Antibodies, Uses, and Methods

Abstract

The present invention relates to antibodies, uses, and methods. Specifically, the present invention relates to anti-human OX40L antibodies, new medical uses, and methods.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580023098.5, filed on March 3, 2015, entitled "Antibody, Use and Method". Technical Field

[0002] This invention relates to anti-human OX40L antibodies, novel medical uses, and methods. Background Technology

[0003] OX40 ligand (OX40L) is a member of the TNF family, a 34 kDa type II transmembrane protein. The crystalline complex of human OX40 and OX40L is a trimer consisting of one OX40L (trimer) and three OX40 monomers. The human extracellular domain is 42% homologous to mouse OX40L.

[0004] OX40L is not constitutively expressed, but it can be induced in professional APCs such as B cells, dendritic cells (DCs), and macrophages. It can also induce expression of OX40L in other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and natural killer (NK) cells. T cells can also express OX40L. The OX40L receptor, OX40, is expressed on activated T cells (CD4 and CD8 T cells, Th2, Th1, and Th17 cells) and CD4+ T cells. + FoxP3 + It is expressed on cells, even when it is not activated.

[0005] The interaction between OX40 and OX40L occurs during T cell-DC interaction 2 or 3 days after antigen recognition. After leaving the DC, OX40-expressing T cells can interact with OX40L-expressing cells, but not the DCs, and receive OX40 signals from these cells. This provides essential signaling for the generation of memory T cells, enhanced Th2 responses, and prolonged inflammatory responses. OX40 signaling entering responding T cells causes them to resist Treg-mediated inhibition.

[0006] Graft-versus-host disease (GVHD) is a leading cause of death following allogeneic bone marrow transplantation. In the acute form of this disease, mature T cells present in the bone marrow graft recognize donor tissue as foreign in a damaged tissue environment. This recognition triggers donor T cell activation and proliferation via host APCs, followed by T cell migration to the liver, spleen, intestine, skin, and lungs, causing tissue damage through CTL effector responses and the release of inflammatory cytokines / chemokines. Acute onset typically occurs within the first 100 days post-transplantation (Hill-Ferrara, Blood, May 1, 2000, Vol. 95, No. 9, pp. 2754-275; Reddy-Ferrara, Blood, Vol. 17, No. 4, December 2003).

[0007] Chronic GvHD typically occurs 100 days after transplantation and is thought to involve several factors, including thymic damage caused by previous acute GvHD leading to reduced clearance of pathogenic T cells (Zhang et al., September 1, 2007, Vol. 179, No. 5, pp. 3305-3314), fibrotic TGF-β upregulation (McCormick et al., J Immuno, November 15, 1999, Vol. 163, No. 10, pp. 5693-5699), and B-cell components driven by elevated B-cell activating factor (BAFF) (Sarantopoulos et al., Clin Cancer Res, October 15, 2007, pp. 13; 6107), as well as autoantibodies against platelet-derived growth factor receptor (Svegliati et al., Blood, July 1, 2007, Vol. 110, No. 1, pp. 237-241).

[0008] Clinical studies have confirmed that OX40 is upregulated in both acute (Morante et al., Clinical and Experimental Immunology, 145:36-43) and chronic (Kotani et al., Blood, November 15, 2001, Vol. 98, No. 10, 3162-3164) GvHD. Administration of antagonistic anti-OX40L improved survival in a lethal acute mouse model of GvHD, with a 70% survival rate in the treated group compared to the untreated group, all of whom died by day 43 (Tsukada et al., Blood, April 1, 2000, Vol. 95, No. 7), while treatment with agonistic anti-OX40 Ab accelerated the disease and mortality (Blazar et al., Blood, May 1, 2003, Vol. 101, No. 9, 3741-3748). Blocking the OX40-OX40L interaction has been shown to be effective in several other inflammatory diseases, including the use of anti-OX40L Ab in treating a mouse model of colitis (Totsuka et al., AJP-GI, April 1, 2003, Vol. 284, No. 4, pp. 595-603), and anti-OX40LAb can block the development of diabetes in NOD mice (Pakala et al., European Journal of Immunology, Vol. 34, No. 11, pp. 3039-3046, November 2004).

[0009] References

[0010] Lamb, LS, Abhyankar, SA, Hazlett, L., O'Neal, W., Folk, RS, Vogt, S., Parrish, RS, Bridges, K., Henslee-Downey, PJ and Gee, AP (1999) Expression of CD134(0X-40) on T-cells during the first 100 days following allogeneic bonemarrow transplantation as a marker for Iymphocyte activation and therapy-resistant graft-versus-host disease. Cytometry, 38: 238-243.

[0011] Xupeng Ge, Julia Brown, Megan Sykes, Vassiliki A.Boussiotis, CD134-Allodepletion Allows Selective Elimination of Alloreactive Human T-cells without Loss of Virus-Specific and Leukemia-Specific Effectors, Biology of Blood and Marrow Transplantation, Volume 14, Issue 5, May 2008, Pages 518-530.

[0012] Naoto Ishii, Takeshi Takahashi, Pejman Soroosh, Kazuo Sugamura, Chapter3-OX40-OX40 Ligand Interaction in T-Cell-Mediated Immunity and Immunopathology, In: Frederick W.Alt, Editor(s), Advances in Immunology, AcademicPress, 2010, Volume 105, Pages 63-98.

[0013] Croft, M., So, T., Duan, W. and Soroosh, P. (2009), The significance of OX40and OX40L to T-cell biology and immune disease. Immunological Reviews, 229: 173-191. Summary of the Invention

[0014] This invention provides anti-human OX40L (hOX40L) antibodies and fragments, and novel medical applications for treating or preventing hOX40L-mediated diseases or symptoms in humans. To this end, this invention provides: -

[0015] In the first mode

[0016] An antibody or fragment thereof that specifically binds to hOX40L, used in a method for treating or preventing hOX40L-mediated diseases or conditions in a human, wherein the antibody or fragment is administered to the human, wherein the antibody or fragment is used to treat or prevent the hOX40L-mediated diseases or conditions by reducing one, several, or all of the following:

[0017] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0018] b. The proliferation of human white blood cells; and

[0019] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells.

[0020] In the second mode

[0021] An antibody or a fragment thereof that specifically binds to hOX40L and competes with antibodies selected from 02D10, 10A07, 09H04 and 19H01 for binding to said hOX40L.

[0022] In the third mode

[0023] Use of antibodies or fragments thereof that specifically bind to hOX40L in the manufacture of pharmaceutical agents intended for administration to humans to treat or prevent hOX40L-mediated diseases or conditions in humans by reducing one, more, or all of the following:

[0024] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0025] b. The proliferation of human white blood cells; and

[0026] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells.

[0027] In the fourth mode

[0028] A method for treating or preventing hOX40L-mediated diseases or symptoms by reducing one, more, or all of the following:

[0029] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0030] b. The proliferation of human white blood cells; and

[0031] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells;

[0032] The method includes administering to the person a therapeutically effective amount of an antibody or fragment that specifically binds to hOX40L.

[0033] In the fifth mode

[0034] An antibody or fragment thereof that specifically binds to hOX40L and competes with antibody O2D10 for binding to hOX40L, wherein the antibody or fragment comprises a VH domain comprising HCDR3 containing the motif VRGXYYY, wherein X is any amino acid.

[0035] In the sixth mode

[0036] An antibody or fragment thereof that specifically binds to hOX40L and competes with antibody 02D10 for binding to hOX40L, wherein the antibody or fragment comprises a VH domain comprising the HCDR3 sequence of SEQ ID NO: 40 or 46 or comprising the HCDR3 sequence of SEQ ID NO: 40 or 46 with fewer than 5 amino acid substitutions.

[0037] In the seventh mode

[0038] A human antibody or fragment thereof comprising 16 to 27 amino acids of HCDR3 and derived from a recombinant human VH gene fragment, a human D gene fragment, and a human JH gene fragment, wherein the human JH gene fragment is IGHJ6, the antibody or fragment specifically binding to hOX40L to treat or prevent autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection.

[0039] In the eighth mode

[0040] Use of a human antibody or fragment thereof comprising 16 to 27 amino acids and derived from a recombinant human VH gene fragment, a human D gene fragment, and a human JH gene fragment in the manufacture of a medicament for administration to humans for the treatment or prevention of hOX40L-mediated diseases or conditions in humans, wherein the human JH gene fragment is IGHJ6, the antibody or fragment specifically binds to hOX40L, and the hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection.

[0041] In the ninth mode

[0042] A method for treating or preventing hOX40L-mediated diseases or conditions, wherein the hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection, the method comprising administering to the person a therapeutically effective amount of a human antibody or fragment thereof comprising 16 to 27 amino acids and derived from a human VH gene fragment, a human D gene fragment, and a human JH gene fragment, wherein the human JH gene fragment is IGHJ6, wherein the antibody or fragment specifically binds to hOX40L, thereby treating or preventing the hOX40L-mediated diseases or conditions.

[0043] The present invention also provides pharmaceutical compositions, reagent kits, nucleic acids, vectors, and hosts. Attached Figure Description

[0044] Figure 1 Characterization analysis of the fully human recombinant anti-OX40L antibody in the HTRF ligand / receptor neutralization assay revealed the role of the anti-OX40L antibody in OX40L / OX40R binding. The data shown represent three replicate experiments.

[0045] Figure 2This study aimed to determine the role of anti-OX40L antibody in allogeneic PBMC / T mixed lymphocyte responses. Data presented are from three independent donor pairs, where each donor was assumed to be a different individual. Figure A shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR percentage inhibition (donor pair 1); Figure B shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR IFNγ (donor pair 1); Figure C shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR percentage inhibition (donor pair 2); Figure D shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR IFNγ (donor pair 2); Figure E shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR percentage inhibition (donor pair 3); and Figure F shows the effect of anti-OX40L antibody relative to IgG-free wells in PBMC / T MLR IFNγ (donor pair 3). Detailed Implementation Plan

[0046] The present invention provides the following aspects 1-113.

[0047] This invention is useful, for example, for the treatment or prevention of transplant rejection, such as graft-versus-host disease (GvHD) or allogeneic transplant rejection. This invention is also useful, for example, for the treatment or prevention of inflammatory bowel disease, such as UC or CD, or for the treatment or prevention of inflammatory airway diseases or symptoms. In one instance, this aspect is useful for the treatment or prevention of asthma. This invention is also useful, for example, for the treatment or prevention of fibrosis. This invention is also useful, for example, for the treatment or prevention of diabetes. This invention is also useful, for example, for the treatment or prevention of uveitis. This invention is also useful, for example, for the treatment or prevention of pyoderma gangrenosa. This invention is also useful, for example, for the treatment or prevention of giant cell arteritis. This invention is also useful, for example, for the treatment or prevention of Schnitzler syndrome. This invention is also useful, for example, for the treatment or prevention of non-infectious scleritis.

[0048] 1. An antibody or fragment thereof that specifically binds to hOX40L, used in a method for treating or preventing hOX40L-mediated diseases or conditions in a human, wherein the antibody or fragment is administered to the human, wherein the antibody or fragment is used to treat or prevent the hOX40L-mediated diseases or conditions by reducing one, several, or all of the following:

[0049] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0050] b. The proliferation of human white blood cells; and

[0051] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells.

[0052] The inventors have thus identified for the first time the reduction of (a), (b) and (c) as a pathway for the treatment and / or prevention of OX40L-mediated diseases and symptoms in humans, and they have provided antibodies and antibody fragments for this purpose.

[0053] In one instance, the secretion is leukocyte secretion. In one instance, (a) is indicated by significantly elevated cytokine levels in human blood, plasma, or serum.

[0054] In one instance, the cytokines are selected from (i) TNFα, (ii) IL-2, and (iii) interferon γ. In one instance, the cytokine is TNFα. In one instance, the cytokine is IL-2. In one instance, the cytokine is interferon γ. In one instance, the cytokines are (i) and (ii); or (i) and (iii); or (ii) and (iii); or (i)-(iii).

[0055] In one instance, the reduction in (a), (b), or (c), or any other reduction disclosed herein, is a reduction of at least 10 or 20% compared to the level in a person at risk of or suffering from an hOX40L-mediated disease or condition. In one instance, the latter is a person prior to administration of the antibody or fragment as described in aspect 1; in another instance, the latter is a different person. In one instance, the reduction is at least 10, 20, 30, 40, 50, or 60%.

[0056] (i) In one instance, the antibody or fragment described in an in vitro assay (as further explained below) is capable of reducing the secretion of related cytokines from leukocytes (e.g., human T cells), and therefore administration of such an antibody or fragment to a human results in a reduction of (a).

[0057] (ii) In one instance, the antibody or fragment described herein is capable of reducing the proliferation of leukocytes (e.g., human PBMCs and / or human T cells) in an in vitro assay (as further explained below), and therefore administration of such an antibody or fragment to a human results in the reduction of (b).

[0058] (iii) In one instance, the antibody or fragment described herein was able to reduce the binding of hOX40 expressed on human T cells to hOX40L expressed on endothelial cells in an in vitro assay (as further explained below), and therefore administration of such an antibody or fragment to a human resulted in a reduction of (c).

[0059] In one instance, (i) and (ii); or (i) and (iii); or (ii) and (iii); or (i)-(iii) apply.

[0060] Alternatively or additionally, the reduction can be assessed using samples from the treated person. For example, see J Clin Immunol. 2004 Jan; 24(1): 74-85; “Increased expression of CCL20 in human inflammatory bowel disease”; Kaser A et al. This publication provides examples of generally applicable techniques for using tissue biopsy and reading out reduced cytokine levels indicating decreased cytokine secretion in vivo following antibody treatment. Similar methods can be used to determine a reduction in the secretion of one or more cytokines in a person who has received the antibody of the present invention. Those skilled in the art will be familiar with techniques for assessing cytokine levels in a patient or patient sample, such as by using one or more of the following: tissue biopsy, immunohistochemistry, immunofluorescence, tissue staining, cytokine mRNA quantification (e.g., using PCR, such as Taqman). TM PCR), cytokine protein detection and quantification (e.g., using cytokine-specific antibody tools and quantification, such as by ELISA or another standard protein quantification technique). For example, when the disease or symptom is a gastrointestinal disease or symptom (e.g., IBD), a biopsy of relevant intestinal tissue from a patient who has received the antibody of the present invention can be performed, followed by quantification of cytokine mRNA and / or cytokine proteins (e.g., using quantitative PCR). The results are compared with cytokine quantification in relevant biopsy tissue from the same patient prior to antibody administration or with another patient with the same disease or symptom but not receiving anti-OX40L treatment or treatment for the disease or symptom. In this way, those skilled in the art can determine that the antibody of the present invention reduces cytokine secretion in human recipients. Instead of assessing intestinal tissue levels, depending on the characteristics and location of the disease or symptom, different tissues or samples from human patients can be used. For example, when the disease or symptom is an airway (e.g., lung) disease or symptom, lung or other tissue samples may be taken for cytokine assessment. Optionally, as will be apparent to those skilled in the art, bronchoalveolar lavage (BAL) samples can be used. In another instance, for some diseases or conditions, the reduction in cytokines in the blood, serum, or plasma of a person who has received the antibody of the present invention can be assessed, and then compared with levels prior to receiving the antibody or with levels in untreated individuals, as discussed above.

[0061] As is known in the art, the term “leukocyte” includes, for example, one or more of lymphocytes, polymorphonuclear leukocytes, and monocytes. As will also be apparent to those skilled in the art, the term “monocyte” includes, for example, peripheral blood mononuclear cells (PBMCs) or cells of monocyte origin, such as dendritic cells (DCs). See, for example, Immunobiology, November 2013; 218(11): 1392-401. doi: 10.1016 / j.imbio.2013.07.005. Epub July 25, 2013; “Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes”, Pfeiffer IA et al.

[0062] As will be apparent to those skilled in the art, tissue biopsy, staining, and histological techniques can be used to assess the proliferation of leukocytes, such as lamina propria lymphocytes (LPL). Hematoxylin and eosin stains (H&E stains or HE stains), for example, are commonly used histologically to look for infiltrating lymphocytes (in various human tissues) and are among the primary histological stains. They are the most widely used stains in medical diagnostics and are often the gold standard, and can also be used to assess leukocyte proliferation according to the present invention. For example, gastrointestinal tissue (e.g., intestinal tissue) from a person with or at risk of hOX40L-mediated disease or condition can be obtained, stained, and the extent of LPL infiltration can be assessed. Comparisons can be made between such tissues from persons who have received the antibodies of the present invention, compared to the extent of infiltration in tissues obtained from the same person prior to administration or from tissues from another person who has not yet received treatment but is at risk of or has the disease or condition. For example, comparisons can be made between intestinal tissues taken from the same (or different) persons with IBD.

[0063] Standard binding assays familiar to the technician can be used, such as ELISA or SPR, to determine whether the antibody or fragment can reduce the binding of the hOX40 receptor expressed on human T cells to the hOX40L expressed on endothelial cells.

[0064] Inflammatory bowel disease (IBD) is a chronic inflammatory condition affecting the gastrointestinal tract, with a significantly increasing incidence and a tendency towards more severe clinical phenotypes. The disease is characterized by an amplified immune response to the luminal flora, suggesting a possible involvement of a deficiency in the gut microbiota barrier function, and research supports this insight (Cucchiara et al., 2012; Josens et al., 2012; Manichanh et al., 2012; Salzman et al., 2007, all cited in Deuring et al., “The cellbiology of the intestinal epithelium and its relation to inflammatory bowel disease,” The International Journal of Biochemistry & Cell Biology 45 (2013) 798-806). IBD comprises two main subtypes: Crohn's disease (CD) and ulcerative colitis (UC). CD patients may have inflammatory lesions throughout their gastrointestinal tract, while inflammation in UC patients is confined to the colon. Also see Hisamatsu et al. (“Immune aspects of the pathogenesis of inflammatory bowel disease”, Pharmacology & Therapeutics 137 (2013) 283-297) and the documents cited therein.

[0065] Granuloma formation is one of the most important pathological features of human Crohn's disease. Mizoguchi et al. demonstrated that F4 / 80-positive immature CD11c + Dendritic cells (DCs) produce IL-23 and contribute to granuloma formation in a murine model of colitis (Mizoguchi et al., 2007). Th1 immune responses are prominent in Crohn's disease. In fact, CD4+ in the LPs of Crohn's disease... + T cells express T-bet and produce large amounts of interferon (IFN)-γ (Matsuoka et al., 2004). Sakuraba et al. demonstrated that dendritic cells (DCs) in the mesenteric lymph nodes of patients with Crohn's disease strongly promote Th1 and Th17 immune responses (Sakuraba et al., 2009). Mesenteric lymph node DCs contribute to the pathogenesis of IBD, particularly Crohn's disease.

