EA039944B1 - Use of anti-cd40 antibodies for treatment of lupus nephritis - Google Patents

Abstract

The invention relates to new humanized antagonistic anti-CD40 antibodies and therapeutic and diagnostic methods and compositions for using these antibodies for treating and/or preventing lupus nephritis.

Description

The field of technology to which the invention belongs

The present invention relates generally to the use of humanized anti-CD40 antibodies for the treatment and/or prevention of lupus nephritis.

Prerequisites for the creation of the invention

CD40 is a 48 kDa type I integral membrane glycoprotein and is a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on a wide variety of cell types, including normal and neoplastic B cells, interdigitated (tangled) cells, carcinoma cells, epithelial cells (eg, keratinocytes), fibroblasts (eg, synoviocytes), and platelets. It is also present on monocytes, macrophages, some types of endothelial cells, and follicular dendritic cells. CD40 is characterized by expression early in B cell ontogeny and appears on B cell progenitors after CD10 and CD19 but before CD21, CD23, CD24 expression and surface immunoglobulin M (sIgM) (Uckun et al., Blood 15, 1990, p. 2449). CD40 has also been found on tonsils and bone marrow-derived plasma cells (PellatDecounynck et al., Blood 84, 1994, p. 2597).

The CD40 ligand is CD40L (also referred to as CD154, gp39 and TRAP), a member of the TNF superfamily. CD40L is a transmembrane protein that is expressed primarily on activated CD4 ⁺ T cells and a small subset of CD8 ⁺ T cells (for review see Van Kooten C. and Banchereau, 2000).

The interaction of CD40 with CD40L induces both humoral and cell-mediated responses. CD40 regulates the activation of B cells and other antigen-presenting cells (APCs), including dendritic cells (DCs), by this ligand-receptor pair (see review (Toubi and Shoenfeld, 2004); (Kiener et al., 1995). extensive studies of CD40 function on B cells Activation of CD40 on B cells induces proliferation, differentiation into antibody-secreting cells and isotype switching in the germinal centers of secondary lymphoid organs In vitro studies have demonstrated the direct effects of CD40 activation on cytokine production (IL- 6, IL-10, TNF-α, LT-α), expression of adhesion molecules and co-stimulatory receptors (ICAM, CD23, CD80, and CD86), and increased expression of MHC class I, MHC class II, and TAP transporter molecules by B lymphocytes (Liu et al. ., 1996) In most of these processes, CD40 acts either in conjunction with other cytokines or in combination with other receptor-ligand interactions.

CD40 signaling on monocytes and DC leads to increased survival as well as the secretion of cytokines (IL-1, IL-6, IL-8, IL-10, IL-12, TNF-α and MIP-1a). Cross-linking of CD40 with these APCs also leads to upregulation of co-stimulatory molecules (such as ICAM-1, LFA-3, CD80 and CD86). Activation of CD40 receptors is one of the critical signals that lead to the full maturation of DC into effective APCs that provide T-cell activation (Banchereau and Steinman, 1998) (Van Kooten C. and Banchereau, 2000).

Recent studies in mouse models have shown that CD40 signaling on dendritic cells also plays an important role in the generation of TH17 cells, which are thought to be mediators of autoimmunity in diseases such as arthritis and multiple sclerosis (Iezzi et al., 2009) (PeronaWright et al. , 2009).

The presence of CD40- and CD40L-knockout mice, as well as agonistic and antagonistic anti-mouse antibodies, creates the opportunity to study the role of CD40-CD40L interactions in several disease models. The beneficial effect of the use of a blocking antibody to CD40L has been demonstrated in several models of autoimmunity, including spontaneous diseases such as lupus nephritis in SNF1 mice or diabetes in NOD mice, or experimentally induced forms of disease such as collagen-induced arthritis (CIA) or experimental autoimmune encephalomyelitis ( EAE) (Toubi and Shoenfeld, 2004). Anti-CD40L mAb inhibited CIA in mice and blocked the development of joint inflammation, serum anti-collagen antibody titers, inflammatory cell infiltration into subsynovial tissue, and cartilage and bone erosion (Durie et al., 1993). It has been demonstrated that in both lupus nephritis and EAE, the anti-CD40L antibody also had the ability to stop the onset of the disease, which confirms the role of CD40-CD40L in the effector phase of the disease (Kalled et al., 1998); (Howard et al., 1999).

Thus, preclinical studies have been conducted that demonstrate the critical role of the CD40-CD40L dyad in providing an effective T-cell dependent immune response. Therefore, blocking CD40 signaling is seen as an acceptable and desirable therapeutic strategy to suppress the pathogenic autoimmune response in diseases such as lupus nephritis.

The International Society of Nephrology (ISN)/Society of Renal Pathology has developed a classification of lupus nephritis based in part on the degree of renal failure (See J. J. Weening et al., The classification of glomerulonephritis in systemic lupus erythematosus revisited, J. Am. Soc. Nephrol 15: 2004 , pp. 241-250).

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Renal biopsies from SLE (systemic lupus erythematosus) patients with lupus nephritis showed a spectrum of vascular, glomerular, and tubulointerstitial lesions. Glomerular damage is determined by the localization of the immune complex on mesangial, endothelial and epithelial cells. Depending on these changes, lupus nephritis can be divided into 6 classes, ranging from relatively mild involvement manifested by immune complex deposition in the mesangial matrix only (class I) or mesangial hypercellularity (class II) to global sclerosis in >90% of the glomerulus (class VI). ). Patients with class I or II lupus nephritis show only asymptomatic proteinuria and no decrease in GFR (glomerular filtration rate). Endocapillary changes are classified as either focal proliferative (class III) or diffuse proliferative (>50% glomerulus affected, class IV). The clinical manifestations in these patients are hematuria, symptomatic proteinuria, and loss of GFR. A pure epithelial lesion (Class V) leads to the deposition of the immunocomplex along the glomerular basement membrane without inflammatory lesions. These patients suffer mainly from nephrotic proteinuria.

There is no need for immunosuppressive therapy for class I and II, and there is no treatment effect after irreversible damage in class VI, while lupus nephritis III-V is treatable with immunosuppressive therapy. About 25% of patients with lupus nephritis have class III lesions, 40% class IV, and 10% class V. About 1/6 of class III and class IV will also have class 5 (overlap).

Immunosuppressive therapy should take into account renal biopsy, and it aims for a complete renal response (proteinuria <0.5 g/24 h with normal or near-normal renal function). Apart from glucocorticoids, there is no approved therapy for the treatment of lupus nephritis, however, cyclophosphamide or mycophenolate (MMF), each in combination with glucocorticoids, has been shown to induce an improvement in the clinical symptoms of active lupus nephritis and reduce the risk of CKD progression. About 20-30% of patients treated with MMF or cyclophosphamide had a complete renal response (proteinuria <0.5 g/s, no evidence of active inflammation, no deterioration in GFR) and another about 20% had at least , partial response (>50% reduction in proteinuria).

Thus, the combination of glycocorticosteroids with either MMF or cyclophosphamide is currently considered the standard treatment for lupus nephritis, which is also in line with the recommendations of the European League Against Rheumatism and the European Kidney Association - Dialysis and Kidney Transplantation (EULAR/ERA-EDTA) . These recommendations also include hydroxychloroquine for all patients with lupus nephritis to reduce the number of exacerbations and the use of ACE inhibitors or ARBs.

However, all treatments available for lupus nephritis may be associated with significant toxicity (eg, infertility, infections, malignancy). Thus, the percentage of patients with a complete objective response remains low and, within patients with a therapeutic effect, there is a high percentage of relapses, justifying long-term maintenance therapy. Recent stage III clinical trials for lupus nephritis (eg, rituximab, abatacept) failed to meet their primary evaluation criteria. Taken collectively, there is currently an urgent unmet need for new therapies for lupus nephritis. This need can be addressed by the humanized anti-CD40 antibodies described in this application and US20110243932 that specifically bind to CD40 and that exhibit antigen-binding specificity, affinity, and pharmacokinetic and pharmacodynamic properties that enable them to be used for therapeutic intervention in lupus nephritis.

Brief summary of the invention

The present invention provides a method for using a humanized monoclonal antibody to treat and/or prevent lupus nephritis (method of the invention).

In exemplary embodiments, the antibody used in the method of the invention comprises a heavy chain sequence selected from the group consisting of any of SEQ ID NO: 1 of SEQ ID NO: 4 and a light chain sequence selected from the group consisting of any of SEQ ID NO: NO: 5 - SEQ ID NO: 8.

In other embodiments, the antibody used in the method of the invention is a humanized antibody or an antigen-binding fragment of an antibody that has a heavy chain variable region amino acid sequence as represented in any of

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SEQ ID NO: 1 - 4, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29, SEQ

ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID

NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID

NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO. 50 SEQ ID NO: 53, SEQ ID

NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID

NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQID

NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQID

NO: 72, or SEQ ID NO: 73.

In other embodiments, the antibody used in the method of the invention is a humanized antibody or an antigen-binding fragment of an antibody that contains the amino acid sequence of the light chain variable region shown in SEQ ID NO: 5 - SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76.

In specific embodiments, the monoclonal antibody used in the method of the invention comprises a heavy chain and a light chain, wherein the heavy chain CDR1 sequence is selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 11, the heavy chain CDR2 sequence is selected from the group , including SEQ ID NO: 12 - SEQ ID NO: 15, and the heavy chain CDR3 sequence is selected from the group including SEQ ID NO: 16 - SEQ ID NO: 17; and wherein the light chain CDR1 sequence has a sequence selected from the group consisting of SEQ ID NO: 18 - SEQ ID NO: 21, a light chain CDR2 sequence shown in SEQ ID NO: 22 - SEQ ID NO: 23, and a light chain CDR3 sequence selected from the group consisting of SEQ ID NO: 24 - SEQ ID NO: 25.

In specific embodiments, the monoclonal antibody used in the method of the invention comprises a heavy chain CDR1 sequence as set forth in SEQ ID NO: 10, a heavy chain CDR2 sequence as set forth in SEQ ID NO: 13, and a heavy chain CDR3 sequence as set forth in SEQ ID NO: 13. NO: 16; and wherein said antibody comprises a light chain CDR1 sequence as set forth in SEQ ID NO: 19, a light chain CDR2 sequence as set forth in SEQ ID NO: 22, and a light chain CDR3 sequence as set forth in SEQ ID NO: 24.

In other specific embodiments, the monoclonal antibody used in the method of the invention comprises a heavy chain CDR1 sequence as set forth in SEQ ID NO: 9, a heavy chain CDR2 sequence as set forth in SEQ ID NO: 14, and a heavy chain CDR3 sequence as set forth in SEQ ID NO: 16; and wherein said antibody comprises a light chain CDR1 sequence as set forth in SEQ ID NO: 20, a light chain CDR2 sequence as set forth in SEQ ID NO: 22, and a light chain CDR3 sequence as set forth in SEQ ID NO: 24.

In another specific embodiment, the monoclonal antibody used in the method of the invention comprises a heavy chain CDR1 sequence as set forth in SEQ ID NO: 9, a heavy chain CDR2 sequence as set forth in SEQ ID NO: 14, and a heavy chain CDR3 sequence as set forth in SEQ ID NO: 16; and wherein said antibody comprises a light chain CDR1 sequence as set forth in SEQ ID NO: 20, a light chain CDR2 sequence as set forth in SEQ ID NO: 22, and a light chain CDR3 sequence as set forth in SEQ ID NO: 24.

