-binding fragment thereof according to claim 5, where the antibody binds residues T176, F177, C178, Q220, S248, H249, G250 and F353 in the second CD154 monomer, where the numbering of the residues corresponds to SEQ ID NO: 1. 8. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain variable region (VH) of SEQ ID NO:59. 9. An antibody or antigen-binding fragment thereof according to claim 8, containing a light chain variable region (VL) with SEQ ID NO:66, 72 or 73. 10. An antibody or antigen-binding fragment thereof according to claim 9, containing VH with SEQ ID NO:59 and VL with SEQ ID NO:66. 11. An antibody or antigen-binding fragment thereof according to claim 9, containing VH with SEQ ID NO:59 and VL with SEQ ID NO:72. 12. An antibody or antigen-binding fragment thereof according to claim 9, containing VH with SEQ ID NO:59 and VL with SEQ ID NO:73. 13. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. 14. An antibody or antigen-binding fragment thereof according to claim 13, containing at least one substitution in the Fc region, where at least one substitution in the Fc region is a substitution L234A, L235A, G237A, P238S, M252Y, S254T, T256E, H268A , A330S or P331S, where the numbering of residues corresponds to the EU index. 15. An antibody or antigen-binding fragment thereof according to claim 14, containing substitutions L234A, L235A, G237A, P238S, H268A, A330S or P331S in the Fc region, where the residue numbering corresponds to the EU index. 16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, containing at least one substitution in the Fc region, where at least one substitution in the Fc region is a substitution V234A, G237A, P238S, M252Y, S254T, T256E, H268A, V309L, A330S or P331S, where the residue numbering corresponds to the EU index. 17. An antibody or antigen-binding fragment thereof according to claim 16, containing substitutions V234A, G237A, P238S, H268A, V309L, A330S and P331S in the Fc region, where the numbering of the residues corresponds to the EU index. 18. An antibody or antigen-binding fragment thereof according to claim 13, containing a heavy chain (HC) and a light chain (LC) with SEQ ID NO: a) 80 and 81 respectively; b) 82 and 81, respectively, or c) 83 and 81, respectively. 19. Multispecific antibody, characterized in that it contains an antibody according to any one of claims 1-3. 20. An isolated antagonistic antibody or antigen-binding fragment thereof specifically binding CD154 with SEQ ID NO:1, containing a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOS: 17, 23, 30, 37, 44 and 52, respectively; b) VH with SEQ ID NO:59 and VL with SEQ ID NO:66 or c) the heavy chain of SEQ ID NO:80 and the light chain of SEQ ID NO:81. 21. An immunoconjugate specifically binding to human CD154 of SEQ ID NO: 1, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-20 associated with a therapeutic agent. 22. An immunoconjugate that specifically binds to human CD154 with SEQ ID NO: 1, containing -