[0066] The role of cytokines in diseases and symptoms

[0067] See Muzes et al., World J Gastroenterol, 7 November 2012; 18(41): 5848-586l ISSN 1007-9327 (print) ISSN 2219-2840 (online), “Changes of the cytokine profile ininflammatory bowel diseases”.

[0068] Cytokines are essential signaling agents of the mucosa-associated immune system for maintaining normal intestinal homeostasis. Imbalances in their pro-inflammatory characteristics can lead to disease states, such as those observed in inflammatory bowel disease (IBD), for example, Crohn's disease (CD) and ulcerative colitis (UC). The roles of pro-inflammatory cytokines such as IL-1α, IL-1β, IL-2, -6, -8, -12, -17, -23, IFN-γ, or TNFα in IBD are associated with the occurrence and progression of UC and CD. CD is often described as a prototype of T-helper cell (Th)1-mediated disease because the main inflammatory mediators are Th1 cytokines such as interleukin (IL)-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α.

[0069] The binding of TNF-like ligands to their receptors triggers intracellular pathways directly involved in cell proliferation, differentiation, and survival. Most members of the TNF / TNF receptor protein superfamily are expressed on immune cells and play key roles in multiple components of the immune response. TNF-α is a major cytokine in the pathogenesis of IBD. It exerts its pleiotropic effects through the expression of adhesion molecules, fibroblast proliferation, procoagulant factors, and the induction of cytotoxicity, apoptosis, and acute phase responses. In IBD, TNF-α is derived from innate immune cells, such as macrophages or monocytes, and can also be differentiated Th1 cells. Serum levels of TNF-α are associated with clinical activity in UC and CD

[31] . It plays a coordinating role in chronic inflammation in IBD. The role of TNF-α in CD has been extensively studied. TNF-α binds to serum soluble TNF receptors 1 and 2 (sTNFR1 and 2) to trigger pro-inflammatory signaling. The levels of sTNFR1 and 2 are elevated in CD.

[0070] Tumor necrosis factor-like factor (TL1A), another member of the TNF family, stimulates IFN-γ secretion by binding to death receptor 3 (DR3). DR3 is expressed at high rates by cells from mucosal biopsies of UC and CD, and elevated IFN-γ levels are observed in IBD patients along with disease activity. The TL1A / DR3 system is involved in the pathogenesis of CD. Macrophages in the lamina propria are the main producers of TL1A, and its expression is significantly enhanced in CD. TL1A and IL-23 have been found to synergistically promote IFN-γ production by mucosal T cells. IFN-γ: Produced by TH1 T cells. IFN-γ is produced once inflammation is triggered and subsequently acts through various molecules and pathways of the immune system to exacerbate the inflammatory process. A large body of literature extensively documents the pro-inflammatory properties of IFN-γ, leading to the mainstream view that IFN-γ is a major pro-inflammatory cytokine in inflammatory and autoimmune diseases. Interferon-γ is causally involved in experimental inflammatory bowel disease in mice (Ito et al., Clinical and Experimental Immunology (2006), 146:330-338). Studies have clearly demonstrated that IFN-γ is involved in the development of inflammatory bowel disease in mice in terms of the degree of weight loss, DAI, histological score, and MPO activity. - / - Mice showed reduced colitis after DSS stimulation. Increased IFN-γ was produced in the colon of wild-type (WT) mice treated with DSS exhibiting severe IBD-like symptoms.

[0071] Interleukin-2 (IL-2) is produced by T cells and is crucial for T cell differentiation into effector T cells. IL-2 is also important for T cell proliferation. This is important for IBD because effector T cells are considered the primary cell type causing damage in IBD.

[0072] IL-8 (interleukin-8; aka CXCL8) primarily mediates neutrophil activation and migration to tissues and sites of inflammation originating from peripheral blood. Tissue levels of IL-8 have been found to be higher in active ulcerative colitis (UC) compared to normal colon tissue, and its serum concentrations have been correlated with endoscopic and histological severity of UC. IL-8 is important for inflammatory conditions and cancer (see, e.g., “The Chemokine CXCL8 in Carcinogenesis and Drug Response”, ISRN Oncol. 2013 Oct 9; 2013: 859154; Gales D et al., and Future Oncol, 2010 Jan; 6(1): 111-6. doi: 10.2217 / fbn.09.128; “CXCL8 and its cognate receptors in melanomaprogression and metastasis”, Singh S et al.). Particularly in cancer, IL-8 is also thought to play a role by supporting angiogenesis.

[0073] The antibodies or fragments described in any mode, aspect or example herein antagonize the binding of hOX40L to the OX40 receptor.

[0074] In any mode, aspect or instance of this document, the antibody or fragment antagonizes the binding of hOX40L to OX40.

[0075] In any mode, aspect, or instance described herein, the OX40L receptor may be human OX40.

[0076] In any mode, aspect, or instance of this article, a person has asthma or is at risk of asthma and the antibody or fragment said therein reduces IgE in the person.

[0077] In any mode, aspect, or instance of this article, a person has asthma or is at risk of asthma and the antibody or fragment described is used to reduce IgE in the person.

[0078] 2. The antibody or fragment according to aspect 1, wherein the antibody or fragment reduces the binding of the hOX40 receptor expressed by human T cells to hOX40L expressed by endothelial cells and reduces the proliferation of human T cells; wherein the antibody or fragment is used to treat or prevent hOX40L-mediated diseases or conditions by reducing the secretion of cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon γ.

[0079] In one instance, the cytokines are selected from (i) TNFα, (ii) IL-2, and (iii) interferon γ. In one instance, the cytokine is TNFα. In one instance, the cytokine is IL-2. In one instance, the cytokine is interferon γ. In one instance, the cytokines are (i) and (ii); or (i) and (iii); or (ii) and (iii); or (i)-(iii).

[0080] 3. The antibody or fragment according to aspect 1, wherein the leukocytes are selected from polymorphonuclear leukocytes, monocytes, peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, antigen-presenting cells (APCs), dendritic cells (DC cells), and natural killer cells (NK cells).

[0081] In one implementation, the white blood cells are peripheral blood mononuclear cells (PBMCs) and T cells (e.g., PBMCs).

[0082] 4. The antibody or fragment according to aspect 3, wherein the leukocytes comprise lamina propria lymphocytes (LPL) and the disease or symptom is a gastrointestinal (GI) disease or symptom.

[0083] 5. The antibody or fragment according to any of the foregoing aspects, wherein the epithelial cells include cells selected from gastrointestinal cells, colon cells, intestinal cells, and airway (e.g., lung) epithelial cells.

[0084] In another embodiment, the epithelial cells include cells selected from gastrointestinal cells, colon cells, intestinal cells, ocular cells, and airway (e.g., lung) epithelial cells. In yet another embodiment, the epithelial cells include ocular cells.

[0085] 6. An antibody or fragment according to any of the foregoing aspects, used to treat or prevent hOX40L-mediated diseases or conditions in the person by reducing T cell proliferation.

[0086] In one instance, the antibody or fragment is capable of reducing T cell proliferation in an in vitro assay (e.g., in a human DC / T cell in vitro assay, as explained further below), and therefore administration of such an antibody or fragment to a human results in a reduction in T cell proliferation in that human.

[0087] 7. An antibody or fragment according to any of the foregoing aspects, used to treat or prevent said hOX40L-mediated disease or condition in said person by antagonizing the interaction between human hOX40L and leukocytes, wherein cell proliferation is reduced.

[0088] In one instance, the antibody or fragment is capable of reducing leukocyte (e.g., monocyte) proliferation in an in vitro assay (e.g., in an MLR in vitro assay, as explained further below), and therefore administration of such an antibody or fragment to a human results in a reduction of leukocyte proliferation in said human.

[0089] 8. An antibody or fragment according to any of the foregoing aspects, used to treat or prevent said hOX40L-mediated disease or condition in said person by reducing human leukocyte proliferation through antagonism of T-cell-mediated OX40L / OX40L receptor interaction in said person.

[0090] In one instance, the antibody or fragment is capable of reducing leukocyte (e.g., monocyte) proliferation in an in vitro assay, wherein the antibody or fragment antagonizes T cell-mediated OX40L / OX40L receptor interaction in the assay, and therefore administration of such an antibody or fragment to a human results in a reduction of leukocyte proliferation in the human.

[0091] 9. An antibody or fragment according to any of the foregoing aspects, used to treat or prevent said hOX40L-mediated disease or condition in said person by reducing the secretion of cytokines selected from TNFα, IL-2 and interferon γ in said person.

[0092] In one instance, the antibody or fragment is intended to treat or prevent hOX40L-mediated diseases, symptoms, or epithelial cell damage in the human body by reducing the secretion of (i) IL-2 and interferon γ, (ii) IL-2 and TNFα, or (iii) interferon γ and TNFα.

[0093] In one instance, the antibody or fragment is capable of reducing the secretion of cytokines selected from IL-2, TNFα, and interferon-γ in an in vitro assay (e.g., in an MLR in vitro assay, as further explained below), and therefore administration of such an antibody or fragment to a human results in a reduction in the secretion of the selected cytokines in the human.

[0094] In one instance, the antibody or fragment is capable of reducing IL-8 secretion in an in vitro assay (e.g., in an MLR in vitro assay, as explained further below), and therefore administration of such an antibody or fragment to a human results in a reduction of IL-8 secretion in said human.

[0095] 10. The antibody or fragment according to aspect 9, used to treat or prevent the disease or condition by reducing the secretion of the cytokines mediated by the interaction between dendritic cells (DC cells) and T cells in humans.

[0096] In one instance, the antibody or fragment is capable of reducing the secretion of the cytokines in an in vitro assay of DC cells / T cells (as explained further below), and therefore administration of such an antibody or fragment to a human results in a reduction in the secretion of the cytokines in that human.

[0097] 11. An antibody or fragment according to any of the foregoing aspects, wherein damage to gastrointestinal cells, colon cells, intestinal cells or airway (e.g., lung) cells is a symptom or cause of the disease or condition described in the person.

[0098] In another embodiment, the epithelial cells include cells selected from gastrointestinal cells, colon cells, intestinal cells, ocular cells, and airway (e.g., lung) epithelial cells. In yet another embodiment, the epithelial cells include ocular cells.

[0099] 12. An antibody or fragment according to any of the foregoing aspects, wherein the person suffers from or is at risk of inflammatory bowel disease (IBD), allogeneic transplant rejection, graft-versus-host disease (GvHD), diabetes, or airway inflammation, and the method treats or prevents IBD, allogeneic transplant rejection, GvHD, diabetes, or airway inflammation in the person.

[0100] 12a. An antibody or fragment according to any of the foregoing aspects, wherein the person has inflammatory bowel disease (IBD), allogeneic transplant rejection, graft-versus-host disease (GvHD), uveitis, pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, diabetes, or airway inflammation or is at risk of these diseases, and the method treats or prevents IBD, allogeneic transplant rejection, GvHD, uveitis, pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, diabetes, or airway inflammation in the person.

[0101] In any of the foregoing instances, the person suffers from or is at risk of an inflammatory or autoimmune disease or condition, or has been diagnosed with such a disease or condition.

[0102] In one instance, an autoimmune disease or condition is selected from the following:

[0103] Acute disseminated encephalomyelitis (ADEM)

[0104] Addison's disease

[0105] Allergic granulomatosis and vasculitis or Churg-Strauss syndrome (CSS)

[0106] Hair loss or alopecia areata (AA)

[0107] Ankylosing spondylitis

[0108] Autoimmune chronic active hepatitis (CAH)

[0109] Autoimmune hemolytic anemia

[0110] Autoimmune pancreatitis (AIP)

[0111] Autoimmune retinopathy (AR), see Retinopathy

[0112] Autoimmune thrombocytopenic purpura

[0113] Autoimmune neutropenia

[0114] Autoimmune inner ear disease (AIED)

[0115] Antiphospholipid syndrome (APS)

[0116] Autoimmune lymphoproliferative syndrome (ALPS)

[0117] Behcet's syndrome

[0118] vesicular pemphigoid

[0119] celiac disease

[0120] Churg-Strauss syndrome (CSS) or allergic granulomatous vasculitis

[0121] Chronic bullous disease in children

[0122] Chronic inflammatory demyelinating polyradiculopathy (CIDP)

[0123] Cica pemphigoid (CP)

[0124] Central nervous system vasculitis

[0125] Crohn's disease

[0126] cryoglobulinemia

[0127] Herpetic dermatitis (DH)

[0128] Discoid lupus erythematosus (DLE)

[0129] Encephalomyelitis

[0130] Acquired epidermolysis bullosa (EBA)

[0131] Giant cell arteritis, see temporal arteritis.

[0132] graft-versus-host disease

[0133] Graves' Disease

[0134] Guillain-Barré syndrome

[0135] For Hanot Syndrome, see Primary Biliary Cirrhosis.

[0136] Hashimoto's thyroiditis, also known as autoimmune thyroiditis and chronic lymphocytic thyroiditis.

[0137] Allergic vasculitis (HV) or small vessel vasculitis

[0138] Immune-mediated infertility

[0139] Inflammatory bowel disease

[0140] Insulin-dependent diabetes mellitus

[0141] Isolated central nervous system vasculitis or CNS vasculitis

[0142] Isaacs' Syndrome: Neuromuscular rigidity

[0143] Kawasaki disease (KD)

[0144] Lambert-Eaton myasthenia gravis (LEMS)

[0145] linear IgA disease

[0146] Lupus - See Systemic Lupus Erythematosus

[0147] Meniere's Disease

[0148] Microscopic polyangiitis (MPA)

[0149] Mixed connective tissue disease or MCTD

[0150] Monoclonal Gamma Globulinosis

[0151] Myasthenia gravis

[0152] Multiple sclerosis

[0153] Multifocal motor neuropathy

[0154] Neuromuscular rigidity or Isaac syndrome

[0155] See also Autoimmune Neutropenia for neutropenia.

[0156] Ovarian inflammation

[0157] Oculoclonus-Myoclonus Syndrome

[0158] orchitis

[0159] Paraneoplastic neurosis

[0160] pemphigus vulgaris

[0161] Pemphigoid foliaceus (PF)

[0162] PG (Pregnancy-related pemphigoid)

[0163] pernicious anemia

[0164] Paraneoplastic pemphigus (PNP)

[0165] Polyangiitis - See Microscopic Polyangiitis

[0166] Polyarteritis nodosa (PAN)

[0167] Polymyositis / Dermatomyositis

[0168] Polymyalgia rheumatica

[0169] Primary biliary cirrhosis (PBC), also known as Arno's syndrome

[0170] Primary sclerosing cholangitis (PSC)

[0171] Raynaud's phenomenon

[0172] Recovery protein-associated retinopathy (RAR), see Retinopathy

[0173] Reactive arthritis, formerly known as Reiter's syndrome,

[0174] Retinopathy

[0175] Rheumatoid arthritis (RA)

[0176] Sarcoidosis

[0177] Sclerosing cholangitis, see primary sclerosing cholangitis.

[0178] Sjogren's syndrome

[0179] Systemic necrotizing angiopathy

[0180] Stiff-person syndrome or Moersch-Woltmann syndrome

[0181] Systemic lupus erythematosus

[0182] Systemic sclerosis (scleroderma)

[0183] Temporal arteritis or giant cell arteritis (GCV)

[0184] Takayasu's arteritis

[0185] Thromboangiitis obliterans or Buerger's disease

[0186] Hypothyroidism

[0187] Hyperthyroidism-related thyroiditis

[0188] Type I autoimmune polyglandular syndrome (PAS)

[0189] Type II autoimmune polygland syndrome

[0190] vasculitis

[0191] Wegener's granulomatosis

[0192] In one instance of any aspect, mode, or implementation, a person suffers from uveitis. For example, the uveitis may actually be non-infectious and / or autoimmune, i.e., non-infectious uveitis or autoimmune uveitis. For example, non-infectious and / or autoimmune uveitis may be caused by or related to Behqet's disease, Fuchs heterochromic iridocyclitis, polyangiitis, HLA-B27-associated uveitis, juvenile idiopathic arthritis, sarcoidosis, spondyloarthritis, sympathetic ophthalmia, tubulointerstitial nephritis, or uveitis syndrome. In one instance, the uveitis may actually be systemic, i.e., systemic uveitis. For example, systemic uveitis is caused by and / or related to ankylosing spondylitis, Behcet's disease, chronic granulomatous disease, enthesitis, inflammatory bowel disease, juvenile rheumatoid arthritis, Kawasaki disease, multiple sclerosis, polyarteritis nodosa, psoriatic arthritis, reactive arthritis, sarcoidosis, systemic lupus erythematosus, Vogt-Koyanagi-Harada syndrome, or Whipple's disease.

[0193] In one instance of any aspect, mode, or implementation, the person suffers from pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, or non-infectious scleritis. In one instance, the person suffers from pyoderma gangrenosa. In one instance, the person suffers from giant cell arteritis. In one instance, the person suffers from Schnitzler syndrome. In one instance, the person suffers from non-infectious scleritis.

[0194] In one instance of any aspect, mode, or implementation, the person suffers from an hOX40L-mediated disease or condition selected from autoimmune diseases or symptoms, systemic inflammatory diseases or symptoms, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD. In another implementation, the person suffers from or is at risk of multi-organ transplant rejection.

[0195] 13. An antibody or a fragment thereof that specifically binds to hOX40L and competes with antibodies selected from 02D10, 10A07, 09H04 and 19H01 for binding to said hOX40L.

[0196] In any aspect, mode, or implementation instance, competition is determined via surface plasmon resonance (SPR), a technique readily apparent to those skilled in the art. SPR can be measured using Biacore. TM Proteon TM Alternatively, another standard SPR technique may be used. Such competition may be due to, for example, the binding of the antibody / fragment to the same or overlapping epitopes of hOX40L. In one example of any aspect, mode, or embodiment, competition is determined by ELISA, a technique readily apparent to those skilled in the art. In one example of any aspect, mode, or embodiment, competition is determined by homogeneous time-resolved fluorescence (HTRF), a technique readily apparent to those skilled in the art. In one example of any aspect, mode, or embodiment, competition is determined by fluorescence-activated cell sorting (FACS), a technique readily apparent to those skilled in the art. In one aspect, the HTRF, ELISA, and / or FACS methods are performed as described in the examples below.