In another specific embodiment, the monoclonal antibody used in the method of the invention comprises a heavy chain CDR1 sequence as set forth in SEQ ID NO: 11, a heavy chain CDR2 sequence as set forth in SEQ ID NO: 15, and a heavy chain CDR3 sequence as set forth in SEQ ID NO: 17; and wherein said antibody comprises a light chain CDR1 sequence as set forth in SEQ ID NO: 21, a light chain CDR2 sequence as set forth in SEQ ID NO: 23, and a light chain CDR3 sequence as set forth in SEQ ID NO: 25.

In another embodiment, the invention relates to the use of an anti-CD40 antibody for the treatment and/or prevention of lupus, wherein the anti-CD40 antibody comprises a heavy chain variable region sequence as set forth in any of SEQ ID NOs: 1-4. In addition, an anti-CD40 antibody is characterized in that it contains a light chain variable region sequence as set forth in any of SEQ ID NO: 5 to SEQ ID NO: 8.

Also within the scope of the invention is the use of an anti-CD40 antibody for the treatment and/or prevention of lupus nephritis, wherein the anti-CD40 antibody comprises a humanized antibody or antibody fragment that has a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO : 27 and SEQ ID NO: 26, respectively; SEQ ID NO: 28 and SEQ ID NO: 26, respectively; SEQ ID NO: 29 and SEQ ID NO: 26, respectively; SEQ ID NO: 30 and SEQ ID NO: 26, respectively; SEQ ID NO: 32 and SEQ ID NO: 31, respectively; SEQ ID NO: 33 and SEQ ID NO: 31, respectively; SEQ ID NO: 34 and SEQ ID NO: 31, respectively;

SEQ ID NO: 35 and SEQ ID NO: 31, respectively; SEQ ID NO: 37 and SEQ ID NO: 36, respectively; SEQ

ID NO: 38 and SEQ ID NO: 36, respectively; SEQ ID NO: 39 and SEQ ID NO: 36, respectively; SEQ ID

NO: 40 and SEQ ID NO: 36, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 27 and SEQ ID NO: 26, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 28 and SEQ ID NO: 26, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 29 and SEQ ID NO: 26, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 30 and SEQ ID NO: 26, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 32 and SEQ ID NO: 31, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 33 and SEQ ID NO: 31, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 34 and SEQ ID NO: 31, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 35 and SEQ ID NO: 31, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 37 and SEQ ID NO: 36, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 38 and SEQ ID NO: 36, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 39 and SEQ ID NO: 36, respectively.

In another embodiment, the humanized antibody or antibody fragment used in the method of the invention comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 40 and SEQ ID NO: 36, respectively.

In another embodiment, the method of the invention relates to the treatment and/or prevention of lupus nephritis using an isolated antibody or antigen-binding fragment that/which specifically binds to human CD40, wherein the isolated antibody or antigen-binding fragment contains a humanized heavy chain variable region that contains a framework region , the amino acid sequence of which is at least 90% identical to the amino acid sequence of the framework region of the human variable region of the heavy chain, the amino acid sequence of which is presented in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, and comprising/comprising a light chain amino acid sequence that is at least 90% identical to the corresponding light chain variable region sequence as set forth in SEQ ID NO: 26.

In another embodiment, the method of the invention relates to the treatment and/or prevention of lupus nephritis using an isolated antibody or antigen-binding fragment that/which specifically binds to human CD40, wherein the isolated antibody or antigen-binding fragment contains a humanized heavy chain variable region that contains a framework region , the amino acid sequence of which is at least 90% identical to the amino acid sequence of the framework region of the human variable region of the heavy chain, the amino acid sequence of which is presented in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and comprising/comprising a light chain amino acid sequence that is at least 90% identical to the corresponding light chain variable region sequence as set forth in SEQ ID NO: 31.

In another aspect, the method of the invention relates to the use of an isolated antibody or antigen-binding fragment as described in the embodiment immediately above, wherein the heavy chain amino acid sequence is shown in SEQ ID NO: 32; in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 33; in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 34; and in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 35.

In another embodiment, the invention relates to the treatment and/or prevention of lupus nephritis using a humanized heavy chain variable region that contains a framework region whose amino acid sequence is at least 90% identical to the amino acid sequence of the human heavy chain variable region framework region, the amino acid sequence which is presented in SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40, and containing/containing a light chain amino acid sequence that is at least 90% identical to the corresponding light chain sequence as shown in SEQ ID NO: 36.

In another aspect, the method of the invention relates to the use of an isolated antibody or antigen-binding fragment as described in the embodiment immediately above, wherein the heavy chain amino acid sequence is shown in SEQ ID NO: 37; in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 38; in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO:39; and in another embodiment, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 40.

The present invention relates to the use of a humanized monoclonal antibody as described herein for the preparation of a medicament for the treatment and/or prevention of lupus nephritis (method of the invention).

In other words, the present invention relates to the use of a humanized monoclonal antibody, as described in this application, for the preparation of a pharmaceutical composition for the treatment and/or prevention of lupus nephritis.

Expressed again differently, the present invention relates to a humanized monoclonal antibody, as described in this application, for use in the treatment and/or prevention of lupus nephritis.

Brief description of some aspects of the figures

In FIG. 1 shows the effect of CD40 ligation on human endothelial cell (HUVEC) cells in medium containing 100 ng/ml sCD40L and either antibody B (·) or isotype control (human IgG1 antibody) (^).

In FIG. 2 shows the effect of CD40 ligation on human proximal tubular epithelial cells (PTECs) from three different donors (D1, D2, D3) in medium containing 50 ng/mL sCD40 antibody B.

Detailed description of the invention

CD40-mediated signaling has now been established to be involved in various targeted disorders. Despite the availability of a number of preclinical studies demonstrating that intervention in these disorders may have therapeutic utility, there remains a need for anti-CD40 antagonist antibodies that can be used to treat autoimmune diseases such as lupus nephritis.

The terms CD40 and CD40 surface antigen refer to an approximately 48 kDa glycoprotein expressed on the surface of healthy and neoplastic B cells that acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., J. Immunol. 138 , 1987, pp. 788-785). The cDNA molecule encoding CD40 was isolated from a library based on the Raji cell line of Burkett's lymphoma (Stamenkovic et al., EMBO J. 8, 1989, p. 1403).

As used herein, a cell that endogenously expresses CD40 is any cell that is characterized by expression of CD40 on their surface, including but not limited to healthy and neoplastic B cells, interdigital cells, epithelial basal cells. cells, carcinoma cells, macrophages, endothelial cells, follicular dendritic cells, tonsil cells and bone marrow-derived plasma cells. In some embodiments, the CD40 molecule is a human CD40 molecule.

The antibodies of the invention bind specifically to human recombinant and native CD40. A humanized monoclonal antibody is an antibody that specifically binds to human CD40, has an antagonist activity with an IC ₅₀ value of less than 1 nM, and, when used at a concentration up to 100 μg/ml, does not have an agonistic effect on B-cell proliferation , and said antibody is also characterized in that the antibody has an in vivo half-life in non-human primates of at least 10 days.

The general structure of an antibody or immunoglobulin is well known to those skilled in the art, these molecules are heterotetrameric glycoproteins, typically about 150,000 Da molecular weight, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to the heavy chain by a single disulfide bond to form a heterodimer, and a heterotetrameric molecule is formed by a covalent disulfide bond between two identical heterodimer heavy chains. Although the light and heavy chains are linked by a single disulfide bond, the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has an amino-terminal variable region (VH) followed by three or four constant domains (C _H1 , CH2, CH3 and C _H4 ) and a hinge region between C _H1 and CH2. Each light chain has two regions, an amino-terminal variable region (VL) and a carboxy-terminal constant domain (CL). The VL region is non-covalently linked to the VH region, and the CL domain is generally covalently linked to the C _H1 domain via a disulfide bond. It is likely that certain amino acid residues form the interface between the variable regions of the light and heavy chains (Chothia et al., J. Mol. Biol. 186, 1985, pp. 651-663).

Certain regions in the variable regions differ significantly among different antibodies, ie. they are hypervariable. These hypervariable regions contain residues that are directly involved in binding and determine the specificity of each particular antibody in relation to its specific antigenic determinant. Hypervariability in both the light chain and heavy chain variable regions is concentrated in three segments, which are referred to as complementarity-determining regions (hypervariable regions) (CDRs) or hypervariable loops (HVLs). CDRs are determined by sequence comparison according to the method described by Kabat et al., in Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991, a HVL structurally determined based on the three-dimensional structure of the variable region according to the method described in Chothia and Lesk, J. Mol. Biol. 196, 1987, pp. 901-917. If these two methods lead to some differences in the identification of CDRs, then structure-based determination is preferred. According to Cabot's nomenclature, CDR-L1 is located at about residues 24-34, CDR-L2 at about residues 50-56, and CDR-L3 at about residues 89-97 in the light chain variable region; CDR-H1 is located at approximately residues 31-35, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102 in the heavy chain variable region. Thus, the CDR1, CDR2, CDR3 of the heavy and light chains determine the unique and functional properties specific to a given antibody.

The three CDRs in each of the heavy and light chains are separated by framework regions (FRs) that contain sequences that tend to be less variable. Starting from the amino terminus towards the carboxy terminus of the heavy and light chain variable regions, FRs and CDRs are ordered as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. To a large extent, the β-sheet conformation of FR allows the CDRs in each of the strands to be in close proximity to each other as well as to CDRs from the other strand. The resulting conformation is inherent in the antigen-binding center (see Kabat et al., NIH Publ. no. 91-3242, vol. I, 1991, pp. 647-669), although it is not a necessary condition that all CDR residues are directly involved in antigen binding .

FR residues and Ig constant domains are not directly involved in antigen binding, but are involved in antigen-associated and/or antigen-mediated effector function. Some FR residues are likely to have a pronounced effect on antigen binding in at least three ways: by binding non-covalently directly to an epitope, by interacting with one or more CDR residues, and by influencing the interface between heavy and light chains. The constant domains are not directly involved in antigen binding but mediate various Ig effector functions.

Vertebrate immunoglobulin light chains are assigned to one or two distinct classes, kappa (k) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, mammalian immunoglobulin heavy chains are classified into one of five major classes based on the sequence of constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 and IgA2. Heavy chain constant domains, which correspond to different classes of immunoglobulins, are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

As used herein, the terms antibody, anti-CD40 antibody, humanized anti-CD40 antibody, and variant humanized anti-CD40 antibody are used in the broadest sense and refer specifically to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies ( e.g., bispecific antibodies) and antibody fragments, such as variable regions and other antibody regions, that have the desired biological activity, such as the ability to bind to CD40.

The term monoclonal antibody (MAb) refers to an antibody from a population of substantially homogeneous antibodies; those. the individual antibodies in this population are identical except for naturally occurring mutations, which may be present in very small amounts. Monoclonal antibodies are highly specific, their target is an individual antigenic determinant, an epitope. Thus, the adjective monoclonal is a term for a substantially homogeneous population of antibodies that target an identical epitope and is not limited to the requirement that antibodies be produced by any particular method. It should be obvious that monoclonal antibodies can be obtained using any technique or methodology known in this field; including, for example, the hybridoma method (Kohler et al., Nature 256, 1975, p. 495), or recombinant DNA methods known in the art (see, for example, US 4816567), or methods for isolating monoclonals obtained using recombinant Antibody DNA using antibody phage libraries as described in Clackson et al., Nature 352, 1991, pp. 624-628 and Marks et al., J. Mol. Biol. 222, 1991, pp. 581-597.