[0197] 14. The antibody or fragment according to aspect 13, wherein the antibody or fragment is according to any one of aspects 1-12.

[0198] 15. An antibody or fragment according to any of the foregoing aspects, comprising a λ light chain variable domain (optionally, of human origin).

[0199] In any aspect, mode, or embodiment of the invention, the variable domain of the antibody or fragment is human or humanized. Optionally, the antibody or fragment also includes a human or humanized constant region (e.g., human Fc and / or human CL). In any aspect of the invention, the variable domain of the antibody or fragment is generated by a transgenic animal (e.g., rodent, mouse, rat, rabbit, chicken, sheep, camel, or shark). In any aspect of the invention, the variable domain of the antibody or fragment is generated or identified by phage display, ribosome display, or yeast display.

[0200] In any aspect, mode, or embodiment of the invention, the antibody or fragment is recombinant.

[0201] In any aspect, mode, or embodiment of the invention, the antibody or fragment is produced from recombinant mammalian, bacterial, insect, plant, or yeast cells. In one example, the mammalian cell is a CHO or HEK293 cell and the antibody or fragment comprises CHO or HEK293 cell glycosylation.

[0202] In any aspect, mode, or embodiment of the invention, the antibody or fragment is isolated.

[0203] 16. An antibody or fragment according to any of the foregoing aspects, comprising a VH domain, said VH domain comprising an HCDR1 sequence selected from the HCDR1 of antibodies:

[0204] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0205] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0206] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0207] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0208] 17. An antibody or fragment according to any of the foregoing aspects, comprising a VH domain, said VH domain comprising an HCDR2 sequence selected from antibodies whose HCDR2 is:

[0209] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0210] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0211] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0212] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0213] 18. An antibody or fragment according to any of the foregoing aspects, comprising a VH domain, said VH domain comprising an HCDR3 sequence selected from antibodies containing HCDR3:

[0214] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0215] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0216] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0217] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0218] 19. An antibody or fragment according to any of the foregoing aspects, comprising a VH domain, said VH domain comprising (i) CDR1 and 2, (ii) CDR1 and 3, (iii) CDR2 and 3 or (iv) CDR1, 2 and 3 sequences:

[0219] a. As described in aspect 16-18(a), and wherein the antibody or fragment competitively binds to the hOX40L with O2D10;

[0220] b. As described in aspect 16-18(b), and wherein the antibody or fragment competitively binds to the hOX40L with 10A07;

[0221] c. As described in aspect 16-18(c), and wherein the antibody or fragment competitively binds to the hOX40L with 09H04; or

[0222] d. As described in aspect 16-18(d), and wherein the antibody or fragment competitively binds to the hOX40L with 19H01.

[0223] 20. An antibody or fragment according to any of the foregoing aspects, comprising a VH domain, said VH domain comprising an amino acid sequence selected from the VH amino acid sequences in the sequence listing.

[0224] On one hand, the present invention provides an anti-hOX40L antibody or fragment (optionally according to any other aspect described herein) comprising a VH domain, said VH domain comprising an amino acid sequence selected from the VH amino acid sequences in the sequence listing. On the other hand, said VH domain comprises an amino acid sequence selected from Seq ID No: 2, Seq ID No: 34, Seq ID No: 66, Seq ID No: 94, Seq ID No: 122, Seq ID No: 124, Seq ID No: 126, Seq ID No: 128, Seq ID No: 132, or Seq ID No: 134.

[0225] In another embodiment of the invention, the antibody or fragment comprises the VH domain amino acid sequence listed in the sequence listing below. Alternatively or optionally, the antibody or fragment comprises the HCDR1 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96, or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42). Alternatively or optionally, the antibody or fragment comprises the HCDR2 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98, or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44). Alternatively or optionally, the antibody or fragment comprises the HCDR3 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 8, Seq ID No: 14, Seq ID No: 40, Seq ID No: 46, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100 or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46).

[0226] In one embodiment of the invention, the antibody or fragment comprises the VL domain amino acid sequence listed in the sequence listing below. Alternatively or additionally, the antibody or fragment comprises the LCDR1 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 18, Seq ID No: 24, Seq ID No: 50, Seq ID No: 56, Seq ID No: 82, Seq ID No: 88, Seq ID No: 110, or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56). Alternatively or additionally, the antibody or fragment comprises the LCDR2 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 20, Seq ID No: 26, Seq ID No: 52, Seq ID No: 58, Seq ID No: 84, Seq ID No: 90, Seq ID No: 112, or Seq ID No: 118, especially Seq ID No: 52 or Seq ID No: 58). Alternatively or optionally, the antibody or fragment comprises the LCDR3 domain amino acid sequence listed in the sequence listing below (i.e., Seq ID No: 22, Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114 or Seq ID No: 120, especially Seq ID No: 54 or Seq ID No: 60).

[0227] In one example of any aspect of this document, the antibody or fragment comprises a heavy chain containing a constant region selected from the heavy chain constant regions SEQ ID NO in the sequence listing (i.e., any one of Seq ID No: 126, 128, 132, or 134, especially the constant region of Seq ID No: 128); and optionally a VH domain as described in aspect 19 or 20. In one example, the antibody or fragment comprises two copies of such a heavy chain. In another example, the heavy chain comprises a rodent, rat, mouse, human, rabbit, chicken, camel, sheep, cattle, non-human primate, or shark constant region (e.g., Fc), especially a mouse constant region.

[0228] In one example of any aspect of this document, the antibody or fragment comprises a heavy chain containing a γ (e.g., human γ) constant region, such as the human γ1 constant region. In another example of any aspect of this document, the antibody or fragment comprises a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, contains a Leu235Glu mutation (i.e., where a wild-type leucine residue is mutated to a glutamate residue). In another embodiment, the heavy chain constant region contains a Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4 containing both the Leu235Glu mutation and the Ser228Pro mutation. This heavy chain constant region is referred to herein as "IgG4-PE".

[0229] In one instance of any aspect of this document, the antibody or fragment is chimeric, for example, containing human variable domains and non-human (e.g., rodent, mouse, or rat, such as mouse) constant regions.

[0230] 21. An antibody or fragment according to any one of aspects 16-20, comprising first and second copies of the VH domain.

[0231] 22. An antibody or fragment according to any of the foregoing aspects, comprising a VL domain, said VL domain comprising an LCDR1 sequence selected from the LCDR1 of antibodies:

[0232] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0233] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0234] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0235] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0236] 23. An antibody or fragment according to any of the foregoing aspects, comprising a VL domain, said VL domain comprising an LCDR2 sequence selected from the LCDR2 of antibodies:

[0237] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0238] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0239] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0240] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0241] 24. An antibody or fragment according to any of the foregoing aspects, comprising a VL domain, said VL domain comprising an LCDR3 sequence selected from the LCDR3 of antibodies:

[0242] a.02D10, and wherein the antibody or fragment competes with 02D10 for binding to the hOX40L;

[0243] b.10A07, wherein the antibody or fragment competes with 10A07 for binding to the hOX40L;

[0244] c.09H04, and wherein the antibody or fragment competes with 09H04 for binding to hOX40L; and

[0245] d.19H01, wherein the antibody or fragment thereon competes with 19H01 for binding to the hOX40L.

[0246] 25. An antibody or fragment according to any of the foregoing aspects, comprising a VL domain, said VL domain comprising (i) CDR1 and 2, (ii) CDR1 and 3, (iii) CDR2 and 3 or (iv) CDR1, 2 and 3 sequences:

[0247] a. As described in aspect 22-24(a), and wherein the antibody or fragment competitively binds to the hOX40L with O2D10;

[0248] b. As described in aspect 22-24(b), and wherein the antibody or fragment competitively binds to the hOX40L with 10A07;

[0249] c. As described in aspect 22-24(c), and wherein the antibody or fragment competitively binds to the hOX40L with 09H04; or

[0250] d. As described in aspect 22-24(d), and wherein the antibody or fragment competitively binds to the hOX40L with 19H01.

[0251] 26. An antibody or fragment according to any of the foregoing aspects, comprising a VL domain comprising an amino acid sequence selected from the VL amino acid sequences in the sequence listing.

[0252] In one aspect of the invention, an anti-hOX40L antibody or fragment (optionally according to any other aspect described herein) is provided, comprising a VL domain comprising an amino acid sequence selected from the VL amino acid sequences in the sequence listing (i.e., Seq ID No: 16, Seq ID No: 48, Seq ID No: 80 or Seq ID No: 108, especially Seq ID No: 48).

[0253] In one example of any aspect thereof, the antibody or fragment comprises a light chain (e.g., a λ light chain) containing a constant region selected from the light chain constant region sequences in the sequence listing (i.e., Seq ID No: 136, Seq ID No: 138, Seq ID No: 140, Seq ID No: 142, Seq ID No: 144, Seq ID No: 146, Seq ID No: 148, Seq ID No: 152, Seq ID No: 154, Seq ID No: 156, Seq ID No: 158, Seq ID No: 160, Seq ID No: 162, Seq ID No: 164, or Seq ID No: 166); and optionally a VL domain (e.g., λVL) as described in aspect 25 or 26. In one example, the antibody or fragment comprises two copies of such a light chain (optionally also comprising two copies of the aforementioned heavy chain). In another instance, the light chain includes rodents, rats, mice, humans, rabbits, chickens, camels, sheep, cattle, non-human primates, or sharks.

[0254] In one example of any aspect thereof, the antibody or fragment comprises a light chain (e.g., a κ light chain) containing a constant region selected from the light chain constant region sequences in the sequence listing (i.e., Seq ID No: 136, Seq ID No: 138, Seq ID No: 140, Seq ID No: 142, Seq ID No: 144, Seq ID No: 146, Seq ID No: 148, Seq ID No: 152, Seq ID No: 154, Seq ID No: 156, Seq ID No: 158, Seq ID No: 160, Seq ID No: 162, Seq ID No: 164, or Seq ID No: 166); and optionally a VL domain (e.g., κVL) as described in aspect 25 or 26. In one example, the antibody or fragment comprises two copies of such a light chain (optionally also comprising two copies of the aforementioned heavy chain). In another instance, the light chain includes rodents, rats, mice, humans, rabbits, chickens, camels, sheep, cattle, non-human primates, or sharks.

[0255] In one instance, the antibody or fragment comprises a λ light chain containing a constant region selected from light chain constant region sequences in the sequence listing (i.e., Seq ID No: 146, Seq ID No: 148, Seq ID No: 152, Seq ID No: 154, Seq ID No: 156, Seq ID No: 158, Seq ID No: 160, Seq ID No: 162, Seq ID No: 164, or Seq ID No: 166); and optionally a λVL domain.

[0256] In one instance, the antibody or fragment comprises a κ light chain containing a constant region selected from light chain constant region sequences in the sequence listing (i.e., Seq ID No: 136, Seq ID No: 138, Seq ID No: 140, Seq ID No: 142, or Seq ID No: 144); and optionally a κVL domain.

[0257] In one example, the VL domain of the antibody or fragment is a λ light chain variable domain. In another example, the VL domain of the antibody or fragment is a κ light chain variable domain.

[0258] 27. An antibody or fragment according to any one of aspects 22-26, comprising first and second copies of the VL domain.

[0259] 28. The antibody or fragment according to any of the foregoing aspects, wherein the hOX40L is hOX40L expressed on the surface of human cells, for example on endothelial cells (e.g., airway or GI tract endothelial cells).

[0260] In another embodiment, the epithelial cells include cells selected from gastrointestinal cells, colon cells, intestinal cells, ocular cells, and airway (e.g., lung) epithelial cells. In yet another embodiment, the epithelial cells include ocular cells.

[0261] 29. An antibody or fragment according to any of the foregoing aspects, wherein, in the presence of hOX40L, the antibody or fragment reduces the proliferation of human PBMCs or T cells by at least 20, 30, 40, 50, or 60% in the presence of hOX40L in an in vitro mixed lymphocyte reaction (MLR) assay compared to the proliferation of human PBMCs or T cells in the absence of an antibody specific to hOX40L in an in vitro control MLR assay. Description of suitable assays is provided in the examples below.

[0262] 30. The antibody or fragment according to aspect 29, wherein the hOX40L in the assay is expressed on the surface of human dendritic cells (DC cells).

[0263] The following examples provide an explanation of appropriate measurements.

[0264] 31. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces NF-κB activity in human HT-1080 cells expressing the hOX40 receptor in vitro in the presence of hOX40L.

[0265] In one instance, by detecting HT-1080 cells in vitro ( The reduction in IL-8 secretion by the antibody or fragment (optionally transfected with hOX40 receptor in the presence of hOX40) was used to determine the decrease in NF-κB activity of the antibody or fragment.

[0266] 32. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces IL-8 secretion in human HT-1080 cells expressing the hOX40 receptor in vitro in the presence of hOX40L.

[0267] 33. The antibody or fragment according to aspect 32, wherein the antibody or fragment reduces IL-8 secretion by at least 20, 30, 40, 50 or 60% compared to the IL-8 production of HT-1080 cells expressing the hOX40 receptor in vitro when an antibody specific to hOX40L is lacking in the presence of hOX40L.

[0268] 34. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces the proliferation of hOX40L-stimulated T cells in vitro.

[0269] 35. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces IL-2 secretion stimulated by hOX40L from human T cells in vitro.

[0270] 36. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces the secretion of cytokines mediated by the interaction between human dendritic cells (DC cells) and human T cells, wherein the cytokines are selected from one, two, more, or all of TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ.

[0271] This can be assessed, for example, using an in vitro MLR assay (e.g., an in vitro MLR assay for DC / T cells). Instructions for suitable assays are provided in the examples below.

[0272] In one instance, as is possible, such as when DC cells originate from a human source different from the human source of T cells, DC cells may mismatch with T cells, for example, due to MHC mismatch. In one instance, DC cells were generated in vitro from human monocytes via GMCSF and IL-4.

[0273] 37. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces interferon γ secretion by at least 20, 30, 40, 50 or 60% compared to the production of interferon γ mediated by the interaction between human dendritic cells (DC cells) and human T cells when there is a lack of antibodies specific to hOX40L.

[0274] 38. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces TNFα secretion by at least 20, 30, 40, 50 or 60% compared to the production of TNFα mediated by the interaction between human dendritic cells (DC cells) and human T cells when there is a lack of antibodies specific to hOX40L.

[0275] 39. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces IL-2 secretion by at least 10, 20, 30, 40, 50 or 60% compared to the production of IL-2 mediated by the interaction between human dendritic cells (DC cells) and human T cells when there is a lack of antibodies specific to hOX40L.

[0276] 40. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces cytokine secretion (e.g., leukocyte cytokine secretion) in a human peripheral blood mononuclear cell (PBMC) mixed lymphocyte (MLR) assay, wherein the cytokine is selected from one, two, more, or all of TNFα, IL-2, IL-4, IL-3, IL-6, IL-8, IL-10, IL-17, RANTES, and interferon γ.

[0277] 41. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces interferon γ secretion by at least 20, 30, 40, 50 or 60% compared to the production of interferon γ in the absence of an antibody specific for hOX40L in a human PBMC MLR assay.

[0278] In one implementation, the comparison is a comparison with the production of interferon-γ in antibody-deficient human PBMC MLR assays.

[0279] 42. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces TNFα secretion by at least 20, 30, 40, 50 or 60% compared to the production of TNFα in the absence of an antibody specific for hOX40L in a human PBMC MLR assay.

[0280] 43. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment reduces IL-2 secretion by at least 10, 20, 30, 40, 50 or 60% compared to the production of IL-2 in the absence of an antibody specific for hOX40L in a human PBMC MLR assay.

[0281] 44. An antibody or fragment according to any one of aspects 36-43, wherein the cell is a primary cell.

[0282] "Primary cells" refers to cells in the human body or such cells that have been removed from a patient for in vitro binding of the antibodies or fragments of the present invention (e.g., potentially useful in methods for diagnosing a person's OX40L state or disease / symptom state). Primary cells as used herein are not typically cells from human cell lines that have undergone numerous in vitro cultures. In this embodiment, the ability of the antibodies or fragments of the present invention to specifically inhibit hOX40L binding to the receptor is advantageous because it provides a direct indication of the applicability of cells to patients suffering from or at risk of hOX40L-mediated diseases or symptoms.

[0283] 45. An antibody or fragment according to any of the foregoing aspects, wherein the antibody or fragment inhibits the binding of hOX40L to the hOX40L receptor (e.g., hOX40) in an HTRF (homogeneous time-resolved fluorescence) assay, IC50 50 1x10 -8 Or lower.

[0284] In one instance, IC 50 In 1x10 -8 Up to 1x10 -11 Within the range or in 1x10 -9 Up to 1x10 -10 Within the range.

[0285] 46. ​​A pharmaceutical composition for treating and / or preventing OX40L-mediated symptoms or diseases, said composition comprising an antibody or fragment as described in any of the foregoing aspects and a diluent, excipient or carrier; and optionally further comprising an anti-inflammatory drug.

[0286] In one instance, the anti-inflammatory drug is independently selected from corticosteroids (e.g., methylprednisolone), anti-IL12 / IL-23 antibodies (e.g., ustekinumab), anti-VLA4 antibodies (e.g., natalizumab), anti-LFA1 antibodies, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., basilixumab), or anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, certolizumab pegol). In one instance, the anti-inflammatory drug was independently selected from corticosteroids (such as methylprednisolone) and anti-LFA1 antibodies.

[0287] 47. A pharmaceutical composition or kit for treating and / or preventing OX40L-mediated symptoms or diseases, said composition or kit comprising an antibody or fragment of the present invention (and optionally an anti-inflammatory drug), optionally combined with a label or instructions for use for treating and / or preventing said disease or symptoms in humans; optionally said label or instructions for use comprising a marketing authorization number (e.g., an FDA or EMA license number); optionally said kit comprising an IV or injection device containing said antibody or fragment.

[0288] 48. A nucleic acid encoding HCDR3 of an antibody described in any one of aspects 1-45.

[0289] In one implementation, the HCDR herein follows the Kabat nomenclature. In another implementation, the HCDR herein follows the IMGT nomenclature.

[0290] 49. The nucleic acid according to aspect 48, comprising a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or 100% identical to the HCDR3 sequence in the sequence listing.

[0291] On one hand, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a VH domain of an anti-hOX40L antibody, wherein the nucleotide sequence comprises an HCDR3 sequence that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical or 100% identical to an HCDR3 sequence in the sequence listing. Optionally, the antibody is according to any other aspect thereof.

[0292] In another embodiment, a nucleic acid according to aspect 48 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR3 sequence in the sequence listing, except for one, two, or three nucleotide substitutions, wherein each substitution does not produce an amino acid change or produces a conserved amino acid change in the corresponding protein sequence (i.e., the nucleotide substitution is a synonymous substitution). Those skilled in the art will be familiar with conserved amino acid changes.

[0293] Amino acid substitution includes changes in which an amino acid is replaced by a different naturally occurring amino acid residue. Such substitutions can be classified as “conservative,” in which an amino acid residue contained in a polypeptide is replaced by another naturally occurring amino acid having similar characteristics regarding polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. Substitutions covered by this invention can also be “non-conservative,” in which an amino acid residue present in a peptide is replaced by an amino acid with different properties, such as a naturally occurring amino acid from a different class (e.g., replacing a charged or hydrophobic amino acid with alanine), or optionally, in which a naturally occurring amino acid is replaced by an unconventional amino acid.

[0294] Alternatively or alternatively, a nucleic acid according to aspect 49 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR3 sequence in the sequence listing except for 1, 2, or 3 nucleotide substitutions that produce conserved amino acid changes on the corresponding protein sequence.

[0295] 50. The nucleic acid encoding HCDR2 of any of the antibodies described in aspects 1-45; optionally, the nucleotides described are according to aspect 48 or 49.

[0296] 51. The nucleic acid according to aspect 50, comprising a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or 100% identical to the HCDR2 sequence in the sequence listing.

[0297] On one hand, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a VH domain of an anti-hOX40L antibody, wherein the nucleotide sequence comprises an HCDR2 sequence that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical or 100% identical to an HCDR2 sequence in the sequence listing. Optionally, the antibody is according to any other aspect thereof.

[0298] In another embodiment, a nucleic acid according to aspect 51 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR2 sequence in the sequence listing except for one, two, or three nucleotide substitutions, wherein each substitution does not produce an amino acid change or produces a conserved amino acid change in the corresponding protein sequence (i.e., the nucleotide substitution is a synonymous substitution). Those skilled in the art will be familiar with conserved amino acid changes.

[0299] Alternatively or alternatively, a nucleic acid according to aspect 50 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR2 sequence in the sequence listing except for 1, 2, or 3 nucleotide substitutions that produce conserved amino acid changes on the corresponding protein sequence.

[0300] 52. The nucleic acid encoding HCDR1 of an antibody described in any of aspects 1-45; optionally, said nucleotide is according to any of aspects 48-51.

[0301] 53. The nucleic acid according to aspect 52, comprising a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or 100% identical to the HCDR1 sequence in the sequence listing.

[0302] On one hand, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a VH domain of an anti-hOX40L antibody, wherein the nucleotide sequence comprises an HCDR1 sequence that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical or 100% identical to an HCDR1 sequence in the sequence listing. Optionally, the antibody is according to any other aspect thereof.

[0303] In another embodiment, a nucleic acid according to aspect 52 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR1 sequence in the sequence listing except for one, two, or three nucleotide substitutions, wherein each substitution does not produce an amino acid change or produces a conserved amino acid change (i.e., the nucleotide substitution is a synonymous substitution) on the corresponding protein sequence. Those skilled in the art will be familiar with conserved amino acid changes.

[0304] Alternatively or alternatively, a nucleic acid according to aspect 52 is provided, comprising a nucleotide sequence that is 100% identical to the HCDR1 sequence in the sequence listing except for 1, 2, 3, 4, 5, 6, or 7 synonymous nucleotide substitutions and without 1, 2, or 3 nucleotide substitutions that produce conserved amino acid changes on the corresponding protein sequence.

[0305] 54. A nucleic acid encoding the VH domain and / or VL domain of an antibody described in any one of aspects 1-45.

[0306] 55. The nucleic acid according to aspect 54, comprising a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or 100% identical to the VH domain nucleotide sequence in the sequence listing.

[0307] In another embodiment, a nucleic acid according to aspect 54 is provided, comprising a nucleotide sequence that is 100% identical to the VH domain nucleotide sequence in the sequence listing, except for one, two, or three nucleotide substitutions, wherein each substitution does not produce an amino acid change or produces a conserved amino acid change (i.e., the nucleotide substitution is a synonymous substitution) on the corresponding protein sequence. Those skilled in the art will be familiar with conserved amino acid changes.

[0308] Alternatively or alternatively, a nucleic acid according to aspect 54 is provided, comprising a nucleotide sequence that is 100% identical to the VH domain nucleotide sequence in the sequence listing except for 1, 2, or 3 nucleotide substitutions that produce conserved amino acid changes on the corresponding protein sequence.

[0309] 56. The nucleic acid according to aspect 54 or 55, comprising a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical or 100% identical to the VL domain nucleotide sequence in the sequence listing.

[0310] In another embodiment, a nucleic acid according to aspect 54 or 55 is provided, comprising a nucleotide sequence that is 100% identical to the VL domain nucleotide sequence in the sequence listing, except for one, two, or three nucleotide substitutions, wherein each substitution does not produce an amino acid change or produces a conserved amino acid change (i.e., the nucleotide substitution is a synonymous substitution) on the corresponding protein sequence. Those skilled in the art will be familiar with conserved amino acid changes.

[0311] Alternatively or additionally, a nucleic acid according to aspect 54 or 55 is provided, comprising a nucleotide sequence that is 100% identical to the VL domain nucleotide sequence in the sequence listing except for 1, 2, 3, 4, 5, 6, or 7 synonymous nucleotide substitutions and without 1, 2, or 3 nucleotide substitutions that produce conserved amino acid changes on the corresponding protein sequence.

[0312] 57. A nucleic acid encoding the heavy or light chain of an antibody described in any one of aspects 1-45.

[0313] 58. The nucleic acid according to aspect 57, comprising a nucleotide sequence as described in any one of aspects 48-56.

[0314] 59. A vector (e.g., a mammalian expression vector) comprising a nucleic acid according to any one of aspects 48-58; optionally, said vector is a CHO or HEK293 vector. In one example, the vector is a yeast vector, such as a yeast (Saccharomyces) or Pichia vector.

[0315] 60. A host comprising a nucleic acid according to any one of aspects 48-58 or a vector according to aspect 59. In one instance, the host is a mammalian (e.g., human, such as CHO or HEK293) cell line or a yeast or bacterial cell line.

[0316] 61. Use of an antibody or fragment thereof that specifically binds to hOX40L in the manufacture of a pharmaceutical agent intended for administration to humans to treat or prevent hOX40L-mediated diseases or symptoms in humans by reducing one, more, or all of the following:

[0317] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0318] b. The proliferation of human white blood cells; and

[0319] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells.

[0320] The features of any of the above aspects, patterns, examples, or implementation schemes may be optionally modified as necessary for this purpose.

[0321] In one instance, a person has asthma or is at risk of asthma and the antibody or fragment is used to reduce IgE in the person, thereby treating, preventing, or alleviating the person's asthma.

[0322] 62. A method for treating or preventing hOX40L-mediated diseases or symptoms by reducing one, more, or all of the following:

[0323] a. Secretion of human cytokines selected from TNFα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, RANTES, and interferon-γ;

[0324] b. The proliferation of human white blood cells; and

[0325] c. Binding of the hOX40 receptor expressed by human T cells to the hOX40L expressed by endothelial cells;

[0326] The method includes administering to the person a therapeutically effective amount of an antibody or fragment that specifically binds to hOX40L.

[0327] The features of any of the foregoing aspects, examples, or implementation schemes may be optionally modified as necessary for this purpose.

[0328] The method of the present invention treats or prevents said disease or condition in humans. The "therapeutic effective amount" of the antibody or fragment is the amount that produces the therapeutic or preventative effect (administered in one or more doses, spaced apart over time, e.g., substantially once a month). This will be apparent to those skilled in the art and can vary depending on the specific human patient and the targeted disease or condition.

[0329] In one instance, a person has asthma or is at risk of asthma and the antibody or fragment reduces IgE in the person, thereby treating, preventing, or alleviating the person's asthma.

[0330] 63. The method or use according to aspect 61 or 62, for treating or preventing hOX40L-mediated diseases, symptoms or epithelial cell damage in the person by reducing T cell proliferation in the person.

[0331] 64. The method or use according to any one of aspects 61-63, for treating or preventing said hOX40L-mediated diseases, symptoms or epithelial cell damage in said person by antagonizing the interaction between human hOX40L and leukocytes, wherein cell proliferation is reduced.

[0332] 65. The method or use according to any one of aspects 61-64, for treating or preventing said hOX40L-mediated diseases, symptoms or epithelial cell damage in said human by antagonizing the T-cell-mediated OX40L / OX40L receptor interaction to reduce human leukocyte proliferation.

[0333] 66. The method or use according to any one of aspects 61-65, for treating or preventing said hOX40L-mediated diseases, symptoms or epithelial cell damage in said human by reducing the secretion of IL-8 cytokine in said human.

[0334] 67. The method according to aspect 66, used to treat or prevent the disease, condition or epithelial cell damage by reducing the secretion of said IL-8 mediated by the interaction between dendritic cells (DC cells) and T cells in a human body.

[0335] 68. The method or use according to any one of aspects 61-67, wherein damage to gastrointestinal cells, colon cells, intestinal cells or airway (e.g., lung) cells is a symptom or cause of the disease or condition described in the person.

[0336] In another embodiment, the epithelial cells include cells selected from gastrointestinal cells, colon cells, intestinal cells, ocular cells, and airway (e.g., lung) epithelial cells. In yet another embodiment, the epithelial cells include ocular cells.

[0337] 69. The method or use according to any one of aspects 61-68, wherein the person suffers from or is at risk of inflammatory bowel disease (IBD), allogeneic transplant rejection, graft-versus-host disease (GvHD), diabetes, or airway inflammation, and the method treats or prevents IBD, allogeneic transplant rejection, GvHD, diabetes, or airway inflammation in the person.

[0338] 69a. The method or use according to any one of aspects 61-68, wherein the person suffers from or is at risk of inflammatory bowel disease (IBD), allogeneic transplant rejection, graft-versus-host disease (GvHD), uveitis, pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, diabetes, or airway inflammation, and the method treats or prevents IBD, allogeneic transplant rejection, GvHD, uveitis, pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, diabetes, or airway inflammation in the person.

[0339] In any aspect, mode, or implementation plan, the person suffers from an hOX40L-mediated disease or condition selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; such as inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD or at risk of these diseases.

[0340] 70. The method or use according to any one of aspects 61-69a, wherein the antibody or fragment is according to any one of aspects 1-45 or any example, pattern, aspect or embodiment described herein.

[0341] 71. The antibody, fragment, composition, kit, method, or use according to any of the foregoing aspects, for the treatment or prevention of inflammatory or autoimmune diseases or conditions in humans, or for the reduction or prevention of angiogenesis in humans.

[0342] 72. The antibody, fragment, composition, kit, method, or use according to any of the foregoing aspects, wherein the disease or condition is selected from inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchiolitis, gingivitis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome (ARDS), septic shock, ulcerative colitis, Sjögren's syndrome, airway inflammation, systemic lupus erythematosus (SLE), diabetes, contact hypersensitivity, multiple sclerosis, and atherosclerosis.

[0343] 72a. The antibody, fragment, composition, kit, method, or use according to any of the preceding aspects, wherein the disease or condition is selected from inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchiolitis, gingivitis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome (ARDS), septic shock, ulcerative colitis, Guelphin syndrome, airway inflammation, systemic lupus erythematosus (SLE), uveitis, pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, diabetes, contact hypersensitivity, multiple sclerosis, and atherosclerosis.

[0344] In any aspect, mode, or implementation plan, the person suffers from an hOX40L-mediated disease or condition selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; such as inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD or at risk of these diseases.

[0345] In one instance, the disease or symptom is an OX40L-mediated disease or symptom disclosed in US7812133 or EP1791869.

[0346] In one instance, the disease or symptom is an inflammatory or autoimmune disease or symptom. In another instance, the disease or symptom is transplant rejection.

[0347] As used herein, an inflammatory disease or condition refers to a pathological state that causes inflammation, such as neutrophil chemotaxis. Examples of such conditions include inflammatory skin diseases, including psoriasis; reactions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); ischemia-reperfusion; adult respiratory distress syndrome; dermatitis; meningitis; encephalitis; uveitis; autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome, vasculitis; diseases involving leukocyte exudation; inflammatory conditions of the central nervous system (CNS), sepsis, or traumatic secondary multiple organ injury syndrome; alcoholic hepatitis, bacterial pneumonia, antigen-antibody complex-mediated diseases; and pulmonary inflammation, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, and cystic fibrosis. Preferred indications are bacterial pneumonia and inflammatory bowel diseases such as ulcerative colitis. Therefore, the invention is provided in one instance for the treatment or prevention of any one or more of such conditions.

[0348] In one instance, the disease or symptom is cancer.

[0349] In one instance, the disease is uveitis, such as systemic uveitis or autoimmune / non-infectious uveitis.

[0350] 73. An antibody or a fragment thereof that specifically binds to hOX40L and competes with antibody O2D10 for binding to said hOX40L, wherein said antibody or fragment comprises a VH domain comprising HCDR3 containing the motif VRGXYYY, wherein X is any amino acid.

[0351] The characteristics of the antibodies described in any aspect, model, example, or embodiment described herein may be optionally modified as necessary to apply to these antibodies; for example, the antibodies may be human antibodies or chimeric antibodies having the functional characteristics described herein. Competition may be determined as described in any aspect, embodiment, example, or model described herein, for example by SPR, ELISA, HTRF, or FACS.

[0352] In one embodiment, the antibody or fragment competes with the variable region of 02D10 (e.g., with an antibody comprising the heavy chain variable region of SEQ ID No: 34 and the light chain variable region of SEQ ID No: 48). In another embodiment, the antibody or fragment competes with 02D10 IgG4-PE having the heavy chain amino acid sequence of SEQ ID No: 62 and the light chain amino acid sequence of SEQ ID No: 64. Thus, for example, an IgG4-PE antibody having the heavy chain amino acid sequence of SEQ ID No: 62 and the light chain amino acid sequence of SEQ ID No: 64 can be used as a reference 02D10 antibody to determine the ability of the antibody or fragment to competitively bind hOX40L with antibody 02D10 by SPR (as described herein).

[0353] In another embodiment, the antibody or fragment may additionally or optionally compete with 10A7. In one embodiment, the antibody or fragment competes with the variable region of 10A7 (e.g., with an antibody comprising the heavy chain variable region of SEQ ID No: 2 and the light chain variable region of SEQ ID No: 16). In another embodiment, the antibody or fragment competes with 02D10 IgG4-PE having the heavy chain amino acid sequence of SEQ ID No: 30 and the light chain amino acid sequence of SEQ ID No: 32.

[0354] In one embodiment, the amino acid is any naturally occurring amino acid.

[0355] 74. The antibody or fragment according to aspect 73, wherein X is a neutral amino acid, optionally P or G.

[0356] In one embodiment, X is P or G. In another embodiment, X is selected from P, N, A, or G. In yet another embodiment, X is selected from P, G, or N.

[0357] 75. Optionally, an antibody or fragment thereof according to aspect 1 or 2, which specifically binds to hOX40L and competes with antibody 02D10 for binding to hOX40L, wherein the antibody or fragment comprises a VH domain comprising the HCDR3 sequence of SEQ ID NO: 40 or 46 or the HCDR3 sequence of SEQ ID NO: 40 or 46 containing fewer than 5 amino acid substitutions.

[0358] The characteristics of the antibodies described in any aspect, model, example, or embodiment described herein may be optionally modified as necessary to apply to these antibodies; for example, the antibodies may be human antibodies or chimeric antibodies having the functional characteristics described herein. Competition may be determined as described in any aspect, embodiment, example, or model described herein, for example by SPR, ELISA, HTRF, or FACS.

[0359] In one embodiment, the HCDR3 sequence of SEQ ID NO: 40 or 46 contains fewer than 4 amino acid substitutions (i.e., 3 or fewer). In one embodiment, the HCDR3 sequence of SEQ ID NO: 40 or 46 contains fewer than 3 amino acid substitutions (i.e., 2 or 1 substitution). In one embodiment, the HCDR3 sequence of SEQ ID NO: 40 or 46 contains fewer than 2 amino acid substitutions (i.e., 1 substitution).

[0360] In one embodiment, the antibody or fragment competes with the variable region of 02D10 (e.g., with an antibody containing the heavy chain variable region of SEQ ID No: 34 and the light chain variable region of SEQ ID No: 48). In another embodiment, the antibody or fragment competes with 02D10 IgG4-PE having the heavy chain amino acid sequence of SEQ ID No: 62 and the light chain amino acid sequence of SEQ ID No: 64.

[0361] In another embodiment, the antibody or fragment may additionally or optionally compete with 10A7. In one embodiment, the antibody or fragment competes with the variable region of 10A7 (e.g., with an antibody comprising the heavy chain variable region of SEQ ID No: 2 and the light chain variable region of SEQ ID No: 16). In another embodiment, the antibody or fragment competes with 02D10 IgG4-PE having the heavy chain amino acid sequence of SEQ ID No: 30 and the light chain amino acid sequence of SEQ ID No: 32.

[0362] 76. The antibody or fragment according to any one of aspects 73-75, wherein the VH domain comprises HCDR3 of 16 to 27 amino acids and is derived from a recombinant human VH gene fragment, a human D gene fragment and a human JH gene fragment, wherein the human JH gene fragment is IGHJ6 (e.g., IGHJ6*02).

[0363] In one embodiment, the human JH gene fragment is selected from IGHJ6*01, IGHJ6*02, IGHJ6*03, and IGHJ6*04. In another embodiment, the human JH gene fragment is selected from IGHJ6*01, IGHJ6*02, and IGHJ6*04. In yet another embodiment, the JH gene fragment is IGHJ6*02.

[0364] In another embodiment, the human VH gene fragment is IGHV3-23, for example selected from IGHV3-23*01, IGHV3-23*02, IGHV3-23*03, IGHV3-23*04, or IGHV3-23*05. In yet another embodiment, the human VH gene fragment is IGHV3-23*01 or IGHV3-23*04, particularly IGHV3-23*04.

[0365] In yet another embodiment, the human DH gene fragment is IGHD3-10, for example selected from IGHD3-10*01 or IGHD3-10*02. In one embodiment, the human DH gene fragment is IGHD3-10*01. In one embodiment, the human DH gene fragment is IGHD3-10*02.