Chimeric antibodies consist of the heavy and light chain variable regions of an antibody from one species (e.g., non-human mammal, such as a mouse) and the light and heavy chain constant regions of an antibody from another species (e.g., human) and can be generated by joining the sequences DNA encoding variable regions of an antibody from a first species (eg, mouse), with DNA sequences of constant regions of an antibody from a second species (eg, human), and transforming the host with an expression vector containing the linked sequences, allowing the host to produce a chimeric antibody. Alternatively, the chimeric antibody may also be an antibody in which one or more heavy and/or light chain regions or domains are identical, homologous, or a variant of the corresponding sequence in a monoclonal antibody from a different immunoglobulin class or isotype or from a consensus or germ line sequence. . Chimeric antibodies may include fragments of these antibodies, provided that the antibody fragment has the desired biological activity of the parent antibody, such as the ability to bind to the same epitope (see, for example, US 4816567; and Morrison et al., Proc. Natl. Acad. Sci USA 81, 1984, pp. 6851-6855).

The terms antibody fragment, anti-CD40 antibody fragment, humanized anti-CD40 antibody fragment, and humanized anti-CD40 antibody variant fragment refer to the portion of a full-length anti-CD40 antibody that retains the variable region or its functionality, such as binding specificity for the CD40 epitope. Examples of antibody fragments include (but are not limited to) Fab-, Fab'-, F(ab') _2- , Fd-, Fv-, scFv- and scFv-Fc fragment, dimeric antibody (diabodi), linear antibody , single chain antibody, minibody, dimeric antibody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

Full length antibodies can be treated with enzymes such as papain or pepsin to produce the desired antibody fragments. Papain digestion is used to produce two identical antigen-binding fragments, referred to as Fab fragments, each with one antigen-binding site, with the remainder referred to as an Fc fragment. The Fab fragment also contains a light chain constant domain and a heavy chain C _H1 domain. After treatment with pepsin, an F(ab') ₂ fragment is obtained which bears two antigen-binding centers and still retains the ability to cross-link with the antigen.

Fab' fragments differ from Fab fragments by the presence of additional residues, including one or more cysteine residues from the antibody hinge region at the C-terminus of the C _H1 domain. F(ab')2 antibody fragments are pairs of Fab' fragments linked via cysteine residues in the hinge region. Other chemical combinations of antibody fragments are known.

The Fv fragment contains a complete antigen-recognition and antigen-binding center, consisting of a dimer including the variable region of one heavy and one light chain in tight non-covalent association. In this configuration, the three CDRs of each variable region interact with a specific antigen-binding site on the surface of the VH-V _L dimer. In total, six CDRs determine the specificity of an antibody for antigen binding.

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A single chain Fv or scFv fragment of an antibody is a single chain Fv variant containing the V _H and V L regions of an antibody, the regions being present on the same polypeptide chain. The single chain Fv has the ability to recognize and bind to an antigen. The scFv polypeptide may optionally also contain a polypeptide linker located between the V _H - and VL regions to facilitate the formation of the three-dimensional structure required for antigen binding using scFv (see, for example, Pluckthun, in: The Pharmacology of monoclonal Antibodies, v. 113 , edited by Rosenburg and Moore, Springer-Verlag, New York, 1991, pp. 269-315).

Other recognizable antibody fragments include fragments that contain a pair of Fd segments arranged in tandem (V _H -C _H1 -V _H -C _H1 ) forming a pair of antigen-binding sites. Said linear antibodies may be bispecific or monospecific as described, for example, in Zapata et al., Protein Eng. 8(10), 1995, pp. 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody that includes an amino acid sequence variant of an immunoglobulin or fragment thereof that has the ability to bind to a predetermined antigen and that contains one or more FRs that has(s) an amino acid sequence, substantially corresponding to the sequence of a human immunoglobulin, and one or more CDRs that(s) have(s) an amino acid sequence that substantially corresponds to the amino acid sequence of a non-human immunoglobulin. Said non-human amino acid sequence, often referred to as an import sequence, is typically derived from the import region of an antibody, in particular the variable region. In general, a humanized antibody comprises at least the CDRs or HVLs of a non-human antibody inserted between the FRs of a human heavy or light chain variable region. The present invention describes specific humanized antibodies to CD40, which contain CDRs derived from mouse monoclonal antibodies, which are presented in table. 3 and 4 inserted between the FR sequences of the human germline heavy and light chain variable regions. It should be apparent that certain mouse FR residues can be imported to provide humanized antibody function, and therefore certain human germline heavy and light chain variable region sequence residues are modified to be the same as in the corresponding mouse sequence.

In another aspect of the invention, a humanized anti-CD40 antibody contains substantially all of at least one and typically two variable regions (such as, for example, those included in Fab-, Fab'-, F(ab') ₂ -, Fabc- and Fv fragments) in which all or substantially all of the CDRs correspond to CDRs of non-human immunoglobulins, and according to the present invention, all CDRs are murine sequences detailed in Tables 1-4 below, and all or substantially all of the FRs are FRs that have a consensus or germline human immunoglobulin sequence. In another aspect of the invention, the humanized anti-CD40 antibody also includes at least a portion of the Fc region of an immunoglobulin, typically a human immunoglobulin. Typically, an antibody will contain both a light chain and at least a heavy chain variable region. The antibody may also optionally include one or more of the following regions: C _H1 , hinge region, C _H2 , C _H3 and/or C _H4 heavy chain.

The humanized anti-CD40 antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG ₂ , IgG ₃ , IgG4, IgA1, and IgA ₂ . Alternatively, a humanized anti-CD40 antibody may comprise sequences from more than one immunoglobulin class or isotype, and selection of specific constant domains to optimize desired effector functions is within the skill of ordinary skill in the art. Particular embodiments of the present invention are antibodies that are IgG1 isotype antibodies, and more specifically IgG1 isotype antibodies, in which effector functions are switched off.

The FR and CDR or HVL of a humanized anti-CD40 antibody need not match exactly the parental sequences. For example, one or more residues in an import CDR or HVL or in a consensus or germline FR sequence can be altered (e.g., by mutagenesis) by substitution, insertion, or deletion such that the resulting amino acid residue is not identical to the original residue at the corresponding position of any parent sequence, but the antibody still retains a function, such as the ability to bind to CD40. Said change, as a rule, should not be extensive and should be conservative changes. Typically, at least 75% of the residues, more often at least 90%, and most often more than 95%, or more than 98%, or more than 99% of the humanized antibody must match the residues of the parental consensus or germline FR sequence and import CDR sequences.

Immunoglobulin residues that affect the interface between the heavy and light chain variable regions (interface V _L -V _H ) are residues that affect

- 8 039944 the proximity or orientation of the two chains relative to each other. Specific residues that may be involved in interstrand interactions are V _L residues 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98 and VH residues 35, 37, 39, 45, 47, 91, 93 , 95, 100, and 103 (according to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). US 6407213 also discusses that residues such as residues V _L 43 and 85 and residues VH 43 and 60 may be involved in this interaction. Although these residues are indicated only for human IgG, they can be considered for other species. Important antibody residues that are expected to be involved in interstrand interactions are selected for replacement in the consensus sequence.

The terms consensus sequence and consensus antibody refer to the amino acid sequence that contains the most frequently occurring amino acid residue at each position in all immunoglobulins of any class, isotype, or subunit structure, such as the variable region of a human immunoglobulin. The consensus sequence may be based on immunoglobulins of specific species or multiple species. By consensus/consensus sequence, structure, or antibody is meant the human consensus sequence specified in specific embodiments of the invention, and refers to the amino acid sequence that contains the most frequently occurring residues at each position in all human immunoglobulins of a particular class, isotype, or subunit structure. Thus, the consensus sequence contains an amino acid sequence that has at each position an amino acid that is present in one or more known immunoglobulins, but which may not be an exact copy of the complete amino acid sequence of any individual immunoglobulin. The consensus sequence variable region is not derived from any naturally occurring antibody or immunoglobulin (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and its variants. The heavy and light chain FR consensus sequences and their variants are valuable sequences for the production of humanized anti-CD40 antibodies (see, for example, US 6,037,454 and 6,054,297).

The human germline sequences are naturally present in the human antibody population. The combination of these germline genes creates a variety of antibodies. The antibody light chain germline sequences are derived from conserved human kappa or lambda germline v and j genes. Similarly, heavy chain sequences are derived from the v, d, and j germline genes (LeFranc M-P and LeFranc G, The Immunoglobulin Facts Book, Academic Press, 2001).

As used herein, a variant, an anti-CD40 antibody variant, a humanized anti-CD40 antibody variant, or a humanized anti-CD40 antibody variant all mean a humanized anti-CD40 antibody that contains at least a murine heavy chain variable region CDR that has any of the sequences set forth in SEQ ID NO: 1-4, or a mouse light chain CDR sequence derived from a mouse monoclonal antibody as shown in any of SEQ ID NO: 5-SEQ ID NO: 8, and FR sequences derived from human consensus sequences. Variants include sequences having one or more amino acid substitutions in the variable regions of one or both of the light chains or heavy chains, provided that the amino acid change does not significantly affect the binding of the antibody to CD40. Examples of humanized antibodies obtained according to the present invention are antibodies designated as antibody A, antibody B and antibody C, and their various heavy and light chain sequences, shown in SEQ ID NO: 26 - SEQ ID NO: 40.

An isolated antibody is an antibody that has been identified and separated and/or purified from a component of its natural environment. Contaminant components of the natural environment of an antibody are substances that may interfere with the diagnostic or therapeutic use of the antibody and may be enzymes, hormones, or other proteinaceous or non-proteinaceous solutes. In one aspect of the invention, the isolated antibody must be an antibody purified to greater than 95% by weight.

An isolated antibody is an antibody in situ within the recombinant cells in which it is produced, if none of the components of the antibody's natural environment is present. However, as a rule, the selected antibody is obtained using at least one stage of purification, during which the recombinant cellular material is removed.

The term characteristic of an antibody refers to factors that describe the recognition of an antigen by an antibody or the performance of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect properties of the antibody, such as folding, and can affect physical factors, such as the initial rate of binding of the antibody to the antigen (k _a ), the dissociation constant of the antibody from the antigen (kd), the affinity constant of the antibody to the antigen ( Kd), antibody conformation, protein stability, and antibody half-life.

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In the context of the present description, the term epitope tagging refers to an anti-CD40 antibody fused to an epitope tag. An epitope tag is a polypeptide consisting of enough amino acids to create an epitope for antibody production, while being designed so that it does not interfere with the desired activity of a humanized anti-CD40 antibody. An epitope tag is typically largely unique, with the result that an antibody that is generated against an epitope tag does not overreact with other epitopes. Acceptable tag polypeptides typically contain at least 6 amino acid residues, and typically contain about 8-50 amino acid residues, or about 9-30 residues. Examples of epitope tags and antibodies that bind to an epitope are the flu HA polypeptide tag (HA of the influenza virus) and its corresponding 12CA5 antibody (Field et al., Mol. Cell. Biol. 8, 1988 2159-2165); c-myc tag and corresponding antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 (Evan et al., Mol. Cell. Biol. 5(12), 1985, pp. 3610-3616); and a label comprising herpes simplex virus glycoprotein D (gD) and an appropriate antibody (Paborsky et al. Protein Engineering 3(6), 1990, pp. 547-553). In specific embodiments, the epitope tag is an antibody-binding rescue epitope. As used herein, an antibody-binding epitope rescuer refers to an epitope in the Fc region of an IgG molecule (such as IgG1, IgG ₂ , IgG ₃ , or IgG4) responsible for prolonging the in vivo serum half-life of an IgG molecule.