[0366] 77. The antibody or fragment according to any one of aspects 73-76, wherein the VH domain comprises the HCDR1 sequence of SEQ ID NO: 36 or 42 or the HCDR1 sequence of SEQ ID NO: 36 or 42 containing fewer than 4 amino acid substitutions.

[0367] In one embodiment, the HCDR1 sequence of SEQ ID NO: 36 or 42 contains fewer than 3 amino acid substitutions (i.e., 2 or 1 substitution). In another embodiment, the HCDR1 sequence of SEQ ID NO: 36 or 42 contains fewer than 2 amino acid substitutions (i.e., 1 substitution).

[0368] 78. The antibody or fragment according to any one of aspects 73-77, wherein the VH domain comprises the HCDR2 sequence of SEQ ID NO: 38 or 44 or the HCDR2 sequence of SEQ ID NO: 38 or 44 containing fewer than 5 amino acid substitutions.

[0369] In one embodiment, the HCDR2 sequence of SEQ ID NO: 38 or 44 contains fewer than 4 amino acid substitutions (i.e., 3 or fewer). In one embodiment, the HCDR2 sequence of SEQ ID NO: 38 or 44 contains fewer than 3 amino acid substitutions (i.e., 2 or 1 substitution). In one embodiment, the HCDR2 sequence of SEQ ID NO: 38 or 44 contains fewer than 2 amino acid substitutions (i.e., 1 substitution).

[0370] 79. An antibody or fragment according to any one of aspects 73-78, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 34, or a heavy chain variable domain amino acid sequence that is at least 80% (e.g., at least 85%) the same as SEQ ID NO: 34.

[0371] In one embodiment, the amino acid sequence of the heavy chain variable domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34.

[0372] 80. An antibody or fragment according to any one of aspects 73-79, comprising first and second copies of the VH domain.

[0373] 81. An antibody or fragment according to any one of aspects 73-80, comprising a VL domain, said VL domain comprising the LCDR1 sequence of SEQ ID NO: 54 or 60 or the LCRD3 sequence of SEQ ID NO: 54 or 60 containing fewer than 5 amino acid substitutions.

[0374] In one embodiment, the LCRD3 sequence of SEQ ID NO: 54 or 60 contains fewer than 4 amino acid substitutions (i.e., 3 or fewer). In one embodiment, the LCRD3 sequence of SEQ ID NO: 54 or 60 contains fewer than 3 amino acid substitutions (i.e., 2 or 1 substitution). In one embodiment, the LCRD3 sequence of SEQ ID NO: 54 or 60 contains fewer than 2 amino acid substitutions (i.e., 1 substitution).

[0375] 82. An antibody or fragment according to any one of aspects 73-81, comprising one or more of the VL domains, the VL domain comprising the LCDR2 sequence of SEQ ID NO: 52 or 58, or the LCRD2 sequence of SEQ ID NO: 52 or 58 containing fewer than two amino acid substitutions.

[0376] 83. An antibody or fragment according to any one of aspects 73-82, comprising one or more of the VL domains, the VL domain comprising the LCDR1 sequence of SEQ ID NO: 54 or 60, or the LCRD1 sequence of SEQ ID NO: 54 or 60 containing fewer than 4 amino acid substitutions.

[0377] In one embodiment, the LCDR1 sequence of SEQ ID NO: 54 or 60 contains fewer than 3 amino acid substitutions (i.e., 2 or 1 substitution). In another embodiment, the LCDR1 sequence of SEQ ID NO: 54 or 60 contains fewer than 2 amino acid substitutions (i.e., 1 substitution).

[0378] 84. An antibody or fragment according to any one of aspects 73-83, comprising one or more of the VL domains, the VL domain comprising the amino acid sequence of SEQ ID NO: 48, or a light chain variable domain amino acid sequence that is at least 80% (e.g., at least 85%) the same as SEQ ID NO: 48.

[0379] In one embodiment, the amino acid sequence of the light chain variable domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 48.

[0380] 85. An antibody or fragment according to any one of aspects 81-84, comprising first and second copies of the VL domain.

[0381] 86. An antibody or fragment according to any one of aspects 81-85, wherein the antibody or fragment comprises a κ light chain.

[0382] In another embodiment, the VL domain is a κVL domain. In one embodiment, the κVL domain is derived from a recombination of a human VL gene fragment and a human JL gene fragment, wherein the human VL gene fragment is IGKV1D-39. In another embodiment, the VL gene fragment is IGKV1D-39*01.

[0383] In yet another embodiment, the human JL gene fragment is IGKJ1 or IGKJ3. In another embodiment, the JL gene fragment is IGKJ1*01. In yet another embodiment, the JL gene fragment is IGKJ3*01.

[0384] 87. An antibody or fragment according to any one of aspects 75-86, wherein the amino acid substitution is a conserved amino acid substitution, optionally wherein the conserved substitution is from one of six groups selected from the following (each group contains amino acids that are conserved substitutions for each other):

[0385] 1) Alanine (A), Serine (S), Threonine (T);

[0386] 2) Aspartic acid (D), glutamic acid (E);

[0387] 3) Asparagine (N), glutamine (Q);

[0388] 4) Arginine (R), Lysine (K);

[0389] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0390] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0391] In one embodiment, the conserved amino acid substitutions are as described herein. For example, F may replace Y, S, or K; T may replace T, A may replace P, D, or Q; E may replace E, D, or G; N may replace N, K; R may replace N, or A; G may replace G, S, or K; T may replace N, or E; D may replace L, or V; I may replace Y, F, T, or A; S may replace R, N, or A; G may replace R, R may replace K, S, K, or P; and A may replace Y. In another embodiment, the conserved amino acid substitutions may be wherein F replaces Y, A, or S replaces T, L, or V replaces I, Y replaces W, L replaces M, D replaces N, A replaces G, A, or S replaces T, N replaces D, L, or V replaces I, Y, or L replaces F, A, or T replaces S, and S, G, T, or V replace A.

[0392] 88. An antibody or fragment according to any one of aspects 73-87, wherein the antibody or fragment comprises a constant region, such as an IgG4 constant region, optionally wherein the constant region is IgG4-PE (Seq ID No: 128).

[0393] In another example of any aspect of this document, the antibody or fragment comprises a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, contains a Leu235Glu mutation (i.e., where a wild-type leucine residue is mutated to a glutamate residue). In another embodiment, the heavy chain constant region contains a Ser228Pro mutation to increase stability.

[0394] 89. The antibody according to any one of aspects 73-88, wherein the antibody comprises a heavy chain and a light chain, the heavy chain amino acid sequence comprising the sequence of SEQ ID No: 62 and the light chain amino acid sequence comprising the sequence of SEQ ID No: 64.

[0395] 90. An antibody or fragment as defined in any of sections 73-89, 98, 99, 101, or 102, for the treatment or prevention of hOX40L-mediated diseases or conditions selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD.

[0396] The antibody and hOX40L-mediated disease characteristics described in any aspect, mode, example, or embodiment herein may be adapted for this use with the necessary modifications. Similarly, any composition, dosing regimen, or administration method described in any aspect, mode, example, or embodiment herein may be adapted for this use with the necessary modifications.

[0397] 91. Use of an antibody or fragment as defined in any of sections 73-89, 98, 99, 101, or 102 in the production of a pharmaceutical agent for the treatment or prevention of hOX40L-mediated diseases or conditions in humans, selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant / host rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD.

[0398] The antibody and hOX40L-mediated disease characteristics described in any aspect, mode, example, or embodiment herein may be adapted for this use with the necessary modifications. Similarly, any composition, dosing regimen, or administration method described in any aspect, mode, example, or embodiment herein may be adapted for this use with the necessary modifications.

[0399] 92. A method for treating or preventing hOX40L-mediated diseases or conditions in a person, wherein the hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD, the method comprising administering to the person a therapeutically effective amount of an antibody or fragment as defined in any one of aspects 73-89, 98, 99, 101, or 102, wherein the hOX40L-mediated disease or condition is thereby treated or prevented.

[0400] The antibody and hOX40L-mediated disease characteristics described in any aspect, mode, example, or embodiment herein, optionally modified as necessary, are applicable to this method. Any composition, dosing regimen, or administration mode described in any aspect, mode, example, or embodiment herein, optionally modified as necessary, is applicable to this method.

[0401] 93. The antibody or fragment according to aspect 90, the use according to aspect 91, or the method according to aspect 92, wherein the hOX40L-mediated disease or symptom is GvHD.

[0402] In another embodiment, the antibody or fragment is capable of treating or preventing GvHD.

[0403] 94. An antibody or fragment, use, or method according to any one of aspects 90-93, wherein the antibody is administered prophylactically.

[0404] In one embodiment, the prevention prevents the onset of the disease or symptom, or the symptoms of the disease or symptom. In one embodiment, the preventive treatment prevents the disease or symptom from worsening or occurring. In one embodiment, the preventive treatment prevents the disease or symptom from worsening.

[0405] In another embodiment, the antibody is administered intravenously. In another embodiment, the antibody is administered at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg). In another embodiment, the antibody is administered at a dose selected from about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg, particularly about 1 mg / kg or about 3 mg / kg.

[0406] In another embodiment, the antibody is administered 1-4 days prior to transplantation, for example, 1-3 days or 1-2 days prior to transplantation. In another embodiment, the antibody is administered weekly, every two weeks, or monthly after transplantation, for example, every two weeks. In yet another embodiment, the antibody is administered prophylactically intravenously at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg) 1-3 days prior to transplantation, and then intravenously at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg) every two weeks.

[0407] In another implementation, the presence of biomarkers predicting the development of GvHD (e.g., acute GvHD) is monitored periodically post-transplant, and the anti-OX40L antibody of the present invention is administered once the biomarker levels indicate that the patient is at risk of developing GvHD (e.g., acute GvHD). This strategy avoids unnecessary drug administration and unnecessary immunosuppression. Examples of biomarkers that can be used as predictive biomarkers for acute GvHD include those identified by Levine et al., “Aprognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study,” Lancet Haematol 2015;2:e21-29. These biomarkers include, but are not limited to, TNFR1, ST-2, elafin, and IL2Rα and Reg3α.

[0408] 95. A human antibody or a fragment thereof comprising 16 to 27 amino acids of HCDR3 and derived from a recombinant human VH gene fragment, a human D gene fragment, and a human JH gene fragment, wherein the human JH gene fragment is IGHJ6 (e.g., IGHJ6*02), wherein the human antibody or fragment thereof specifically binds to hOX40L to treat or prevent hOX40L-mediated diseases or conditions selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; such as inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD (e.g., wherein the antibody is used for GvHD prevention).

[0409] The characteristics of the antibody and hOX40L-mediated disease in any aspect, mode, example, or embodiment may be optionally modified as necessary for this use. Any composition, dosing regimen, or administration method described in any aspect, mode, example, or embodiment herein may be optionally modified as necessary for this use.

[0410] 96. Use of a human antibody or fragment thereof comprising 16 to 27 amino acids and derived from a recombinant human VH gene fragment, a human D gene fragment, and a human JH gene fragment in the production of a medicament for administration to humans for the treatment or prevention of hOX40L-mediated diseases or conditions in humans, wherein the human JH gene fragment is IGHJ6 (e.g., IGHJ6*02), and the human antibody or fragment thereof specifically binds to hOX40L, wherein the hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD.

[0411] The characteristics of the antibody and hOX40L-mediated disease in any aspect, mode, example, or embodiment may be optionally modified as necessary for this use. Any composition, dosing regimen, or administration method described in any aspect, mode, example, or embodiment herein may be optionally modified as necessary for this use.

[0412] 97. A method for treating or preventing hOX40L-mediated diseases or conditions in humans, wherein the hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, or atherosclerosis, especially GvHD, the method comprising administering to the human a therapeutically effective amount of a human antibody or fragment thereof comprising 16 to 27 amino acids and derived from a human VH gene fragment, a human D gene fragment, and a human JH gene fragment, wherein the human JH gene fragment is IGHJ6 (e.g., IGHJ6*02), the human antibody or fragment thereof specifically binding to hOX40L, thereby treating or preventing the hOX40L-mediated diseases or conditions.

[0413] The characteristics of the antibody and hOX40L-mediated disease in any aspect, mode, example, or embodiment may be optionally modified as necessary to apply to this method. Any composition, dosing regimen, or administration method described in any aspect, mode, example, or embodiment herein may be optionally modified as necessary to apply to this method.

[0414] In one embodiment of any of aspects 95-97, the human JH gene fragment is selected from IGHJ6*01, IGHJ6*02, IGHJ6*03, and IGHJ6*04. In another embodiment of any of aspects 95-97, the human JH gene fragment is selected from IGHJ6*01, IGHJ6*02, and IGHJ6*04. In yet another embodiment of any of aspects 95-97, the human JH gene fragment is IGHJ6*02.

[0415] In another embodiment of any of aspects 95-97, the human VH gene fragment is IGHV3-23, for example selected from IGHV3-23*01, IGHV3-23*02, IGHV3-23*03, IGHV3-23*04, or IGHV3-23*05. In yet another embodiment of any of aspects 95-97, the human VH gene fragment is IGHV3-23*01 or IGHV3-23*04, particularly IGHV3-23*04.

[0416] In another embodiment of any of aspects 95-97, the human DH gene fragment is IGHD3-10, for example selected from IGHD3-10*01 or IGHD3-10*02. In one embodiment of any of aspects 95-97, the human DH gene fragment is IGHD3-10*01. In one embodiment of any of aspects 95-97, the human DH gene fragment is IGHD3-10*02.

[0417] In one embodiment of any of aspects 90-97, the antibody is capable of treating or preventing GvHD. In another embodiment of any of aspects 90-97, the antibody or fragment is used to treat or prevent diseases other than GvD, but the antibody or fragment is capable of treating or preventing GvHD.

[0418] 98. An antibody or fragment according to aspect 86, or an antibody or fragment according to aspect 95, for use according to aspect 96, or for method according to aspect 97, wherein the antibody or fragment comprises a κ light chain, for example, wherein the VL domain of the light chain is derived from a recombinant human VL gene fragment and a human JL gene fragment, wherein the human VL gene fragment is IGKV1D-39 (e.g., IGKV1D-39*01), and optionally the human JL gene fragment is IGKJ1 (e.g., IGKJ1*01) or IGKJ3 (e.g., IGKJ3*01).

[0419] In another embodiment, the VL domain is a κVL domain. In one embodiment, the κVL domain is derived from a recombination of a human VL gene fragment and a human JL gene fragment, wherein the human VL gene fragment is IGKV1D-39. In another embodiment, the VL gene fragment is IGKV1D-39*01.

[0420] In yet another embodiment, the human JL gene fragment is IGKJ1. In another embodiment, the JL gene fragment is IGKJ1*01. In yet another embodiment, the human JL gene fragment is IGKJ3. In yet another embodiment, the JL gene fragment is IGKJ3*01.

[0421] 99. An antibody or fragment according to any one of aspects 73-89, 98, 101 or 102, or an antibody or fragment, use or method according to any one of aspects 90-98, wherein the antibody or fragment enables greater than 80% stem cell donor chimerism in a rhesus monkey model of haploidentical hematopoietic stem cell transplantation by day 12, optionally wherein the antibody is used for GvHD prophylaxis.

[0422] On the other hand, antibodies or fragments, uses, or methods according to any one of aspects 95-98 are provided, wherein the antibodies or fragments are used to treat or prevent transplant rejection (e.g., GvHD) in the person by enabling higher than 80% stem cell donor chimerism in the person up to day 12 after donor hematopoietic stem cell transplantation.

[0423] In another embodiment, an antibody or fragment according to any one of aspects 73-89, 98, 101 or 102 is provided, wherein the antibody or fragment enables greater than 80% stem cell donor chimerism in a rhesus monkey model of haploidentical hematopoietic stem cell transplantation by day 12.

[0424] In one implementation, chimeric T cells (CD3) are used. + / CD20 - Chimerism. In another embodiment, chimerism is peripheral blood chimerism. In another embodiment, chimerism is peripheral blood or T cell (CD3) chimerism. + / CD20 - ) Inlay.

[0425] In one implementation, microsatellite markers with discrete donor and recipient-specific MHC linkages are used to determine stem cell donor chimerism (e.g., peripheral blood or T cell (CD3)) by comparing the peak heights of donor and recipient-specific amplicon. + / CD20 -(Chimerism). In another embodiment, as described by Kean, LS et al., “Induction of chimerism in rhesusmacaques through stem cell transplant and costimulation blockade-based immunosuppression”, Am J Transplant. 2007 Feb; 7(2): 320-35, stem cell donor chimerism is determined. In another embodiment, stem cell donor chimerism is determined as described in Example 7.

[0426] In one implementation, a rhesus monkey model of haploidentical hematopoietic stem cells is achieved by the following: the transplantation (HSCT) recipient animal undergoes a conditioning procedure and administration of anti-OX40L antibody, followed by infusion of peripheral blood products isolated from a half-sib donor animal, after which the animal continues to receive weekly doses of the anti-OX40L antibody of the present invention, and blood samples are taken and chimerism is analyzed.

[0427] In another implementation, in the HSCT model, recipient animals receive a modulated radiation dose of 1020 cGy in four doses over two days (experimental day-2 and day-1) to restore the recipient’s immune system before intravenous administration of the anti-OX40L antibody of the present invention (following intravenous doses on day-2, days 5, 12, 19, 26, 33, 40, and 47) and transplantation of leukemia cells and stem cell-rich peripheral blood from MHC half-matched (half-sibling) donor animals, while providing ongoing supportive care, blood collection, and monitoring for GVHD signs.

[0428] In one implementation, the antibody or fragment, its use, or method is used for GvHD prevention.

[0429] In one embodiment, the anti-hOX40L antibody of the present invention is administered prophylactically. In another embodiment, prophylactic treatment prevents the disease or symptoms from worsening or flaring up.

[0430] In another embodiment, the antibody is administered intravenously. In another embodiment, the antibody is administered at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg). In another embodiment, the antibody is administered intravenously. In another embodiment, the antibody is administered at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg). In another embodiment, the antibody is administered at a dose selected from about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg, particularly at about 1 mg / kg or about 3 mg / kg.

[0431] In another embodiment, the antibody is administered 1-4 days prior to transplantation, for example, 1-3 days or 1-2 days prior to transplantation. In another embodiment, the antibody is administered weekly, every two weeks, or monthly after transplantation, for example, every two weeks. In yet another embodiment, the antibody is administered prophylactically intravenously at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg) 1-3 days prior to transplantation, and then intravenously at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg) every two weeks.