Antibodies can also be conjugated to prodrugs. A prodrug is a precursor or derivative of the pharmaceutically active substance See, for example, Wilman in: Prodrugs in Cancer Chemotherapy, Biochemicals Society Transactions, 14, 1986, cc. 375-382, 615th Belfast Symposium (1986); and Stella et al. in Prodrugs: A Chemical Approach to Targeted Drug Delivery, Directed Drug Delivery, ed. Borchardt et al., Humana Press, 1985, pp. 247-267.

For diagnostic purposes, as well as for monitoring treatment, the antibodies used in the method according to the invention can also be labeled, either labeled alone or labeled in combination with an additional second agent (prodrug, etc.). The label, unlike the second additional agent, is a substance that is a detectable compound or a detectable composition, and it can be conjugated directly or indirectly with a humanized antibody of the present invention. The label itself can be a detectable substance (eg, radioisotope labels or fluorescent labels) or, as in the case of an enzymatic label, it can catalyze a chemical change in a compound or composition that is/is a substrate that can be detected. The labeled humanized anti-CD40 antibody can be prepared and used for a variety of purposes, including in vitro and in vivo diagnostics.

The antibodies used in the method of the invention can be formulated to be part of a liposome formulation for efficient in vivo administration. A liposome is a small vesicle composed of various types of lipids, phospholipids and/or a surfactant that can be used to administer a compound or composition to a mammal, such as the humanized anti-CD40 antibody described herein, in combination or in combination with one or several pharmaceutical active ingredients and/or labels. The components of liposomes are usually ordered in a bilayer structure similar to the lipid structure of biological membranes.

The term treated mammal refers to any animal that belongs to the class of mammals, including humans, domestic and farm animals, and zoo animals intended for sports or pets, such as dogs, horses, cats, cows, etc. .d. Preferably the mammal is a human.

In the context of the present description, the term violation refers to lupus nephritis.

The term intravenous bolus or intravenous pulse refers to the administration of a drug to an animal or human such that the drug enters the body in about 15 minutes or less, typically 5 minutes or less.

Subcutaneous administration refers to the introduction of an agent under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by relatively slow, sustained administration from a drug-containing reservoir. Pinching or pulling the skin up and down from the underlying tissue can create a pocket.

The term subcutaneous infusion refers to the introduction of a drug under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by relatively slow prolonged administration from a reservoir containing the drug over a period of time of (but not limited to) 30 minutes. or less or 90 minutes or less. Optionally, infusion can be performed by subcutaneous implantation of a drug pump implanted under the skin of a diseased animal or human, the pump delivering drug in a predetermined amount over a predetermined time period of 30 minutes, 90 minutes, or a period of time corresponding to duration of the treatment regimen.

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The term subcutaneous bolus refers to the administration of a drug under the skin of a sick animal or human, wherein the duration of drug administration in the form of a bolus is less than about 15 minutes; in another aspect of the invention for less than 5 minutes, and in another aspect of the invention for less than 60 seconds. In a further aspect of the invention, insertion is made into a pocket between the skin and underlying tissue, where the pocket can be created by pinching or pulling the skin up and down from the underlying tissue.

In the context of the present description, the concept of a therapeutically effective amount refers to the amount of active substance, the use of which is the weakening or alleviation of one or more symptoms of the disorder being treated. The active substance is applied in such a way that in the indicated amount it has a beneficial effect on the patient, for example, an effect that manifests itself in the cessation of growth or leading to the elimination of cells. In one aspect of the invention, the active substance, taken in a therapeutically effective amount, has apoptotic activity or the ability to induce cell death. In another aspect of the invention, a therapeutically effective amount refers to the concentration in the patient's serum at which the active substance is found to be effective in retarding the progress of the disease. Efficacy can be assessed in conventional ways depending on the condition being treated. For example, in neoplastic diseases or disorders characterized by the presence of cells that express CD40, efficacy can be assessed by time to disease progression or response rates.

In the context of the present description, the concepts of treatment and therapy, etc. refers to therapeutic, as well as preventive or suppressive measures for a disease or disorder that result in any clinically important or beneficial effect, including (but not limited to) alleviation or amelioration of one or more symptoms, regression, slowing or cessation of the development of the disease or violations. Thus, for example, treatment encompasses the administration of an agent before or after the onset of a symptom of a disease or disorder, which results in the prevention or elimination of one or more signs of the disease or disorder. In another example, the term refers to the administration of an agent after a clinical manifestation of a disease to control the symptoms of the disease. In addition, in the context of the present description, the term treatment or therapy refers to the administration of an agent after the onset and after the development of clinical symptoms, where the administration affects the clinical parameters of the disease or disorder, such as the degree of tissue damage or the number and spread of metastases, regardless of whether whether or not the treatment results in alleviation of the disease. In addition, if the use of the compositions of the invention, either alone or in combination with another therapeutic agent, relieves or alleviates at least one of the symptoms of the disorder being treated in comparison with the specified symptom without the use of a composition containing a humanized anti-CD40 antibody, the result should be considered as an effective treatment for the desired disorder, whether or not all symptoms of the disorder are alleviated.

The term package insert refers to the instructions that are commonly included in the packaging of therapeutic products intended for sale, which contain information about the indications, methods of use, administration, contraindications and / or precautions for the use of these therapeutic products.

Unless specifically stated otherwise in this application, the terms pharmaceutical composition, pharmaceutical product and drug can be used interchangeably.

Antibodies

Humanized anti-CD40 antibodies and binding agents can be used to treat and/or prevent various diseases or disorders characterized by the proliferation of cells that express the CD40 surface antigen, such as lupus nephritis. The humanized anti-CD40 antibody and CD40 binding agent each/each includes at least a region that specifically recognizes a CD40 epitope (ie, an antigen-binding fragment).

Methods for producing an anti-CD40 antibody have previously been described in US 20110243932, the entire content of which is incorporated herein by reference.

As previously described in US 20110243932, initially characterized mouse antibodies were selected based on CD40 binding characteristics.

Based on these initial studies, mouse antibodies were selected that had the heavy chain variable regions shown in Table 1 below and the light chain variable regions shown in Table 2 below.

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Table 1. Mouse anti-CD40 leader antibodies - VH sequences

EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYVHWVKQRPEKGLEWIGR IDPEDGDSKYAPKFQGKATMTADTSSNTAYLHLSSLTSEDTAVYYCTTSY 2H11 YVGTYGYWGQGTTLTVSS (SEQ ID NO:1)

EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYIHWVKQRPEKGLEWIGR IDPEDGDTKYDPKFQGKATMTADTSSNTAYLHLSSLTSEDTAVYYCTTSY 10F2 YVGTYGYWGQGTTLTVSS(SEQ ID NO:2)

EVQLQQSGAELVRPGASVQLSCTASGFNIKDYYVHWVKQRPEKGLEWIGR

IDPED GDTKF APKFQGKATMT ADT S SNT VYLHL S SLT SEDT AVYYCTT SY

19B10 YVGTYGYWGQGTTLTVSS(SEQ ID NO:3)

EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAY ISSGNRIIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTALYYCARQD

20E2 GYRYAMDYWGQGTSVTVSS(SEQ ID NO:4)

Table 2. Mouse anti-CD40 leader antibodies - VK sequences

2H11 TKLEIK (SEQ ID NO:5)

QIVLTQSPTIMSASPGEKVIITCSATSSVSYILWFQQKPGTSPKLWIYST

SNLASGVPARFSGSGSGASYSLTISRMEAEDAATYYCQQRTFYPYTFGGG

10F2 TKLEIK (SEQ ID NO:6)

QIVLTQSPAIMSASPGEKVTITCSASSSVSYMLWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRTFYPYTFGGG 19B10 TKLEIK (SEQ ID NO:7)

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWHQQKPGQPP KLLIYWTSTRESGVPDRFTGSGSGTDFTLTISNLQAEDLAVYYCQNDYTY 20E2 PLTFGAGTKLELK (SEQ ID NO:8)

Human framework sequences were selected for each mouse leader antibody based on framework homology, CDR structure, conserved canonical residues, conserved interface residues, and other parameters.

Selected mouse heavy chain and light chain CDRs of various mouse antibodies are presented in Table 3 and Table 4, respectively.

Table 3. Heavy chain CDR sequences

Designation

designs 2H11 H-CDR1 GFNIKD OOOO H-CDR2 RIDPEDGDSKYAPKFOG H-CDR3 SYYVGTYGY SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:16 10F2 GFNIKD YU/Z RIDPEDGD TKYDPKFOG SYYVGTYGY SEQ ID NO:10 SEQ ID NO:13 SEQ ID NO:16 19B10 GFNIKD OOOO RIDPEDGD TKFAPKFOG SYYVGTYGY SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:16 20E2 GFTF SDYGMH YISSGNRIYYAD TVKG QDGYRYAMDY SEQ ID NO:11 SEQ ID NO:15 SEQ ID NO:17

H-CDR1.

The H-CDR1 sequences shown above are defined according to the Hotia nomenclature (A1 Lazikani et al., JMB 273, 1997, pp. 927-948). CDR1 and CDR2 sequences defined according to the Cabot nomenclature are in bold and italics, and residues defined according to the IMGT nomenclature are underlined in the above table. The H-CDR3 sequences for each of the 2H11, 10F2 and 19B10 antibodies is TTSYYVGTYGY [p NO: 77) and for 20E2 is ARQDGYRYAMDY [p NO: 78).

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Table 4. Light chain CDR sequences

Designation

designs L-CDR1 L-CDR2 L-CDR3 2H11 SASSSVSYML STSNLAS OORTFYPYT SEQ ID NO:18 SEQ ID NO:22 SEQ ID NO:24 10F2 SATSSVSYIL STSNLAS OORTFYPYT SEQ ID NO:19 SEQ ID NO:22 SEQ ID NO:24 19B10 SASSSVSYML STSNLAS OORTFYPYT SEQ ID NO:20 SEQ ID NO:22 SEQ ID NO:24 20E2 KSSOSLLNSGNOKNYL T WTSTRES ONDYTYPLT SEQ ID NO:21 SEQ ID NO:23 SEQ ID NO:25

Again, in Table 4, Hotia nomenclature was used, with sequences defined according to Cabot nomenclature in bold and italics, and those defined according to IMGT nomenclature underlined.

Fab fragments found to have improved or equivalent binding compared to the chimeric parent Fab fragment were selected for conversion to IgG. Clones from the 20E2 series were converted into two different IgG formats: a) IgG4DM (double mutant) had two mutations in the Fc/hinge region, namely the Ser228Pro mutation, which leads to a decrease in the formation of halves of the molecules, and the Leu235Glu mutation, which further reduces FcyR binding. b) IgGIKO (knock-out effector functions) had two mutations in the Fc region, namely Leu234Ala and Leu235Ala, which reduce effector function such as FcyR and complement binding. Both IgG formats are described in the literature. Example 1 describes in more detail the humanization of three candidate antibodies. As a result of this humanization, it was possible to create sequences of humanized antibodies, the sequences of light and heavy chains of which are presented below.

Designation Subsequence SEQID NO: Antibody A (light chain) DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTW 26 HQQKPGQPPKLLIYWTSTRESGVPDRFSGSGS GTDFTLTIS SLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC Antibody A (heavy chain, IgGIKO) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDY GMHWVRQAP 27 GKGLEWVAYIS SGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNS LRAED TALYYCARQDGYRYAMDYWAQGTLVTVSSAS ТК GP S VF PLAP S S KS Т S GGTAALGCLVKDY F РЕ PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDY GMHWVRQAP 28 GKGLEWVAYIS SGNRIIYYADTVKGRFTISRDNAKNSLYLQ

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IgGl) MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSAS ТК GP SVF FLAPS SKS ТSGGTAALGCLVKDYFPE PVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody A (heavy chain, IgG4DM) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP 29 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSAS TK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody A (severe, IgGlKOb) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP thirty GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSSAS TK GP SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (light chain) DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYL 31 TWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKS GTASWCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQ GLS S PVTKS FNRGE C Antibody B (heavy chain, IgGlKO) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP 32 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS SAS TK GP SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgGl) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP 33 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS SAS TK GP SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

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KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody B (heavy chain, IgG4 DM) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP 34 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS SAS TK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody B (heavy chain, IgGlKOb) EVQLVE S GGGLVKPGGS LRLS CAAS GFTFSDYGMHWVRQAP 35 GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS SAS TK GP SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (light chain) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQ QKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIKRT VAAP SVFIFPPSDEQLKS GTAS WCLLNNF Y PREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLTL SKADYEKHKNSVYACEVEKRT 36 Antibody C (heavy chain, IgGlKO) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 37 GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG P SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GAL T S GVHT F PAVLQ S S GLY S LS S WTVP S S S LGTQ T YICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 38 GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG P SVF PLAP S S KS T S GGTAALGCLVKDY F PE PVTVSWNS GAL T S GVHT F PAVLQ S S GLY S LS S WTVP S S S LGTQ TYI CNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgG4 DM) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 39 GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG

- 15 039944

PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody C (heavy chain, IgGlKOb) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP 40 GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG P SVF PLAPS SKS TSGGTAALGCLVKDYFPE PVTVSWNSGAL T S GVHT F PAVLQ S S GLY S LS S WTVP S S S LGTQ T YICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGWFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the humanized anti-CD40 antibodies, including their antigen-binding fragments such as heavy and light chain variable regions, comprise an amino acid sequence that includes residues derived from the CDRs of antibody A (heavy chain sequence set forth in SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29 or SEQ ID NO: 30; light chain sequence shown in SEQ ID NO: 26), antibody B (heavy chain sequence shown in SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34 or SEQ ID NO: 35; light chain sequence shown in SEQ ID NO: 31) and antibody C (heavy chain sequence shown in SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40; the light chain sequence shown in SEQ ID NO: 36), as described above, and amino acid residues derived from human immunoglobulin framework regions. Humanized anti-CD40 antibodies optionally include specific amino acid substitutions in consensus framework regions or germline framework regions.

Specific substitution of amino acid residues in these framework regions can improve various aspects of antibody performance, such as binding affinity and/or stability, as compared to those found in humanized antibodies obtained by direct exchange of CDRs or HVL with human germline framework regions, as demonstrated below in examples.

Some embodiments of the present invention are other monoclonal antibodies whose heavy chain (VH) sequences are shown in SEQ ID NO: 1-SEQ ID NO: 4 and whose light chain (VL) sequences are shown in SEQ ID NO: 5-SEQ ID NO : 8 (see Tables 1 and 2 above). The CDR sequences of these mouse antibodies are shown in Tables 3 and 4, the introduction of these CDRs into the FRs of human heavy and light chain consensus variable regions can result in acceptable humanized antibodies of the present invention.

In some specific embodiments, the humanized anti-CD40 antibodies provided herein comprise at least a heavy or light chain variable region that contains the CDRs or HVLs of the mouse monoclonal antibodies set forth in Tables 1-4 above and the FRs of the heavy or light chain variable regions. and human germline light chains. In exemplary embodiments of the invention, the humanized antibodies generated are: Antibody A, Antibody B, and Antibody C and their various heavy and light chain sequences as set forth in SEQ ID NO: 26-SEQ ID NO: 40.

Specific embodiments of the invention are antibodies that have a heavy chain sequence as set forth in any of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30 in combination with a light chain sequence as set forth in SEQ ID NO: 26. In another embodiment, the antibodies have the heavy chain sequence shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35 in combination with the light chain sequence shown in SEQ ID NO: 31. Additional embodiments of the invention are humanized antibodies that have the heavy chain sequence shown in SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40, in combination with the light chain sequence shown in SEQ ID NO: 36.

The CDRs of these sequences are shown in Tables 3 and 4. In specific embodiments of the invention, it is understood that chimeric antibodies with CDR region substitutions (i.e., for example, the replacement of one or two CDRs in antibody A with similar CDRs from antibody C) between these exemplary immunoglobulins can produce useful antibodies.

In specific embodiments, the humanized anti-CD40 antibody is an antibody fragment. Various antibody fragments are generally described above, as well as methods developed to obtain antibody fragments. Fragments can be obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24, 1992, pp. 107-117 and Brennan et al., Science 229, 1985, p. 81). Alternatively, fragments can be generated directly from recombinant host cells. For example, Fab'-SH fragments can be isolated directly from E. coli and chemically linked to form F(ab') ₂ fragments (see, e.g., Carter et al., Bio/Technology 10, 1992, pp. 163- 167). According to another approach, F(ab') ₂ fragments can be isolated directly from a recombinant host cell culture. Other techniques for obtaining antibody fragments should be obvious to a person skilled in the art.

Specific embodiments of the invention include an F(ab')2 fragment of a humanized anti-CD40 antibody that has a heavy chain sequence as set forth in any of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO : 30, in combination with the light chain sequence shown in SEQ ID NO: 26. Another option is antibodies that have the heavy chain sequence shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35, in combination with the light chain sequence shown in SEQ ID NO: 31. Further embodiments of the invention are humanized antibodies that have the heavy chain sequence shown in SEQ ID NO: 37, SEQ ID NO: 38; SEQ ID NO: 39 or SEQ ID NO: 40, in combination with the light chain sequence shown in SEQ ID NO: 36. Said embodiments of the invention may include an intact antibody containing the indicated F(ab') ₂ fragments.

The effector domain of an antibody can be derived from any suitable vertebrate species and any isotype. Isotypes from different animal species differ in their ability to mediate effector functions. For example, the ability of a human immunoglobulin to mediate CDC and ADCC/ADCP generally follows the order: IgM=IgG ₁ =IgG ₃ >IgG ₂ >IgG ₄ „IgG^IgGpIgG^IgM/IgG, respectively.

The ability of mouse immunoglobulins to mediate CDC and ADCC/ADCP is generally in the following order:

mouse IgM^IgG ₃ » IgG _2b > IgG _2a » IgGi and IgG _2b > IgG _2a > IgG ₁ » IgG _3, respectively.

In another example, mouse IgG _2a mediates ADCC and mouse IgG _2a and IgM mediate CDC.

Antibody modifications

Humanized anti-CD40 antibodies and agents may include modifications to the humanized anti-CD40 antibody or antigen-binding fragment thereof.

Humanized anti-CD40 antibody conjugates can be prepared by known methods using a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(paraazidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(para-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2, 6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared according to the method described in Vitetta et al., Science, 238, 1987, p. 1098). ^C14 labeled 1-isothiocyanatobenzyl3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating a radionucleotide to an antibody. Conjugates can also be made using a cleavable linker.

The humanized anti-CD40 antibodies provided herein can be formulated into immunoliposome-type forms.

Antibody-containing liposomes are prepared using methods well known in the art, such as those described in Epstein et al., Proc. Natl. Acad. sci. USA: 82, 1995, p. 3688; Hwang et al., Proc. Natl. Acad. sci. USA: 77, 1980, p. 4030; US 4,485,045 and 4,544,545. Liposomes with extended circulation life are described in US 5,013,556.

The most preferred liposomes can be obtained by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters with a certain pore size, obtaining liposomes of the required diameter. Fab' fragments of an antibody of the present invention can be incorporated into liposomes according to the method described by Martin et al., J. Biol. Che., 257, 1982, pp. 286-288 via a disulfide bond formation reaction.

In other embodiments, covalent modifications of the humanized anti-CD40 antibody can also be used. Covalent modifications include the cysteia modification

- 17 039944 nyl residues, histidyl residues, lysinyl and amino terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl or threonyl residues. Another type of covalent modification involves the chemical or enzymatic cross-linking of glycosides with an antibody. These modifications can be carried out by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if possible. To introduce other types of covalent modifications into the antibody molecule, the interaction of labeled amino acid residues of the antibody with an organic derivatizing agent that can interact with selected side chains or amino-terminal or carboxy-terminal residues can be used.

Removal of any carbohydrate moieties present on the antibody can be done chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem. Biophys. 259, 1987, p. 52 and in Edge et al., Anal. Biochem., 118, 1981, p. 131. Enzymatic cleavage of carbohydrate fragments on antibodies can be performed using endo- and exoglycosidases according to the method described in Thotakura et al., Meth. Enzymol., 138, 1987, p. 350.

Another type of suitable covalent modification involves linking the antibody to one of numerous non-protein polymers, such as, for example, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, using the methods described in one or more of US 4640835, US 4496689, US 4301144, US 4670417, US 4,791,192 and US 4,179,337.

Humanization and amino acid sequence variants

Amino acid sequence variants of an anti-CD40 antibody can be generated by introducing appropriate nucleotide changes into the DNA of an anti-CD40 antibody or by peptide synthesis. These variants include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the anti-CD40 antibodies exemplified herein. Any combination of deletions, insertions and substitutions is used to obtain the final construct, provided that the final construct has the required characteristics. Amino acid changes can also alter the post-translational processing of the humanized anti-CD40 antibody or variant, such as changing the number or position of glycosylation sites.

A useful method for identifying specific residues or regions in an anti-CD40 antibody that are preferred sites for mutagenesis is so-called alanine-scanning mutagenesis, described in Cunningham and Wells, Science, 244, 1989, pp. 1081-1085). It identifies a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) and replaces it with a neutral or negatively charged amino acid (usually alanine) to affect the interaction of amino acids with antigen CD40. Those amino acid positions that have been shown to be functionally sensitive to substitutions are then improved by the introduction of additional or other variants at the substitution sites or substitution sites. Thus, although the site for the introduction of amino acid sequence variation is predetermined, the nature of the mutation itself is not predetermined. For example, to analyze the efficiency of a mutation at a site in question, an alanine scan or non-specific mutagenesis of the target codon or target region is performed and the resulting anti-CD40 antibody variants are screened for the desired activity.

Insertions into an amino acid sequence include amino- and/or carboxy-terminal fusions of various lengths from one residue to polypeptides that contain one hundred or more residues, as well as insertions into the sequence of one or more amino acid residues. An example of a terminal insertion is an anti-CD40 antibody, an epitope-tagged bullion. Other insertional variants of the anti-CD40 antibody molecule include fusion to the N- or C-terminus of the anti-CD40 antibody of an enzyme or polypeptide that increases the serum half-life of the antibody.