[0432] In another implementation, the presence of biomarkers predicting the development of GvHD (e.g., acute GvHD) is monitored periodically post-transplant, and the anti-OX40L antibody of the present invention is administered once the biomarker levels indicate that the patient is at risk of developing GvHD (e.g., acute GvHD). This strategy avoids unnecessary drug administration and unnecessary immunosuppression. Examples of biomarkers that can be used as predictive biomarkers for acute GvHD include those identified by Levine et al., “Aprognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study,” Lancet Haematol 2015;2:e21-29. These biomarkers include, but are not limited to, TNFR1, ST-2, elastin, and IL2Rα and Reg3α.

[0433] In another implementation, HSCT models, such as those by Miller, Weston P. et al., "GVHD after haploidentical transplantation: a novel, MHC-defined rhesus macaque model identifies CD28," are used. - CD8 + The implementation is as described in Blood, 116, 24(2010): 5403-5418, which describes T cells as a reservoir of breakthrough T-cell proliferation during costimulation blockade and sirolimus-based immunosuppression. In another embodiment, the HSCT model is implemented as described in Example 7.

[0434] 100. An antibody or fragment, use or method according to any one of aspects 95-99, wherein the antibody is defined as in any one of aspects 73-89, 98, 99, 101 or 102.

[0435] 101. An antibody or fragment according to any one of aspects 73-89, 98, 99 or 102, or an antibody or fragment, use or method according to any one of aspects 90-100, wherein said antibody or fragment is expressed as a stable transfection library in Lonza GS-Xceed™ at a level higher than 1.5 g / L in a batch overgrowth culture using a Lonza 8 version feeding system for an overgrowth period of 14 days.

[0436] In one implementation, the expression level is higher than 1.0 g / L, higher than 1.1 g / L, higher than 1.2 g / L, higher than 1.3 g / L, or higher than 1.4 g / L.

[0437] 102. An antibody or fragment according to any one of aspects 73-89, 98, 99 or 101, or an antibody or fragment, use or method according to any one of aspects 90-101, wherein in a rhesus monkey model of haploidentical hematopoietic stem cell transplantation, the antibody or fragment remains > total CD4 on day 12. + 20% of the T cell population is naturally CD4 + T cell population

[0438] On the other hand, it provides an antibody or fragment according to any one of aspects 73-89, 98, 99 or 101, or an antibody or fragment, use or method according to any one of aspects 90-101, wherein the antibody or fragment is used to maintain human > total CD4 on day 12 after donor hematopoietic stem cell transplantation. + 20% of the T cell population are natural donors of CD4 + T-cell populations can be used to treat or prevent transplant rejection in the individuals in question.

[0439] In one implementation, the HSCT model is as described in any of the implementations considered above, such as in conjunction with aspect 99.

[0440] In another embodiment, the natural population is measured by evaluating the relative proportions of specific T cell phenotypes using flow cytometry, wherein cell subsets are identified by labeling with fluorescent antibody probes, and thus the natural CD4 or CD8 T cells are labeled CD4 T cells, respectively. + / CD28 + / CD95 - or CD8 + / CD28 + / CD95 - Central memory CD4 or CD8 T cells are labeled CD4+ and CD8+, respectively. + / CD28 + / CD95 + or CD8 + / CD28 + / CD95 + Furthermore, the effector memory CD4 or CD8 T cells are labeled CD4... + / CD28 - / CD95 + or CD8 + / CD28 - / CD95 + .

[0441] 103. The antibody or fragment, use, or method according to any one of aspects 90-102, further comprising administering to the person another therapeutic agent, optionally said other therapeutic agent being independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, and mycophenolate mofetil. Anti-CD28 antibody, anti-IL12 / IL-23 antibody (e.g., ustekinumab), anti-CD20 antibody (e.g., rituximab), anti-CD30 antibody (e.g., brentuximab), CTLA4-Fc molecule (e.g., abatacept), CCR5 receptor antagonist (e.g., maraviroc), anti-CD40L antibody, anti-VLA4 antibody (e.g., natalizumab), anti-LFA1 antibody, fludarabine, anti-CD52 antibody (e.g., alemtuzumab), anti-CD45 antibody Cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibody (e.g., eculizumab), anti-a4b7 integrin antibody (e.g., vedolizumab), anti-IL6 antibody (e.g., tocilizumab), anti-IL2R antibody (e.g., basilixumab), anti-CD25 antibody (e.g., daclizumab), anti-TNFα / TNFα-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, or certolizumab) pegol) and vorinostat, especially rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, CTLA4-Fc molecule (e.g., abatacept), anti-CD40L antibody, anti-LFA1 antibody, anti-CD52 antibody (e.g., alemtuzumab), cyclophosphamide, and anti-thymocyte globulin.

[0442] In one embodiment, the other therapeutic agent is an anti-inflammatory drug. In another embodiment, the anti-inflammatory drug is independently selected from corticosteroids (e.g., methylprednisolone), anti-IL12 / IL-23 antibodies (e.g., uterotumab), anti-VLA4 antibodies (e.g., natezumab), anti-LFA1 antibodies, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., baliximab), or anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, sertozumab). In one example, the anti-inflammatory drug is independently selected from corticosteroids (e.g., methylprednisolone) and anti-LFA1 antibodies.

[0443] 104. The antibody or fragment, use, or method according to aspect 103, wherein the other therapeutic agent is administered sequentially or simultaneously with the anti-hOX40L antibody or fragment.

[0444] 105. A pharmaceutical composition comprising an antibody or fragment as defined in any one of aspects 73-89, 98, 99, 101, or 102 and a pharmaceutically acceptable excipient, diluent, or carrier, and optionally further comprising another therapeutic agent independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, anti-IL12 / IL-23 antibody (e.g., uterotium bromide), anti-CD20 antibody (e.g., rituximab), anti-CD30 antibody (e.g., butuximab), CTLA4-Fc molecule (e.g., abatacept), CCR5 receptor antagonist (e.g., malaviro), anti-CD40L antibody, anti-VLA4 antibody (e.g., natetzumab), anti-LFA1 antibody, fludarabine, anti-CD52 antibody (e.g., abatacept), and other similar antibodies. Anti-CD45 antibodies, cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., baliximab), anti-CD25 antibodies (e.g., daliximab), anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, or sertozumab), and vorinostats, especially rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibodies, CTLA4-Fc molecules (e.g., abatacept), anti-CD40L antibodies, anti-LFA1 antibodies, anti-CD52 antibodies (e.g., alemtuzumab), cyclophosphamide, and anti-thymocyte globulin.

[0445] Pharmaceutically acceptable excipients, diluents, or carriers as described herein, with necessary modifications, are suitable for these compositions.

[0446] In one embodiment, the other therapeutic agent is an anti-inflammatory drug. In another embodiment, the anti-inflammatory drug is independently selected from corticosteroids (e.g., methylprednisolone), anti-IL12 / IL-23 antibodies (e.g., uterotumab), anti-VLA4 antibodies (e.g., natezumab), anti-LFA1 antibodies, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., baliximab), or anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, sertozumab). In one example, the anti-inflammatory drug is independently selected from corticosteroids (e.g., methylprednisolone) and anti-LFA1 antibodies.

[0447] 106. The pharmaceutical composition according to aspect 105, or a kit comprising the pharmaceutical composition as defined in aspect 105, wherein the composition is for the treatment and / or prevention of hOX40L-mediated diseases or conditions selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD.

[0448] The hOX40L-mediated diseases described in this article may be optionally modified as necessary to suit this combination.

[0449] 107. A combination of a pharmaceutical composition according to aspect 105 or aspect 106 with a kit containing a label or instructions according to aspect 106, or a kit containing a label or instructions for treating and / or preventing said disease or condition in humans; optionally, wherein said label or instructions contain a marketing authorization number (e.g., FDA or EMA license number); optionally, wherein said kit includes an IV or injection device containing said antibody or fragment.

[0450] The labels, instructions, hOX40L-mediated diseases and symptoms described herein may be optionally modified as necessary for this combination.

[0451] 108. A nucleic acid encoding HCDR3 of an antibody or fragment as defined in any of aspects 73-89, 98, 99, 101 or 102.

[0452] 109. A nucleic acid encoding a VH domain and / or VL domain of an antibody or fragment as defined in any of aspects 73-89, 98, 99, 101 or 102.

[0453] 110. The nucleic acid according to aspect 109, comprising at least 80% identical nucleotide sequences to those of SEQ ID NO: 33 and / or SEQ ID NO: 47.

[0454] In one instance, the nucleotide sequence is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 33 and / or SEQ ID NO: 47.

[0455] 111. A nucleic acid encoding the heavy or light chain of an antibody described in any of the aspects 73-89, 98, 99, 101 or 102.

[0456] 112. A vector comprising a nucleic acid according to any one of aspects 108-111; optionally wherein the vector is a CHO or HEK293 vector.

[0457] 113. A host comprising a nucleic acid of any one of aspects 108-111 or a vector of aspect 112.

[0458] As illustrated in the examples, the inventors devised a series of criteria that are particularly useful for identifying the antibodies and fragments of the present invention. These criteria are: -

[0459] (a) The ability of the antibody or fragment described in the HTRF assay to bind to cell surface hOX40L on CHO-S cells (optionally transfected with full-length human OX40L) and / or to bind to recombinant hOX40L;

[0460] (b) The ability of the antibody or fragment to neutralize human OX40 (e.g., neutralize the binding of human OX40L to the human OX40 receptor) in receptor and HTRF assays and / or flow cytometry receptor and assays; and

[0461] (c) The ability of the antibody or fragment to specifically bind to human and rhesus monkey OX40L (useful, enabling the evaluation of the PK, PD, efficacy and other parameters of the antibody or fragment in a rhesus monkey model as a human substitute).

[0462] Therefore, in one embodiment of the present invention, the antibody or fragment satisfies criteria (a), (b) and (c).

[0463] In one instance, a criterion (a) is set such that FACS assays show <70% receptor binding to hOX40L expressed on CHO-S cells.

[0464] In one instance, a standard (a) is set such that the antibody or fragment shown in an HTRF assay exhibits <90% receptor binding to OX40L.

[0465] In one instance, a standard (a) is set such that the antibody or fragment shows at least 20% efficacy in an HTRF assay.

[0466] In one instance, OX40 is used in standard (b).

[0467] In one embodiment, the assay or test of the antibody or fragment of the present invention is performed at or substantially at pH 7 (e.g., for in vitro assays and determinations) and at or substantially at room temperature.

[0468] Optionally, the antibody or fragment specifically binds to hOX40L, for example, under the SPR conditions disclosed herein, by SPR determination, with an affinity (apparent affinity, Kd) below 1 μM, ranging from 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, below 200 pM, ranging from 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, for example, in the range of 1 mM to 1 pM (e.g., 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM). Alternatively or additionally, the antibody or fragment specifically binds to rhesus monkey OX40L, for example, under the SPR conditions disclosed herein, by an SPR assay, with an affinity (apparent affinity, Kd) below 1 μM, ranging from 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, below 200 pM, ranging from 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, for example, in the range of 1 mM to 1 pM (e.g., 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM). Such binding measurements can be performed using various binding assays known in the art, such as surface plasmon resonance (SPR), for example using Biacore. TM Or use ProteOn XPR36 TM (Bio- ),use (Sapidyne Instruments, Inc.) or use ForteBio Octet (Pall ForteBio Corp.).

[0469] OX40L binding capacity, specificity, and affinity (Kd, K off and / or K on This can be determined by any conventional method in the art, such as surface plasmon resonance (SPR). The term "Kd," as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0470] In one embodiment, surface plasmon resonance (SPR) is performed at 25°C. In another embodiment, SPR is performed at 37°C.

[0471] In one implementation, SPR is performed at a physiological pH, such as about pH 7 or at pH 7.6 (e.g., using Hepes buffered saline at pH 7.6, also known as HBS-EP).

[0472] In one implementation, SPR is performed at physiological saline levels, such as 150 mM NaCl.

[0473] In one embodiment, SPR is at a detergent level not exceeding 0.05% by volume, for example at 0.05% of P20 (polysorbate 20; for example, Tween-20). TM (Tween-20 TM The procedure was carried out in the presence of 3 mM EDTA.

[0474] In one example, SPR was performed at 25°C or 37°C in a buffer solution at pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA. The buffer solution may contain 10 mM Hepes. In another example, SPR was performed at 25°C or 37°C in an HBS-EP. HBS-EP is available from Teknova Inc. (Califomia; catalog number H8022).

[0475] In one instance, the affinity of the antibody or fragment was determined using SPR in the following manner:

[0476] 1. For example, by primary amine coupling to match the constant region of anti-mouse (or other relevant human, rat, or non-human vertebrate antibody species) IgG (e.g., Biacore) TM BR-1008-38) is coupled to a biosensor chip (e.g., a GLM chip);

[0477] 2. Expose anti-mouse IgG (or other matching species antibody) to the test IgG antibody to capture the test antibody on the chip;

[0478] 3. Pass the test antigen through the capture surface of the chip at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM, and 0 nM (i.e., separate buffer solutions); and

[0479] 4. For example, under the SPR conditions discussed above (e.g., in physiological buffer at 25°C), the binding affinity of the test antibody to the test antigen is determined using surface plasmon resonance. Any standard SPR apparatus can be used, such as Biacore. TM Or use ProteOn XPR36 TM (Bio- Perform SPR.

[0480] Regeneration of the captured surface can be performed using 10 mM glycine at pH 1.7. This removes the captured antibody and makes the surface available for another interaction. Standard techniques, such as those using ProteOn XPR36, can be used. TM The analysis software's inherent model will be fitted to the data to form a 1:1 inherent model.

[0481] In one example, the antibody or fragment of the present invention is contained in a medical container, such as a vial, syringe, IV container, or injection device (e.g., intraocular or intravitreal injection device). In one example, the antibody or fragment is in vitro, for example, in a sterile container. In one example, the present invention provides a kit comprising the antibody or fragment of the present invention, packaging, and instructions for use in humans for the treatment, prevention, or diagnosis of OX40L-mediated diseases or symptoms. In one example, the instructions indicate that the OX40L variant sequence of the present invention should be genotyped in humans before administration of the antibody or fragment. In one example, the instructions indicate that the OX40L variant of the present invention should be phenotypically analyzed in humans before administration of the antibody or fragment. In one example, the human is Chinese (e.g., Han or CHS) and the instructions are in Chinese (e.g., Mandarin).

[0482] In one instance, the binding sites of the antibody or fragment are selected from multiple binding sites (e.g., a library). For example, the multiple binding sites comprise or consist of multiple 4-chain antibodies or fragments thereof, such as dAb, Fab, or scFv. Suitable methods for generating multiple binding sites for screening include phage display (generating a phage display library of antibody binding sites), ribosome display (generating a ribosome display library of antibody binding sites), yeast display (generating a yeast display library of antibody binding sites), or immunization of non-human vertebrates (e.g., rodents, such as mice or rats, e.g., Velocimouse) with hOX40L or hOX40L epitopes. TM Kymouse TM Xenomouse TM Aliva Mouse TM HuMab Mouse TM Omnimouse TM Omnirat TM Or MeMo Mouse TM And antibody-generating cell libraries (e.g., B cell, plasma cell, or plasmablastic cell libraries) and / or libraries of isolated antibodies, fragments, or binding sites.

[0483] The term "epitope" is a region of an antigen that is bound to an antibody or fragment. Epitopes can be defined as structural or functional. Functional epitopes are typically subsets of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0484] With respect to any aspect of the invention, the term "isolated," for example, of an antibody or fragment, means that the tested antibody or fragment (1) does not contain at least some other proteins that would normally be found with it, (2) is substantially free of other proteins from the same source, such as from the same species, (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other substances associated with it in nature, (5) is operatively associated with polypeptides (through covalent or non-covalent interactions) that are not associated with it in nature, or (6) is not found in nature. Typically, "isolated" antibodies, fragments, etc., constitute at least about 5%, at least about 10%, at least about 25%, or at least about 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or >99% of a given sample. Such isolated antibodies, fragments, etc., may be encoded by genomic DNA, cDNA, mRNA or other RNA of synthetic origin or any combination thereof. Preferably, the isolated antibodies, fragments, etc., are substantially free of proteins or peptides or other contaminants found in their natural environment that would interfere with their therapeutic, diagnostic, preventative, research, or other uses.

[0485] For example, an “isolated” antibody is an antibody that has been identified as being separated and / or recovered from components of its production environment (e.g., naturally or recombinantly). Preferably, the isolated peptide does not associate with any other components from its production environment, for example, such that the antibody has been isolated to FDA-approved or approved standards. Contaminant components of its production environment, such as contaminants generated by recombinant transfected cells, are substances that typically interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In a preferred embodiment, the peptide will be purified to: (1) greater than 95% by weight of antibody, and in some embodiments, greater than 99% by weight of antibody, for example, by Lowry assay; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a turn-cup sequencer; or (3) homogeneity achieved by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Because at least one component of the antibody’s natural environment will be absent, isolated antibodies include in situ antibodies from recombinant cells. However, typically, isolated peptides or antibodies are prepared through at least one purification step.

[0486] Immunoconjugates

[0487] This invention covers antibodies or fragments conjugated to therapeutic components (“immunoconjugates”), such as cytotoxins, chemotherapeutic agents, immunosuppressants, or radioisotopes. Cytotoxic agents include any reagents that are harmful to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see, for example, WO 05 / 103081, which is incorporated herein by reference in its entirety.

[0488] Bispecific

[0489] The antibodies and fragments of the present invention can be monospecific, bispecific, or multispecific. A multispecific mAb may be specific to different epitopes of a target polypeptide or may contain antigen-binding domains specific to more than one target polypeptide. See, for example, Tutt et al., (1991) J. Immunol. 147: 60-69. Human anti-hOX40L antibodies or fragments may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to produce bispecific or multispecific antibodies with a second binding specificity.

[0490] Exemplary bispecific antibody forms that can be used in the context of this invention involve the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and the difference of at least one amino acid residue reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without amino acid residues. In one embodiment, the first Ig CH3 domain binds to protein A while the second Ig CH3 domain contains a mutation that reduces or eliminates protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R, according to EU numbering). The second CH3 domain may also contain a Y96F modification (according to IMGT; Y436F, according to EU). Other modifications that may be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V821 (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I, according to EU) in the case of IgG1 antibody; N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I, according to EU) in the case of IgG2 antibody; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I, according to EU) in the case of IgG4 antibody. These modifications to the form of the bispecific antibodies are covered within the scope of this invention.

[0491] In some embodiments, the antibody or its OX40L binding fragment contains fewer than 6 CDRs. In some embodiments, the antibody or its antigen-binding fragment contains or consists of 1, 2, 3, 4, or 5 CDRs selected from HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.In a specific implementation, the antibody or its antigen-binding fragment comprises or consists of 1, 2, 3, 4, or 5 CDRs selected from the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the sequence listing (i.e., for HCDR1, Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96, or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42; for HCDR2, Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98, or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44; for HCDR3, Seq ID No: 8, Seq ID No: 96, ... Seq ID No: 36, Seq ID No: 42, Seq ID No: 36, Seq ID No: 42, Seq ID No: 42, Seq ID No: 62, Se For Seq ID No: 14, Seq ID No: 40, Seq ID No: 46, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46; for LCDR1, Seq ID No: 18, Seq ID No: 24, Seq ID No: 50, Seq ID No: 56, Seq ID No: 82, Seq ID No: 88, Seq ID No: 110, or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56; for LCDR2, Seq ID No: 20, Seq ID No: 26, Seq ID No: 52, Seq ID No: 58, Seq ID No: 84, Seq ID No: 90, Seq ID No: 112, or Seq ID No: 118, especially Seq ID No: 52 or Seq ID No: 106. IDNo: 58; and for LCDR3, Seq ID No: 22, Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114 or Seq ID No: 120, especially Seq ID No: 54 or Seq ID No: 60).

[0492] In specific embodiments, the antibody of the present invention is a fully human antibody, a monoclonal antibody, a recombinant antibody, an antagonistic antibody, an hOX40L neutralizing antibody, or any combination thereof, or the present invention provides its hOX40L binding fragment. In one example, the antibody is a chimeric antibody comprising a human variable domain and a non-human (e.g., mouse, rat, or rabbit) constant domain. In a particular embodiment, the antibody is a fully human antibody, such as a fully human monoclonal antibody or its antigen-binding fragment, which specifically binds to hOX40L. In a preferred embodiment, the antibody is an antagonistic antibody. In a preferred embodiment, the antibody is a neutralizing antibody.

[0493] In one instance, the antibody or fragment is a λ-type antibody or fragment (i.e., its variable domain is a λ-variable domain). Optionally, the antibody or fragment also includes a λ-constant domain.

[0494] In some embodiments, the antibody competitively (e.g., in a dose-dependent manner) binds to hOX40L, such as cell surface-expressed hOX40L or soluble hOX40L, against OX40 or its fusion protein (e.g., Fc:OX40). Exemplary competitive blocking assays are provided in the embodiments herein.

[0495] On the other hand, this article provides isolated nucleic acids that encode antibodies that bind to hOX40L peptides (e.g., cell surface-expressed or soluble hOX40L), hOX40L peptide fragments, or hOX40L epitopes. In some embodiments, the nucleic acid encodings are as disclosed in the sequence: VH strand, VL strand, VH domain, VL domain, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (i.e., Seq ID No. 30 or Seq ID No. 62 for the VH strand; Seq ID No. 32 or Seq ID No. 64 for the VL strand; Seq ID No. 2, Seq ID No. 34, Seq ID No. 66, or Seq ID No. 94 for the VH domain, especially Seq ID No. 34; Seq ID No. 16, Seq ID No. 48, Seq ID No. 80, or Seq ID No. 108 for the VL domain, especially Seq ID No. 48; Seq ID No. 4, Seq ID No. 10, Seq ID No. 36, Seq ID No. 42, and Seq ID No. 64 for the HCDR1). For HCDR2, the Seq ID No: 68, Seq ID No: 74, Seq ID No: 96, or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42; for HCDR2, the Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98, or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44; for HCDR3, the Seq ID No: 8, Seq ID No: 14, Seq ID No: 40, Seq ID No: 46, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46; for LCDR1, the Seq ID No: 18, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96, or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42; for HCDR2, the Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98, or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44; for HCDR3, the Seq ID No: 8, Seq ID No: 14, Seq ID No: 40, Seq ID No: 46, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: IDNo: 24, SeqIDNo: 50, SeqID No: 56, Seq ID No: 82, Seq ID No: 88, Seq ID No: 110 or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56;For LCDR2, SeqIDNo: 20, SeqIDNo: 26, SeqIDNo: 52, SeqIDNo: 58, SeqIDNo: 84, SeqIDNo: 90, SeqIDNo: 112, or SeqIDNo: 118, especially Seq ID No: 52 or Seq ID No: 58; and for LCDR3, Seq ID No: 22, Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114, or Seq IDNo: 120, especially Seq ID No: 54 or Seq ID No: 60).

[0496] On the other hand, this article provides vectors and host cells containing nucleic acids encoding antibodies or fragments of the present invention.

[0497] In some embodiments, the antibody specifically binds to one or more single nucleotide polymorphism (SNP) variants of hOX40L. In examples of any aspect of the invention, hOX40L is a trimer of the monomer.

[0498] On the one hand, this article provides a method for reducing (e.g., at least 20, 30, 40, 50, or 60%, or 70%, 80%, 90%, 95%, or >90%) or completely inhibiting the binding of hOX40L to OX40 in a subject (e.g., a human subject), comprising administering to the subject an effective amount of an antibody or fragment thereof of the present invention that specifically binds to hOX40L (e.g., cell surface-expressed or soluble hOX40L).

[0499] On one hand, this article provides a method for treating or preventing hOX40L-mediated diseases or conditions in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of an antibody or fragment of the present invention that specifically binds to hOX40L (e.g., cell surface-expressed or soluble hOX40L), wherein the disease or condition is treated or prevented by said antibody or fragment. In one example, the method comprises reducing or inhibiting the secretion of one, more, or all of the subject's hOX40L bioactivity, such as IL-2, IL-8, TNFα, and interferon γ. In one example, the bioactivity is selected from the secretion of one, more, or all of IL-2, TNFα, and interferon γ. In one example, the bioactivity is selected from the secretion of one, more, or all of IL-8, CCL20, and RANTES.

[0500] On one hand, this article provides a method for reducing or inhibiting the secretion of one, more, or all of the hOX40L biological activity, such as IL-2, IL-8, TNFα, and interferon γ, in a subject (e.g., a human subject). The method comprises administering to the subject an effective amount of an antibody or fragment of the present invention that specifically binds to hOX40L (e.g., cell surface-expressed or soluble hOX40L), wherein the hOX40L biological activity is reduced by said antibody or fragment. In one example, the biological activity is selected from the secretion of one, more, or all of IL-2, TNFα, and interferon γ. In another example, the biological activity is selected from the secretion of one, more, or all of IL-8, CCL20, and RANTES.

[0501] The term “about” or “approximately” means within 20%, preferably 10%, and more preferably 5% (or 4%, 3%, or 2%, or in one instance 1% or less) of a given value or range.

[0502] As used herein, “administration” means the act of injecting or otherwise physically delivering a substance (e.g., the anti-hOX40L antibody provided herein) into a patient as it is present in vitro, such as via mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. In treating a disease or its symptoms, administration of a substance is typically performed after the onset of the disease or its symptoms. In preventing a disease or its symptoms, administration of a substance is typically performed before the onset of the disease or its symptoms.

[0503] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of shared positions (i.e., identity % = identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length.

[0504] The determination of percentage identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be achieved using mathematical algorithms. Preferred, non-limiting examples of mathematical algorithms for comparing two sequences are those described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215: 403. BLAST nucleotide searches can be performed using NBLAST nucleotide procedure parameters set, for example, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using XBLAST procedure parameters set, for example, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain vacancy alignments for comparative purposes, vacancy BLAST can be used, as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389 3402. Alternatively, PSIBLAST can be used for an iterative search to detect distant relationships between molecules (Id.). When using the BLAST, vacancy BLAST, and PSI BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm for sequence alignment is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used.

[0505] Similar techniques to those described above can be used to determine the percentage identity between two sequences, allowing or disallowing gaps. In calculating percentage identity, typically only exact matches are counted.

[0506] As used herein, an "antagonist" or "inhibitor" of hOX40L refers to a ligand (e.g., an antibody or fragment) capable of inhibiting or otherwise reducing, for example, the biological activity of hOX40L in cells expressing hOX40L or in cells expressing hOX40L ligands. For example, in some embodiments, the antibody of the present invention is an antagonist that inhibits or otherwise reduces the secretion of CCL20, IL-8, and / or RANTES from cells having OX40 expressed on their cell surface (when the antibody is contacted with the cells). In some embodiments, an antagonist of hOX40L (e.g., the antagonistic antibody of the present invention) may, for example, act by inhibiting or otherwise reducing the activation and / or cell signaling pathways of cells expressing OX40L, thereby inhibiting the hOX40L-mediated biological activity of cells relative to the biological activity of hOX40L in the absence of the antagonist. In some embodiments, the antibody provided herein is a fully human, antagonistic anti-hOX40L antibody, preferably a fully human, monoclonal, antagonistic anti-hOX40L antibody.

[0507] The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein. Antibodies or fragments thereof that specifically bind to the hOX40L antigen may cross-react with the relevant antigen. Preferably, antibodies or fragments thereof that specifically bind to the hOX40L antigen do not cross-react with other antigens (but may optionally cross-react with OX40L from different species, such as rhesus monkeys or mice). Antibodies or fragments thereof that specifically bind to the hOX40L antigen can be assessed, for example, by immunoassay, BIAcore, etc. TM Or other techniques known to those skilled in the art for identification. An antibody or fragment thereof specifically binds to the hOX40L antigen when it binds to the hOX40L antigen with a higher affinity than to any cross-reactive antigen, as measured using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction will be at least twice the background signal or noise, and more often more than 10 times the background. For a discussion of antibody specificity, see, for example, Paul ed., Fundamental Immunology 2nd ed., 1989, Raven Press, New York, pp. 332-336.

[0508] The antibodies of the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above. Specifically, the antibodies of the present invention comprise immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen-binding domains or molecules containing antigen-binding sites that specifically bind to hOX40L antigens (e.g., one or more complementarity-determining regions (CDRs) of anti-hOX40L antibodies). The antibodies of the present invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, especially IgG4), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. In a preferred embodiment, the hOX40L antibody is fully human, such as a fully human monoclonal hOX40L antibody. In some embodiments, the antibodies of the present invention are IgG antibodies, or one of their classes (e.g., human IgG1 or IgG4) or subclasses. In some embodiments, the antibodies of the present invention contain a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, contains the Leu235Glu mutation. In another embodiment, the heavy chain constant region contains the Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4-PE.

[0509] The terms "antigen-binding domain," "antigen-binding region," "antigen-binding fragment," and similar terms refer to an antibody moiety (e.g., a complementarity-determining region (CDR)) containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen upon the binder. The antigen-binding region can be derived from any animal species, such as rodents (e.g., rabbits, rats, or hamsters) and humans. Preferably, the antigen-binding region will be of human origin.

[0510] As used herein, the term "composition" is intended to cover products containing (optionally) specified amounts of specified ingredients (e.g., antibodies of the present invention), and products directly or indirectly produced by combinations of (optionally) specified amounts of specified ingredients.

[0511] In the case of peptides, as used herein, the term "derivative" refers to a peptide comprising an hOX40L peptide, an hOX40L peptide fragment, or an antibody that specifically binds to an hOX40L peptide, the amino acid sequence of which has been altered by introducing substitutions, deletions, or additions of amino acid residues. As used herein, the term "derivative" refers to, for example, an hOX40L peptide, an hOX40L peptide fragment, or an antibody that specifically binds to an hOX40L peptide, which has been chemically modified by covalently linking the peptide to any type of molecule. For example, but not in a limited manner, the hOX40L peptide, hOX40L peptide fragment, or hOX40L antibody may be chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / capping groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatives may be modified at the type or position of the linked molecule in a manner different from that of naturally occurring or initiating peptides or peptides. Derivatives also include the deletion of one or more chemical groups naturally present on said peptide or peptide. Derivatives of hOX40L peptides, hOX40L peptide fragments, or hOX40L antibodies can be chemically modified using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. Furthermore, the derivatives of hOX40L peptides, hOX40L peptide fragments, or hOX40L antibodies may contain one or more non-classical amino acids. The peptide derivatives have similar or identical functions to the hOX40L peptides, hOX40L peptide fragments, or hOX40L antibodies described herein.

[0512] As used herein, the term "effective amount" refers to an amount of therapy (e.g., the antibody or pharmaceutical composition provided herein) sufficient to reduce and / or improve the severity and / or duration of a given disease and / or associated symptoms. The term also covers amounts necessary to reduce or improve the progression or development of a given disease, reduce or improve relapses, development, or onset of a given disease, and / or enhance or strengthen the preventive or therapeutic effect of another therapy (e.g., a therapy other than the anti-hOX40L antibody provided herein). In some embodiments, the effective amount of the antibody of the present invention is from about 0.1 mg / kg (mg antibody / kg subject weight) to about 100 mg / kg. In some embodiments, the effective amount of the antibody provided is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg (or a range thereof). In some embodiments, as used herein, “effective amount” also refers to the amount of the antibody of the present invention used to achieve a specified result (e.g., inhibition of cellular hOX40L biological activity, such as inhibition of CCL20, IL-8, or RANTES, or INF-γ, TNF-α, or IL-2, especially INF-γ secreted from cells).

[0513] As used herein, the term "epitope" refers to a localized region on the surface of an antigen, such as an hOX40L polypeptide or a fragment of hOX40L polypeptide, which is capable of being bound by one or more antigen-binding regions of an antibody and has antigenic or immunogenic activity in animals, preferably mammals and most preferably humans, capable of inducing an immune response. An epitope with immunogenic activity is a portion of a polypeptide that induces an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide specifically bound by an antibody by any method well known in the art, such as by an immunoassay described herein. An antigenic epitope is not necessarily immunogenic. Epitopes typically consist of chemically active surface molecules such as amino acids or sugar side chains and have specific three-dimensional structural features and specific charge features. The polypeptide region contributing to the epitope may be a continuous amino acid of the polypeptide or the epitope may be derived together from two or more discontinuous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of an antigen. In some embodiments, an hOX40L epitope is a three-dimensional surface feature of an hOX40L polypeptide (e.g., in the form of a trimer of the hOX40L polypeptide). In other embodiments, the hOX40L epitope is a linear characteristic of the hOX40L polypeptide (e.g., in trimer or monomeric form). The antibodies provided herein can specifically bind to the epitope in the monomeric (denatured) form of hOX40L, the epitope in the trimeric (natural) form of hOX40L, or the epitope in both the monomeric (denatured) and trimeric (natural) forms of hOX40L. In a specific embodiment, the antibodies provided herein specifically bind to the epitope in the trimeric form of hOX40L, but not specifically to the monomeric form of hOX40L.

[0514] As used herein, the term "excipient" refers to an inert substance commonly used as a diluent, medium, preservative, binder, or stabilizer in pharmaceuticals and includes, but is not limited to, proteins (e.g., serum albumin), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

[0515] In the case of peptides or polypeptides, the term "fragment" as used herein refers to a peptide or polypeptide containing less than the full-length amino acid sequence. Such fragments can be generated, for example, by truncation at the amino terminus, truncation at the carboxyl terminus, and / or deletion of residues from the interior of the amino acid sequence. Fragments can be generated, for example, by selective RNA splicing or in vivo protease activity. In some embodiments, the hOX40L fragment comprises a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues. In one specific embodiment, the fragment of the hOX40L polypeptide or antibody that specifically binds to the hOX40L antigen retains at least one, at least two, or at least three functions of the polypeptide or antibody.

[0516] The terms “fully human antibody” or “human antibody” are used interchangeably herein and refer to an antibody that comprises a human variable region and, most preferably, a human constant region. In specific embodiments, the term refers to an antibody comprising both a human-derived variable region and a constant region. In some embodiments, a “fully human” anti-hOX40L antibody may also encompass an antibody that binds to an hOX40L polypeptide and is encoded by a nucleic acid sequence of a somatic variant of a human germline immunoglobulin nucleic acid sequence. In one specific embodiment, the anti-hOX40L antibody provided herein is a fully human antibody. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences, as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication 91-3242). For example, exemplary methods for generating fully human antibodies are provided in the embodiments herein, but any method known in the art may be used.

[0517] The phrase “recombinant human antibody” includes human antibodies prepared, expressed, produced, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from recombinant or combined human antibody libraries, antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cattle) that are transgenic with respect to the human immunoglobulin gene (see, for example, Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing a human immunoglobulin gene sequence onto another DNA sequence. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242). However, in some implementations, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic cell mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody, although derived from and associated with human germline VH and VL sequences, are not sequences that are naturally present in the in vivo human antibody germline library.

[0518] As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of an antibody and the amino acid sequence of a heterologous peptide or protein (i.e., a peptide or protein that is not typically part of an antibody (e.g., a non-anti-hOX40L antigen antibody). The term "fusion," when used with respect to hOX40L or anti-hOX40L antibodies, refers to the linking of a peptide or polypeptide or fragment thereof, variant, and / or derivative thereof to a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of the hOX40L or anti-hOX40L antibody. In some embodiments, the fusion protein comprises an hOX40L antibody VH domain, a VL domain, VH CDRs (one, two, or three VH CDRs), and / or VL CDRs (one, two, or three VL CDRs), wherein the fusion protein specifically binds to the hOX40L epitope.

[0519] The term "heavy chain," when used in relation to antibodies, refers to the amino acid sequence based on the heavy chain constant domain, designated as five distinct types: α, δ, ε, γ, and μ. These different types of heavy chains are well-known and generate five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. Preferably, the heavy chain is a human heavy chain. In one instance, the heavy chain is a disabling IgG isotype, such as disabling IgG4. In some embodiments, the antibodies of the present invention contain a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, contains the Leu235Glu mutation. In another embodiment, the heavy chain constant region contains the Ser228Pro mutation to increase stability. In yet another embodiment, the heavy chain constant region is IgG4-PE.

[0520] As used herein, the term "host" refers to an animal, preferably a mammal, and more preferably a human.

[0521] As used in this article, the term "host cell" refers to a specific test cell transfected with a nucleic acid molecule and its progeny or potential progeny. Due to mutations that may occur in the next generation, environmental influences, or the integration of nucleic acid molecules into the host cell genome, the progeny of such cells may differ from the transfected parent cell.