Another type of variant is a variant comprising an amino acid substitution. In these embodiments, at least one amino acid residue in the anti-CD40 antibody molecule is removed and another residue is inserted in its place. The sites of greatest interest for mutagenesis by substitution are the hypervariable regions, but changes to the FR can also be made. Conservative substitutions are presented in table. 5 titled preferred substitutions. If such substitutions result in a change in biological activity, then more significant substitutions, referred to as Examples of substitutions, or other substitutions described below with reference to the amino acid class, can be introduced, and then the products can be screened.

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Table 5

original balance Substitution examples Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; gin; Asn Lys Asn(N) gin; His; asp; Lys; Arg gin Asp(D) Glu; Glu Cys(C) Ser; Ala Ser Gin (Q) asn; Glu Asn Glu(E) asp; gin asp Gly (G) Ala Ala His(H) asn; gin; Lys; Arg Arg He(I) Leu; Val; met; Ala; Ph; norleucine Leu Leu(L) norleucine; lie; Val; met; Ala; Phe lie Lys (K) Arg; Ph; lie Arg Met(M) Leu; Ph; lie Leu Phe(F) Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser TRP(W) Tyr; Phe Tyr Tyr (Y) trp; Ph; Thr; Ser Phe Val(V) lie; Leu; met; Ph; Ala; norleucine Leu

It is generally accepted in protein chemistry that the biological properties of an antibody can be modified by selecting substitutions that vary widely in their effect on maintaining (a) the backbone structure of the polypeptide at the site of the substitution, such as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule in target site or (in) side chain size. Naturally occurring residues are divided into groups based on the common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: Asn, Gln, His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions involve replacing a representative of one of these classes with a representative of another class.

Any of the cysteine residues not involved in maintaining the proper conformation of the humanized anti-CD40 antibody or variant can also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal crosslinking, or to create points for conjugation to the target compound. . Conversely, cysteine(s) bond(s) can be introduced into the antibody to improve its stability (especially if the antibody is an antibody fragment, such as an Fv fragment).

One type of variant generated by substitution involves substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibody). As a rule, the resulting(e) variant(s) selected(s) for further improvement should(s) have improved biological properties relative to the parent antibody from which it (they) derived(s). A suitable way to generate such substitution-based variants involves affinity maturation using phage presentation. In general, the method is as follows: several hypervariable region sites (eg, 6-7 sites) are mutated to give all possible amino acid substitutions at each site. The antibody variants thus obtained are presented in monovalent form on the surface of the filamentous phage particles as fusions with the gene III product of the M13 phage that is present in each particle. Variants that are presented by the phage are then screened for their biological activity (eg, binding affinity). In order to identify hypervariable region sites that are promising for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that are most important for antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex in order to identify points of contact between the antibody and human CD40. Such contact residues and adjacent residues are candidates for replacement using the methods developed in the creation of the present invention. Once such variants have been generated, a panel of variants is screened as described herein and antibodies with improved properties in one or more of the relevant assays are selected for further development.

Another way to obtain an amino acid variant of an antibody is to change the original glycosylation pattern of the antibody. By alteration is meant the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more glycosylation sites not present in the antibody.

In some embodiments of the invention, it may be required to modify the antibodies of the invention by adding glycosylation sites. Glycosylation of antibodies usually occurs through either N-linking or O-linking. N-linking involves the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognizable sequences designed to enzymatically attach a carbohydrate moiety to the side chain of an asparagine residue. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Linked glycosylation involves the attachment of one of the sugars, such as Nacetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Thus, to glycosylate a given protein, such as an antibody, an amino acid sequence of the protein is created that contains one or more of the tripeptide sequences described above (for N-glycosylation sites). The change may also be the addition or replacement of one or more serine or threonine residues in the sequence of the original antibody (for Oglycosylation sites).

Nucleic acid molecules that encode variants of the amino acid sequences of an anti-CD40 antibody are prepared using a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide mutagenesis (or site-directed mutagenesis), PCR mutagenesis, and cassette mutagenesis of a previously generated variant or a non-previous variant. version of the anti-CD40 antibody.

In the context of the present description, the concept of identical or percent identity regarding two or more nucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acid residues when compared and aligned for maximum match. To determine percent identity, sequences are aligned for optimal comparison (eg, gaps can be introduced into the sequence of the first amino acid or nucleotide sequence for optimal primary structure comparison with a second amino acid or nucleotide sequence). Amino acid residues or nucleotides are then compared at the corresponding amino acid positions or nucleotide positions. When a position in the first sequence has the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions in the sequences (ie, % identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In some embodiments, the two sequences being compared are the same length after introducing gaps in the sequence if necessary (eg, excluding additional sequence extending beyond the sequences to be compared). For example, when variable region sequences are compared, leader and/or constant domain sequences are not considered. When comparing the sequences of two sequences, the corresponding CDR refers to the CDR located in the same position in both sequences (eg, CDR-H1 of each sequence).

Determining the percent identity or percent similarity between two sequences can be done using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used to compare two sequences is (but is not limited to) the algorithm of Karlin and Altschul, Proc. Natl. Acad. sci. USA 87, 1990, pp. 2264-2268 modified as described by Karlin and Altschul, Proc. Natl. Acad. sci. USA 90, 1993, pp. 5873-5877. This algorithm is included in the NBLAST and XBLAST programs described by Altschul et al., J. Mol. Biol. 215, 1990, pp. 403-410. Nucleotide analysis with BLAST can be performed using the NBLAST program, in which score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. Protein analysis using BLAST can be performed using the XBLAST program, in which

- 20 039944 score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To perform comparative analyzes of the primary structure, including gaps, you can use the Gapped BLAST program described in Altschul et al., Nucleic Acids, 25, 1997, cc. 3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search to determine distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the respective programs (eg XBLAST and NBLAST) can be used. Another preferred example (but not limited to) of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Myers and Miller, CABIOS, 1989. This algorithm is included in the ALIGN program (version 2.0), which is part of the software package for comparative analysis of the primary structure of GCG sequences. When the ALIGN program is used to compare amino acid sequences, a PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. . Appl. biosci. 10, 1994, pp. 3-5; and FASTA, described by Pearson and Lipman, Proc. Natl. Acad. sci. USA 85, 1988, pp. 2444-2448. In the FASTA program, the ktup parameter denotes a control parameter that sets the sensitivity and speed of the search. If ktup = 2, then similar regions in the two sequences to be compared are identified by looking at pairs of aligned residues; if ktup = 1, then the individual aligned amino acids are evaluated. For protein sequences, ktup can be set to 2 or 1, and for DNA sequences, values ranging from 1 to 6. Alternatively, comparative analysis of the primary structure of protein sequences can be performed using the CLUSTAL W algorithm described in Higgins et al., Methods Enzymol. 266, 1996, pp. 383-402.

Therapeutic Applications

The humanized anti-CD40 antibody or agent is administered by any suitable route, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes of administration and, if topical immunosuppressive treatment is required, by administration to the affected area (including perfusion or other route of contacting the graft with the antibody prior to transplantation) . The humanized anti-CD40 antibody or agent can be administered, for example, by infusion or bolus. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. In addition, the humanized anti-CD40 antibody can be administered by pulsatile infusion, primarily in lower doses of the antibody. According to one aspect of the invention, dosing is carried out by injection, most preferably intravenous or subcutaneous injection, depending on whether the treatment is short-term or prolonged.

To prevent or treat a disease, the appropriate dose of an antibody will depend on a number of factors such as the type of disease being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody, and from the prescription of the attending physician. The antibody can be administered to the patient once or through a series of treatments.

Depending on the type and severity of the disease, from about 1 μg/kg to 20 mg/kg (e.g., about 0.1-15 mg/kg/dose), antibodies can be initially administered to the patient at the initial intended dose, by, for example, one or multiple injections or by continuous infusion. Typically, the daily dose will be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of the present symptoms of the disease. However, other dosing regimens may be used. The success of this therapy is easily assessed by conventional methods and assays. An example of a dosing regimen is provided in WO 94/04188.

In the context of the present description, the concept of suppression has the same meaning as the concepts of relief and relief, and refers to the weakening of one or more characteristics of the disease.

The antibody composition can be formulated, dosed and administered in accordance with good clinical practice. Factors of importance in this regard include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to the medical practitioner. In this regard, the therapeutically effective amount of the antibody to be administered is adjusted to be the minimum amount necessary to prevent, alleviate, or treat a disorder associated with CD40 expression.

The antibody is optional, but in certain cases, included in the formulation in combination with one or more agents currently used to prevent or treat lupus nephritis. The effective amount of said other agents depends on the amount of humanized anti-CD40 antibody present in the formulation and other specified

- 21 039944 above factors. They are generally used at the same doses and using the same routes of administration in which they were previously used, or used at a dose of from about 1 to 99% of their accepted for use doses.

To treat or prevent lupus nephritis using the methods provided herein, an effective amount of an anti-CD40 antibody or an agent is administered to an individual in need of such treatment or prevention, or an agent wherein antibody (I) binds to activated immune cells that express CD40 and which are associated with the disease state, and (II) has an immunosuppressive effect on activated immune cells.

Pharmaceutical Compositions and Their Administration

A composition containing a CD40 binding agent (eg, an anti-CD40 antibody) may be administered to an individual who suffers from or is at risk of developing lupus nephritis. The invention also relates to the use of a CD40 binding agent (eg an anti-CD40 antibody) for the preparation of a medicament for the prevention or treatment of lupus nephritis. The term "individual" as used herein means any mammalian patient to whom a CD40 binding agent can be administered, including, for example, humans and non-human mammals such as primates, rodents, and dogs. Individuals, including humans, in particular, can be treated using the methods presented in the present description. Antibodies or agents can be administered alone or in combination with other compositions for the prevention or treatment of lupus nephritis.

Preferred antibodies for use in said pharmaceutical compositions are those that are a humanized antibody or antibody fragment that has a heavy chain variable region amino acid sequence as set forth in any of

SEQ ID NO: 1-4, SEQ ID NO: 27, SEQ ID NO: 28,

SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO:

40.

In specific embodiments, the CD40 binding agent composition is administered by injection, by catheter, by suppository, or by an implant, wherein the implant consists of a porous, non-porous, or gel-like material, including a membrane, such as a silastic membrane, or fiber. As a rule, when the composition is administered, substances are used that do not absorb the anti-CD40 antibody or agent.

In other embodiments, the anti-CD40 antibody or agent is administered via a controlled release system. In one embodiment, a pump may be used (see, for example, Langer, Science 249, 1990, pp. 1527-1533; Sefton, CRC Crit. Ref. Biomed. Eng. 14, 1989, p. 201; Buchwald et al., Surgery 88, 1980, p.507; Saudek et al., N. Engl. J. Med., 321, 1989, p.574). In another embodiment of the invention, polymeric materials can be used (see, for example, Medical Applications of Controlled Release, ed. Langer and Wise, CRC Press, Boca Raton, Florida, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance, ed. Smolen and Ball, Wiley, New York, 1984); Ranger and Peppas, Macromol. sci. Rev. macromol. Chem. 23, 1983, p. 61; see also Levy et al., Science 228, 1985, p. 190; During et al., Ann. Neurol. 25, 1989, p. 351; Howard et al., J. Neurosurg. 81, 1989, p. 105). Langer, supra, discusses other controlled release systems.

A CD40 binding agent (eg, an anti-CD40 antibody) may be administered as pharmaceutical compositions containing a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingredients.

According to typical embodiments of the invention, the pharmaceutical composition is prepared according to conventional procedures to obtain a pharmaceutical composition suitable for intravenous or subcutaneous administration to humans. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical agent may also contain a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Typically, the ingredients are either supplied individually or mixed to form a unit dose of the drug, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active ingredient. Where the pharmaceutical agent is to be administered by infusion, it may be filled into infusion bottles containing pharmaceutical grade sterile water or saline. Where the pharmaceutical agent is administered by injection, an ampoule of sterile water for injection or saline may be used so that the ingredients can be mixed prior to administration.

In addition, the pharmaceutical composition may be included in a pharmaceutical kit that

- 22 039944 contains (a) a container with a CD40 binding agent (eg, an anti-CD40 antibody) in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (eg, sterile water) for injection. A pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized anti-CD40 antibody or agent. Optionally, the specified container(s) may be accompanied by a notice in a form approved by an official agency authorizing the manufacture, use, or sale of pharmaceuticals or biological products, where the notice reflects the agency's approval for manufacture, use, or sale for the purpose of administration to humans.

In one embodiment, the pharmaceutical composition comprises an aqueous composition that has an anti-CD40 antibody concentration of about 10 mg/mL to about 200 mg/mL; or from about 100 mg/ml to about 200 mg/ml; or from about 120 mg/ml to about 180 mg/ml; or about 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/mL.

In addition to the anti-CD40 antibody, the pharmaceutical composition may further comprise a buffer, a stabilizing agent, and optionally a pH adjusting agent. Non-limiting examples of buffering agents include one or more salts such as sodium chloride, arginine hydrochloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride, and sodium acetate; or salts of suitable acids such as acetic acid and amino acids. The buffering agent is added in an amount sufficient to provide a viscosity suitable for administering the drug to a patient, for example by injection. The pharmaceutical composition may contain from about 100 mm up to about 200 mm salt or buffer, or from about 120 mm up to about 180 mm salt or buffer. In one embodiment, the pharmaceutical composition contains a buffer containing sodium acetate at a concentration of from about 20 mm to about 30 mm and sodium chloride at a concentration of from about 120 mm to about 140 mm. In another embodiment, the pharmaceutical composition contains a buffer containing sodium acetate at a concentration of approximately 25 mm and sodium chloride at a concentration of approximately 130 mm.

A non-limiting example of a suitable stabilizing agent is Polysorbate 20 (Tween 20). The stabilizing agent is present in an amount sufficient to maintain the chemical and physical stability of the pharmaceutical composition. The pharmaceutical composition may contain from about 0.001% to about 0.1% (w/v) of a stabilizing agent; or from about 0.0015% to about 0.015% (w/v) of a stabilizing agent; or about 0.01% (w/v) stabilizing agent.

In one embodiment, the pharmaceutical composition contains an anti-CD40 antibody in an amount of from about 120 mg/ml to about 180 mg/ml; a buffer containing sodium acetate at a concentration of from about 20 mm to about 30 mm and sodium chloride at a concentration of from about 120 mm to about 140 mm; and a surfactant that is polysorbate 20 at a concentration of from about 0.0015 to about 0.015% (w/v). In another embodiment, the anti-CD40 antibody preparation contains an anti-CD40 antibody in an amount of about 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/mL; buffer containing sodium acetate at a concentration of approximately 25 mm and sodium chloride at a concentration of approximately 130 mm; and a surfactant, which is polysorbate 20 at a concentration of approximately 0.01% (w/v).

In another embodiment, each of the pharmaceutical compositions described above may contain from about 70 mg to about 250 mg of an anti-CD40 antibody; or about 80 to 240 mg of an anti-CD40 antibody. In another embodiment, the pharmaceutical compositions described above contain 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 mg antibodies to CD40.

Each of the pharmaceutical compositions described above has a pH of from about 4.0 to about 12.0; or from about 5 to about 6.0; or about 5.5. The pH can be adjusted by adding a sufficient amount of a suitable pH adjusting agent such as an acid (eg hydrochloric acid) or a base (eg sodium hydroxide).

In another embodiment, the invention relates to a method of using any of the pharmaceutical compositions containing an anti-CD40 antibody as described herein for the treatment or prevention of lupus nephritis.

The amount of a CD40 binding agent (eg, an anti-CD40 antibody) that is effective in treating or preventing lupus nephritis characterized by CD40 expression can be determined by conventional clinical testing methods. Additionally, in vitro assays may optionally be used, the results of which may help determine optimal dosage ranges. The exact dosage to be used in the formulation should also depend on the route of administration and the stage of lupus nephritis characterized by CD40 expression, and should be taken based on the advice of the attending physician and the circumstances of each patient.

- 23 039944

Effective doses can be determined by extrapolation from dose-response curves obtained from in vitro experiments or animal model test systems.

For example, the toxicity and therapeutic efficacy of an anti-CD40 antibody or agent can be determined in cell cultures or experimental animals using standard pharmaceutical methods used to determine ED50 (therapeutically effective dose for 50% of the population). A CD40 binding agent (eg, an anti-CD40 antibody) that has a high therapeutic index is preferred. In cases where toxic side effects are found with the use of a CD40 binding agent, an administration system that provides targeted delivery of the CD40 binding agent to the affected tissue can be used to minimize potential damage to cells that do not express CD40, and thereby to reducing side effects.

Data obtained from cell culture assays and animal studies can be used in determining the dose limit intended for human use. Typically, the dose of the CD40 binding agent is in the range of circulating concentrations that include the ED50 while having little or no toxicity. The dose can be varied within the indicated limits depending on the dosage form used and the route of administration used. For any CD40 binding agent used according to the method, the therapeutically effective dose may initially be determined from the results of cell culture assays. The dose can be determined from data obtained in animal models by reaching plasma concentration limits that include the IC50 (i.e. the concentration of the test compound at which symptom inhibition is achieved, which is half the maximum) according to cell culture data. . Such information can be used to more accurately determine suitable doses for humans. Plasma concentrations can be determined, for example, by high performance liquid chromatography, ELISA, and the like.

Typically, the dose of an anti-CD40 antibody or CD40 binding agent that is administered to a patient with lupus nephritis characterized by CD40 expression is about 0.1 to about 100 mg/kg of the individual's body weight. The dose administered to an individual is about 0.1 to about 50 mg/kg, about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, or about 1 to about 10 mg/kg of an individual's body weight.

Examples of doses are (but are not limited to) doses ranging from 1 ng/kg to 100 mg/kg. In some embodiments, the dosage is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg , about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg or about 16 mg/kg. The dose can be administered, for example, every day, once a week (every week), twice a week, three times a week, four times a week, five times a week, six times a week, once every two weeks or monthly, every two months or every three months. In specific embodiments, the dosage is about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/kg/week, approximately 6 mg/kg/week, approximately 7 mg/kg/week, approximately 8 mg/kg/week, approximately 9 mg/kg/week, approximately 10 mg/kg/week, approximately 11 mg /kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week, or about 16 mg/kg/week. In some embodiments, the dosage range is from about 1 mg/kg/week to about 15 mg/kg/week.

In another embodiment, the dose is from about 70 mg to about 250 mg per week; or about 80 to 240 mg per week. In another embodiment, the dose is about 80 mg per week, 120 mg per week, 130 mg per week, 140 mg per week, 160 mg per week, 170 mg per week, 180 mg per week, 200 mg per week, 210 mg per week, 220 mg per week, mg per week, 240 mg per week, or 250 mg per week.

In some embodiments of the invention, pharmaceutical compositions containing a CD40 binding agent may further include a therapeutic agent conjugated or unconjugated with a binding agent. The anti-CD40 antibody or CD40 binding agent may be co-administered with one or more therapeutic agents used to treat or prevent lupus nephritis.

Such combination therapy may have an additive or synergistic effect in relation to the signs of the disease (for example, the severity of the symptom, the number of symptoms, or the frequency of relapses).

In combination administration regimens, in a particular embodiment, the anti-CD40 antibody or CD40 binding agent is co-administered with the therapeutic agent. In another specific embodiment, the therapeutic agent is administered before or after administration of the anti-CD40 antibody or CD40 binding agent for at least 1 hour to several months, such as at least 1 hour, 5 hours, 12 hours, days , weeks, months, or three months before or after administration of an anti-CD40 antibody or CD40 binding agent.

- 24 039944

In some embodiments, the additional therapeutic agent is an immunosuppressant. The immunosuppressant may be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, mycophenolate (MMF), cyclophosphamide (CyP), azathioprine, hydroxychloroquine, mizoribine, mycophenolate mofetil, or methotrexate. Alternatively, the immunosuppressant may be, for example, a glucocorticoid (eg cortisol or aldesterone) or a glucocorticoid analogue (eg prednisone or dexamethasone). In another embodiment, the immunosuppressant may be an angiotensin converting enzyme (ACE) inhibitor (eg captopril, quinapril or enalapril) or an angiotensin II receptor blocker (ARB) (eg losartan or candesartan).

In another embodiment, the additional therapeutic agent is selected from the group consisting of mycophenolate (MMF), cyclophosphamide (CyP), a glucocorticoid (GC), and corticosteroids, or any combination thereof.

In one embodiment, the additional therapeutic agent is mycophenolate (MMF).

In another embodiment, the additional therapeutic agent is cyclophosphamide (CyP).

In another embodiment, the additional therapeutic agent is a glucocorticoid (GC).

In another embodiment, the additional therapeutic agent is a corticosteroid.

Products

Another object of the invention is an article containing substances suitable for the treatment of the disorders described above. The product includes a container and a label. Acceptable containers are, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass or plastic. The container contains a composition that is effective for treating a particular condition and may have a sterile access port. For example, the container may be an intravenous solution bag or vial having a closure device that is permeable to a hypodermic injection needle. The active substance in the composition is a humanized anti-CD40 antibody. The label on or associated with the container indicates that the composition is used to treat the selected condition. The article of manufacture may further include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. It may additionally include other substances whose presence is desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The invention is described below with the help of the presented examples, which are not intended to limit the scope of the invention.

Examples

Example 1. Obtaining a humanized antibody to CD40.

Humanized anti-CD4 antibodies according to the invention can be obtained in accordance with the procedures described in US 20110243932. Antibody A, Antibody B and Antibody C were humanized antibodies derived from mouse antibody 20E2 (Antibody A and Antibody B) or from 2H11 ( Antibody C) cloned in a human IgG1-KO (KO=knockout)/kappa framework. IgG1-KO has two mutations in the Fc region, Leu234Ala and Leu235Ala, which decrease FcyR and complement binding.

As a result of this humanization, various humanized heavy and light chain variable region sequences were obtained, which are presented below.

- 25 039944

SEQ ID NO: 41 (light chain variable region sequence):

DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO: 42 (heavy chain variable region sequence):

EVQLVKSGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA DTVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO:43 (light chain variable region sequence)

DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO: 44 (heavy chain variable region sequence)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA DTVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS

SEQ ID NO: 45 (light chain variable region sequence)

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIK

SEQ ID NO: 46 (heavy chain variable region sequence)

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA DTVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS.

- 26 039944

SEQ ID NO: 47 (light chain variable region sequence)

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO: 48 (heavy chain variable region sequence)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA DTVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO: 49 (light chain variable region sequence)

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGAGTKVEIK

SEQ ID NO: 50 (light chain variable region sequence)

EVQLVESGGGLVKPGGSRRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA DTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO: 51 (light chain variable region sequence)

DIVMTQSPDSLAVSLGEKVTMNCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDYTYPLTFGAGTKVEIK.

SEQ ID NO: 52 (light chain variable region sequence)

DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYLTWHQQKPGQPPKLLIYWTSTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIK

SEQ ID NO: 53 (heavy chain variable region sequence)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGNRIIYYA

DTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVSS

SEQ ID NO: 54 (light chain variable region sequence)

QIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLWIYSTSNLASGVPARF SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:55 (light chain variable region sequence)

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPARF SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:56 (light chain variable region sequence)

DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQQKPGKAPKLLIYSTSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO:57 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

- 27 039944

SEQ ID NO:58 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:59 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDSKYA PKFQGKVTMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS.

SEQ ID NO:60 (Heavy Chain Variable Region Sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDSKYA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:61 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDSKYA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:62 (heavy chain variable region sequence):

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:63 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQRPGQGLEWMGRIDPEDGDTKFA PKFQGKVTMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:64 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWIGRIDPEDGDTKFA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:65 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:66 (heavy chain variable region sequence)

QVQLVQSGAEVKKPGASVKVSCTASGFNITDYYVHWVKQAPGQGLEWMGRIDPEDGDTKFA PKFQGKATMTADTSTSTVYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS

SEQ ID NO:67 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYD PKFQGRVTMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:68 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYD PKFQGRVTMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

- 28 039944

SEQ ID NO:69 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYD

PKFQGKVTMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:70 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWMGRIDPEDGDTKYD

PKFQGKATMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:71 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQRPGKGLEWMGRIDPEDGDTKYD

PKFQGKATMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:72 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCTVSGFNIKDYYIHWVKQAPGKGLEWIGRIDPEDGDTKYD

PKFQGKATMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:73 (heavy chain variable region sequence)

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVQQAPGKGLEWMGRIDPEDGDTKYD

PKFQGRVTMTADTSTDTAYMELSSLRSEDTAVYYCTTSYYVGTYGYWGQGTTVTVSS

SEQ ID NO:74 (light chain variable region sequence) 1 from 10F2Hum antibody:

DIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO: 75 (light chain variable region sequence) 2 from 10F2Hum antibody:

DIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLLIYSTSNLASGVPARF

SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

SEQ ID NO: 76 (light chain variable region sequence)

QIQMTQSPSSLSASVGDRVTITCSATSSVSYILWFQQKPGKAPKLWIYSTSNLASGVPARF

SGSGSGTDFTLTISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIK

Examples of humanized antibodies of the present invention are antibodies that have the heavy and light chain sequences shown in the table below. The underlined sequences in bold in the table below correspond to the variable regions, and the ununderlined sequences in normal font correspond to the constant regions.

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Designation Subsequence SEQID NO: Antibody A (light chain) DIVMTQSPDSLAVSLGERATMSCKSSQSLLNSGNQKNYLTW HQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVAAPSVFI F R R S DEQ LKS GTAS WCLLNNF Y PREAKVQWKVDNALQ S GN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 26 Antibody A (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS AS ТК GP SVF PLAP S SKS Т S GGTAALGCLVKDY F РЕ PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 27 Antibody A (heavy chain, IgGl) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS AS TK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28 Antibody A (heavy chain, IgG4DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS AS TK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKS RWQ E GNVF S CSVMHEALHNHY TQKSLSLSLGK 29 Antibody A (heavy chain, IgGlKOb) EVQLVE S GGGLVKPGGS LRL S CAAS GFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCARQDGYRYAMDYWAQGTLVTVSS AS TK GP SVF PLAP S SKS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK thirty Antibody B (light chain) DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYL TWHQQKPGQPPKLLIYWTSTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQNDYTYPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKS GTASWCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKDSKDSTYSLSSTLTLSKADYEKSSFKVYACE 31

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Antibody B (heavy chain, IgGlKO) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS SAS ТК GP SVF PLAPS SKS ТSGGTAALGCLVKDYFРЕ PVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 32 Antibody B (heavy chain, igGi) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS S AS TK GP SVF PLAP S SKS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33 Antibody B (heavy chain, IgG4 DM) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS S AS TK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKS RWQ E GNVF S CSVMHEALHNHYTQKSLSLSLGK 34 Antibody B (heavy chain, IgGlKOb) EVQLVE S GGGLVKPGGS LRL S CAAS GFTFSDYGMHWVRQAP GKGLEWVAYISSGNRIIYYADTVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARQDGYRYAMDYWGQGTLVTVS S AS TK GP SVF PLAP S SKS T S GGTAALGCLVKDY F PE PVTVSWNS GA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 35 Antibody C (light chain) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMLWFQ QKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQRTFYPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL S KADYVE CHKVYACE S KADYVE CHKVYACE S 36 Antibody C (heavy chain, IgGlKO) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSASTKG P SVF PLAP S S KS T S GGTAALGCLVKDYF PE PVTVSWNS GAL 37

- 31 039944

TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGWFSCSVMHEALHNHYTQKSLSLSPGK Antibody C (heavy chain, IgGl) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSS AS TKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 38 Antibody C (heavy chain, IgG4 DM) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS TKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQ FNS TYRWSVLTVLHQDWLNGKE YKCKVSNKGLP S SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 39 Antibody C (heavy chain, IgGlKOb) QVQLVQSGAEVKKPGASVKVSCTASGFNIKDYYVHWVKQAP GQGLEWMGRIDPEDGDSKYAPKFQGKATMTADTSTSTVYME LSSLRSEDTAVYYCTTSYYVGTYGYWGQGTLVTVSSAS TKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL T S GVHT F PAVLQ S S GLY S LS S WTVP S S S LGTQ T YICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 40

The variable regions were subcloned into one or two different IgG suitable expression vectors.

A) Format: human IgG1-KO (knockout)/kappa with Leu234A1a, Leu235A1a double mutation in the Fc region resulting in reduced effector function such as FcyR binding. and complement.

B) Format: human IgG4-DM (double mutant)/kappa with a Ser228Pro mutation in the hinge that reduces the occurrence of IgG4 halves and a Leu235Glu mutation further reduces FcyR binding.

Antibody A and Antibody B were purified and evaluated according to the following criteria:

appearance, CCF (haze);

filtration capacity, CCF;

yield on recombinant protein A;

turbidity in evaluation and neutralization;

soluble aggregates (SEC);

purity / pollution characteristics (SDS);

Charge Distribution (IEF).

Example 2

SLE is a systemic autoimmune disease characterized by a loss of B cell tolerance to various self-antigens, especially nucleic acids and their binding proteins. These autoantibodies form immune complexes that are deposited in various tissues in the body, and in particular with other factors, trigger the uptake of inflammatory cells and mediators into the kidneys, leading to lupus nephritis. The formation of autoantibodies depends on the mutual

- 32 039944 interaction of T cells with B cells in the lymph node and requires CD40-CD40L to trigger B cell proliferation and germinal center formation. At the germinal center, CD40-CD40L interactions mediate B cell maturation by Ig isotype switching, somatic mutation, clonal expansion of high affinity B cells, and eventual differentiation into plasma cells (Tarlington, 1998). Aberrant expression of CD40L on T cells and platelets in patients with lupus (Koshy et al., 1996 and Mapea et al., 2009) may also serve as a mechanism for triggering autoreactive B cells. Finally, overexpression of CD40L in SLE B cells leads to spontaneous production of autoantibodies in a T cell independent manner (Grammer et al., 2003). See also US 20110243932, the entire content of which is incorporated into this application by reference.

Further evidence for the involvement of the CD40 pathway in lupus nephritis is elevated levels and expression of CD40 and CD40L in affected patients. Along with elevated membrane bound CD40L, soluble CD40L is elevated in the circulation of patients with (Goules et al., 2006) and this increase correlates with disease activity. Increased expression of CD40 in monocytes also correlates with disease activity in lupus nephritis (Kuroiwa et al., 2003). In addition to the role of the CD40 pathway in the formation of autoantibodies and inflammation in the kidney, the findings indicate the involvement of this pathway in the modulation of non-immune cells that contribute to inflammation in lupus nephritis. For example, activation of endothelial cells through the CD40-CD154 interaction triggers the production of pro-inflammatory cytokines and chemokines, matrix metalloproteinase and tissue factor expression and increases the density of leukocyte adhesion molecules CD62E, CD106, and CD54 (Pulivenet et al., 2008). These events play an important role in stimulating hemorrhage and the accumulation of activated T cells at sites of inflammation. Human renal mesangial cells express low levels of CD40 and activate this receptor in response to treatment with IFN-γ and activated CD154+ platelets from SLE patients. (Delams et al., 2005). Similarly, in proximal tubular epithelial cells (PTEC), induction of CD40L mediated cytokines such as IL-6, IL-8 is observed.

The effects of CD40 ligation on human endothelial cells (HUVEC) and human proximal tubular epithelial cells (PTEC) can be assessed by stimulating these cells with CD40L. HUVEC was obtained from Lonza Allendale, NJ HUVEC P901 (Cat. No. C2519A, Lot 0000220212). Cells were cultured in HUVEC medium (Lonza EBM-2 medium with single bullet supplements) and subculture reagents: (Lonza Allendale, NJ cat. CC-3156, lot 0000243756). Cells were grown in BD Biocoat Collagen I T75 flasks (BD Biosciences San Jose, CA cat. 356485, lot 1108951). Cells were subcultured using a Lonza subculture kit according to the manufacturer's instructions.

In FIG. 1 shows that soluble CD40L induces inflammatory cytokines such as MCP-1 in HUVEC, which is effectively blocked by typical Antibody B (·) or isotype control (human IgG1 antibody) (^), depending on the dose.

In FIG. 2 shows the response to sCD40L in PTEC from three different human donors. PTECS was obtained from Lonza, cat no. CC-2553, and included three different donors (D1, D2, D3). Cells were grown in renal cell growth medium (Renal Cell Growth Medium (REGM)) containing 50 ng/ml sCD40L (recombinant human mega CD40L, Alexis Biochemical-Enzo Life Science Farmingdale, NY cat. 522-110-C010, lot 26945 , 10 μg/ml). The REGM SingleQuot kit (Lonza, Cat # CC-4127, Lot # 0000193027) provided a medium containing 0.5% fetal bovine serum (FBS), hydrocortisone, human epidermal growth factor (hEGF), epinephrine, insulin, triiodothyronine, transferrin, gentamicin/amphotericin-B. The results in FIG. 2 indicate that PTECs from three different human donors produce IL-6 in response to sCD40L stimulation, and this response can be dose-dependently inhibited by antibody B (antibody A concentrations from 0 to 2000 ng/mL).

The Applicant believes that these results and other disclosures as described herein establish a compelling rationale for the CD40 pathway playing a central role in the pathogenesis of lupus nephritis.

Claims (5)

CLAIM 1. Use of a humanized monoclonal antibody that specifically binds to human CD40 for the prevention or treatment of lupus nephritis, wherein said antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 53 and a light chain variable region as set forth in SEQ ID NO: 52 . 2. Use of a humanized monoclonal antibody according to claim 1, wherein the antibody is an IgG1 antibody. 3. Use of a humanized monoclonal antibody according to claim 1 or 2, wherein the antibody has a Leu234A1a and Leu235A1a mutation. 4. The use of a humanized monoclonal antibody according to one of claims 1 to 3, containing the heavy chain of SEQ ID NO: 35 and the light chain of SEQ ID NO: 31. - 33 039944 5. Use of a humanized monoclonal antibody according to one of claims 1 to 4, wherein said antibody is administered parenterally, intravenously, or subcutaneously.