[0522] As used herein, the term "immunomodulator" and its variations, including but not limited to immunomodulators, refers to agents that modulate the host's immune system. In some embodiments, the immunomodulator is an immunosuppressant. In some other embodiments, the immunomodulator is an immunostimulant. According to the present invention, the immunomodulators used in the combination therapies of the present invention do not include anti-hOX40L antibodies or antigen-binding fragments. Immunomodulators include, but are not limited to, small molecules, peptides, polypeptides, proteins, fusion proteins, antibodies, inorganic molecules, mimics, and organic molecules.

[0523] As used herein, the term "combination" in the context of administering other therapies means the use of more than one therapy. The use of the term "combination" does not restrict the order in which therapies are administered to the infected subject. The first therapy may be administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), simultaneously with (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the second therapy is administered to a subject who has, has, or is susceptible to hOX40L-mediated disease. Any additional therapy may be administered in any order along with other additional therapies. In some embodiments, the antibodies of the present invention may be administered in combination with one or more therapies (e.g., therapies not of the present invention, but administered concurrently to prevent, treat, manage, and / or improve hOX40L-mediated disease). Non-limiting examples of therapies that may be administered in combination with the antibodies of the present invention include analgesics, anesthetics, antibiotics, or immunomodulators, or any other agents listed in the United States Pharmacopeia and / or the Physician's Desk Reference.

[0524] "Isolated" or "purified" antibodies (e.g.) are substantially free of cellular material or other contaminating proteins from the cell or tissue from which the antibody originates, or are substantially free of chemical precursors or other chemicals when synthesized by chemical methods. The term "substantially free of cellular material" includes antibody preparations in which the antibody is separated from the cellular components from which it is isolated, or produced by recombinant methods. Therefore, antibodies substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). When antibodies are produced by recombinant methods, they are also preferably substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, or 5% of the volume of the protein preparation. When antibodies are produced by chemical synthesis, they are preferably substantially free of chemical precursors or other chemicals, i.e., separated from the chemical precursors or other chemicals involved in protein synthesis. Therefore, such antibody preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the target antibody. In a preferred embodiment, the antibody of the present invention is isolated or purified.

[0525] "Isolated" nucleic acid molecules are nucleic acid molecules that are separated from other nucleic acid molecules present in natural sources of nucleic acid molecules. Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when synthesized by chemical methods. In one specific embodiment, the nucleic acid molecule encoding the antibody of the present invention is isolated or purified.

[0526] The terms “human OX40L,” “hOX40L,” or “hOX40L polypeptide,” and similar terms refer to polypeptides that include a sequence listing and related polypeptides, including the amino sequence in its SNP variants (“polypeptide,” “peptide,” and “protein” are used interchangeably herein). Related polypeptides include allelic variants (e.g., SNP variants); splicing variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, which preferably retain hOX40L activity and / or are sufficient to produce an anti-hOX40L immune response. Soluble forms of hOX40L sufficient to produce an anti-hOX40L immune response are also covered. As those skilled in the art will recognize, the anti-hOX40L antibodies of the present invention can bind to hOX40L polypeptides, polypeptide fragments, antigens, and / or epitopes, since an epitope is part of a larger antigen, a larger antigen is part of a larger polypeptide fragment, and a larger polypeptide fragment is part of a larger polypeptide hOX40L, which can exist in trimer (natural) or monomer (denatured) form.

[0527] The term "Kabat number" and similar terms are recognized in the art and refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy chain variable region of the antibody or its antigen-binding moiety (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region typically ranges from amino acid position 31 to 35 for CDR1, from amino acid position 50 to 65 for CDR2, and from amino acid position 95 to 102 for CDR3.

[0528] The term "monoclonal antibody" refers to an antibody obtained from a group of homologous or substantially homologous antibodies, and each monoclonal antibody typically recognizes a single epitope on an antigen. In a preferred embodiment, "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell, wherein the antibody specifically binds only to the hOX40L epitope, as determined, for example, by an ELISA or other antigen-binding or competitive binding assay known in the art or provided herein. The term "monoclonal" is not limited to any particular method of preparing the antibody. For example, the monoclonal antibody of the present invention can be prepared by the hybridoma method described, such as in Kohler et al.; Nature, 256:495 (1975), or isolated, for example, from a phage library using techniques as described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed therefrom are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th edition, eds. Ausubel et al., John Wiley and Sons, New York). Other exemplary methods for producing other monoclonal antibodies are provided in the embodiments herein.

[0529] The terms “natural” or “natural”, when used in conjunction with biological materials such as nucleic acid molecules, peptides, and host cells, refer to those found in nature and not manipulated by humans.

[0530] As used in this article, "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically for use in humans.

[0531] As used herein, the term "polyclonal antibody" refers to a group of antibodies with numerous epitopes generated in an immune response to a protein and includes various different antibodies targeting the same and different epitopes within a protein. Methods for generating polyclonal antibodies are known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th edition, eds. Ausubel et al., John Wiley and Sons, New York).

[0532] As used in this article, the terms “polynucleotide,” “nucleotide,” “nucleic acid,” “nucleic acid molecule,” and other similar terms are used interchangeably and include DNA, RNA, mRNA, etc.

[0533] As used herein, the term “prevention” refers to complete or partial suppression of the development, recurrence, onset, or spread of hOX40L-mediated disease and / or associated symptoms resulting from the application of the therapies or combinations of therapies provided herein (e.g., preventive or therapeutic agents, such as combinations of antibodies of the present invention).

[0534] As used herein, the term "prophylactic agent" means any agent that can completely or partially inhibit the development, recurrence, onset, or spread of hOX40L-mediated disease and / or related symptoms in a subject. In some embodiments, the term "prophylactic agent" refers to the antibody of the present invention. In some other embodiments, the term "prophylactic agent" refers to an agent other than the antibody of the present invention. Preferably, the prophylactic agent is an agent known for, or has been or is currently used for, preventing or inhibiting the onset, development, progression, and / or severity of hOX40L-mediated disease and / or related symptoms. In a specific embodiment, the prophylactic agent is a fully human anti-hOX40L antibody, such as a fully human anti-hOX40L monoclonal antibody.

[0535] In one embodiment, the prevention prevents the onset of the disease or symptom, or the symptoms of the disease or symptom. In one embodiment, the preventive treatment prevents the disease or symptom from worsening or occurring. In one embodiment, the preventive treatment prevents the disease or symptom from worsening.

[0536] In another embodiment, the anti-OX40L antibody of the present invention is administered intravenously (e.g., before or simultaneously with transplantation, such as blood or organ transplantation). In another embodiment, the antibody is administered at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg). In another embodiment, the antibody is administered at a dose selected from about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg, particularly at about 1 mg / kg or about 3 mg / kg.

[0537] In another embodiment, the antibody is administered 1-4 days prior to transplantation (e.g., blood or organ transplantation), for example, 1-3 days or 1-2 days prior to transplantation. In another embodiment, the antibody is administered weekly, every two weeks, or monthly after transplantation, for example, every two weeks. In yet another embodiment, the antibody is administered prophylactically intravenously at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg) 1-3 days prior to transplantation, followed by intravenous administration every two weeks at a dose of about 5-10 mg / kg (e.g., about 8 mg / kg).

[0538] In another implementation, the presence of biomarkers predicting transplant rejection or GvHD (e.g., acute GvHD) is monitored periodically post-transplant, and the anti-OX40L antibody of the present invention is administered once the biomarker levels determine that the patient is at risk of developing transplant rejection or GvHD (e.g., acute GvHD). This strategy avoids unnecessary drug administration and unnecessary immunosuppression. Examples of biomarkers that can be used as predictive biomarkers for acute GvHD include those identified by Levine et al., “A prognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study,” Lancet Haematol 2015;2:e21-29. These biomarkers include, but are not limited to, TNFR1, ST-2, elastin, and IL2Rα and Reg3α.

[0539] The hOX40L region that contributes to an epitope can be a continuous amino acid of the polypeptide, or the epitope can originate from two or more discontinuous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of the antigen. A localized region on the surface of an hOX40L antigen that can elicit an immune response is an hOX40L epitope. An epitope may or may not be a three-dimensional surface feature of the antigen.

[0540] The terms "hOX40L-mediated disease" and "hOX40L-mediated symptom" are used interchangeably and refer to any disease or symptom that is wholly or partially caused by or is a result of hOX40L. In some embodiments, hOX40L is aberrantly (highly) expressed on the cell surface. In some embodiments, hOX40L may be aberrantly upregulated in specific cell types. In other embodiments, binding of hOX40L to its ligand causes normal, aberrant, or excessive cell signaling. In some embodiments, the hOX40L ligand is, for example, OX40 expressed on the cell surface, such as the surface of colonic epithelial cells. In some embodiments, the hOX40L-mediated disease is inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC). In other embodiments, the hOX40L-mediated disease is graft-versus-host disease (GVHD). In other embodiments, hOX40L-mediated diseases are selected from pyoderma gangrene, giant cell arteritis, Schnitzler syndrome, non-infectious scleritis, and uveitis (non-infectious / autoimmune and / or systemic). In other embodiments, hOX40L-mediated diseases or conditions are selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; for example, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD.

[0541] The terms "hOX40L receptor" or "hOX40L-binding receptor" are used interchangeably herein and refer to a receptor polypeptide that binds to hOX40L. In a specific embodiment, the hOX40L receptor is Hox40. In some embodiments, the hOX40L receptor is expressed on the cell surface, such as the surface of colonic epithelial cells; or on graft or transplanted tissue; or on host tissue.

[0542] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), most preferably a human. In one embodiment, the subject is a mammal suffering from an hOX40L-mediated disease, preferably a human. In another embodiment, the subject is a mammal at risk of developing an hOX40L-mediated disease, preferably a human.

[0543] As used in this article, “substantially all” means at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0544] As used herein, “substantially free of surfactant” refers to an antibody formulation that specifically binds to the hOX40L antigen and contains less than 0.0005%, less than 0.0003%, or less than 0.0001% of surfactant and / or less than 0.0005%, less than 0.0003%, or less than 0.0001% of surfactant.

[0545] As used herein, “substantially salt-free” refers to an antibody formulation that specifically binds to the hOX40L antigen and contains less than 0.0005%, less than 0.0003%, or less than 0.0001% of inorganic salts.

[0546] As used herein, the term "surfactant" refers to an amphoteric organic substance; that is, it consists of groups with opposite solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be classified into anionic, cationic, and nonionic surfactants based on the charge of their active moieties. Surfactants are commonly used as wetting agents, emulsifiers, solubilizers, and dispersants in the preparation of various pharmaceutical compositions and biomaterials.

[0547] As used herein, the term "tag" refers to any type of portion attached to, for example, a polypeptide and / or a polynucleotide encoding hOX40L or an hOX40L antibody or its antigen-binding fragment. For example, a polynucleotide encoding hOX40L, an hOX40L antibody, or its antigen-binding fragment may contain one or more additional tag-encoding nucleotide sequences encoding, for example, a detectable portion or a portion that facilitates affinity purification. During translation, the tag and antibody may be in the form of a fusion protein. The terms "detectable" or "detectable" with respect to a tag refer to any tag that is visible or in which the presence of the tag can be further determined and / or measured (e.g., by quantification). A non-limiting example of a detectable tag is a fluorescent tag.

[0548] As used herein, the term "therapeutic agent" refers to any agent that can be used to treat, manage, or improve hOX40L-mediated diseases and / or associated symptoms. In some embodiments, the term "therapeutic agent" refers to the antibody of the present invention. In some other embodiments, the term "therapeutic agent" refers to an agent other than the antibody of the present invention. Preferably, the therapeutic agent is an agent known for, or has been or is currently used for, the treatment, management, or improvement of one or more symptoms of hOX40L-mediated diseases or associated symptoms. In a specific embodiment, the prophylactic agent is a fully human anti-hOX40L antibody, such as a fully human anti-hOX40L monoclonal antibody.

[0549] Combinations of therapies (e.g., the use of preventative or therapeutic agents) are more effective than the additive effect of any two or more single therapies. For example, the synergistic effect of a combination of preventative and / or therapeutic agents allows for the use of one or more agents at lower doses and / or less frequent administration to subjects with hOX40L-mediated disease. The ability to utilize lower doses of preventative or therapeutic therapies and / or less frequent administration of the therapy reduces the toxicity associated with administering the therapy to subjects without reducing the efficacy of the therapy in the prevention, management, treatment, or improvement of hOX40L-mediated disease. Furthermore, synergistic effects can lead to enhanced efficacy of the therapy in the prevention, management, treatment, or improvement of hOX40L-mediated disease. Finally, the synergistic effect of combinations of therapies (e.g., preventative or therapeutic agents) can avoid or reduce adverse or harmful side effects associated with the use of any single therapy.

[0550] In one embodiment, the combination comprises the anti-OX40L antibody of the present invention and another therapeutic agent, said other therapeutic agent being independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, anti-IL12 / IL-23 antibody (e.g., uterotumab), anti-CD20 antibody (e.g., rituximab), anti-CD30 antibody (e.g., butuximab), CTLA4-Fc molecule (e.g., abatacept), CCR5 receptor antagonist (e.g., malaviro), anti-CD40L antibody, anti-V LA4 antibodies (e.g., nastatin), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies (e.g., alenzab), anti-CD45 antibodies, cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., baliximab), anti-CD25 antibodies (e.g., daliximab), anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, or sertozumab), and vorinostat. In another embodiment, the combination comprises the anti-OX40L antibody of the present invention and another therapeutic agent independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, CTLA4-Fc molecule (e.g., abatacept), anti-CD40L antibody, anti-LFA1 antibody, anti-CD52 antibody (e.g., alemtuzumab), cyclophosphamide, and anti-thymocyte globulin.

[0551] As used herein, the term "therapy" means any regimen, method, and / or agent that can be used for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases (e.g., IBD or GVHD). In some embodiments, the term "therapy" means a biological therapy, supportive therapy, and / or other therapy that is useful in the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases as known to those skilled in the art as medical personnel.

[0552] As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity, and / or duration of hOX40L-mediated disease (e.g., IBD or GVHD) caused by the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the antibodies of the present invention). In specific embodiments, such terms refer to a reduction or inhibition of hOX40L binding to OX40, a reduction or inhibition of CCL20 production or secretion from cells expressing hOX40 or hOX40L, a reduction or inhibition of IL-8 production or secretion from cells expressing hOX40 or hOX40L, a reduction or inhibition of RANTES production or secretion from cells expressing hOX40 or hOX40L, and / or inhibition or relief of one or more symptoms associated with hOX40L-mediated diseases such as IBD or GVHD. In specific embodiments, such terms refer to the reduction or inhibition of hOX40L binding to OX40, the reduction or inhibition of INF-γ production or secretion from cells expressing hOX40 or hOX40L, the reduction or inhibition of TNF-α production or secretion from cells expressing hOX40 or hOX40L, the reduction or inhibition of IL-2 production or secretion from cells expressing hOX40 or hOX40L, and / or the inhibition or mitigation of one or more symptoms associated with hOX40L-mediated diseases such as IBD or GVHD (especially GvHD). In one instance, the cell is a human cell. In specific embodiments, the prophylactic agent is a fully human anti-hOX40L antibody, such as a fully human anti-hOX40L monoclonal antibody.

[0553] The term "variable region" or "variable domain" refers to a portion of the OX40L and heavy chain, typically about 120 to 130 amino acids from the N-terminus of the heavy chain and about 100 to 110 amino acids from the light chain, which are widely different in sequence among antibodies and are responsible for the binding and specificity of each specific antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called backbone regions (FRs). The CDRs of the OX40L and heavy chain are primarily responsible for antibody-antigen interactions. The amino acid positions used herein are numbered according to the EU index, as described in Kabat et al. (1991), Sequences of proteins of immunological interest (USD Department of Health and Human Services, Washington, DC), 5th edition ("Kabat et al."). In a preferred embodiment, the variable region is a human variable region.

[0554] Antibody

[0555] The antibodies of the present invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0556] Specifically, the antibodies provided herein comprise immunoglobulin molecules and the immunoactive portions of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to the hOX40L antigen. The immunoglobulin molecules provided herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. In one specific embodiment, the antibody provided herein is an IgG antibody, preferably IgG1 or IgG4. In some embodiments, the antibody of the present invention contains a human γ4 constant region. In another embodiment, the heavy chain constant region does not bind to the Fc-γ receptor and, for example, contains the Leu235Glu mutation. In another embodiment, the heavy chain constant region contains the Ser228Pro mutation to increase stability. In yet another embodiment, the heavy chain constant region is IgG4-PE.

[0557] Antibody variants and derivatives include antibody fragments that retain the ability to specifically bind to epitopes. Preferred fragments include Fab fragments; Fab′ (antibody fragments containing a single anti-binding domain comprising Fab and a heavy chain appendage via a hinge region); F(ab′)2 (two Fab′ molecules linked by interchain disulfide bonds within the heavy chain hinge region; Fab′ molecules may target the same or different epitopes); bispecific Fab (Fab molecules having two antigen-binding domains, each of which can target a different epitope); single-chain Fab chains containing variable regions, also known as sFv; disulfide-linked Fv or dsFv; camel-derived VH (antibodies where some amino acids at the VH interface are amino acids found in the heavy chain of naturally occurring camel antibodies). The invention includes: a single heavy chain variable antigen-binding determinant region (SFR); a bispecific sFv (sFv or dsFv molecule having two antigen-binding domains, each of which can target different epitopes); a double-chain antibody (a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding domains of the double-chain antibody can target the same or different epitopes); and a triple-chain antibody (a trimerized sFv formed in a manner similar to that of a double-chain antibody, but wherein three antigen-binding domains are generated in a single complex; the three antigen-binding domains can target the same or different epitopes). Antibody derivatives also include one or more CDR sequences at the antibody binding site. When two or more CDR sequences are present, the CDR sequences can be linked together on the backbone. In some embodiments, antibodies used with the invention comprise a single-chain Fv (“scFv”). scFv is an antibody fragment containing the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Typically, scFv peptides also contain peptide linkers located between the VH and VL domains, enabling scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0558] The antibodies of this invention can be derived from any animal source, including birds and mammals (e.g., humans, rodents, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In some embodiments, the antibodies of this invention are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of human immunoglobulins and include antibodies isolated from a human immunoglobulin library or from mice expressing antibodies derived from human genes.

[0559] In a preferred embodiment, the antibody of the present invention is a fully human antibody, such as a fully human antibody that specifically binds to the hOX40L polypeptide, the hOX40L polypeptide fragment, or the hOX40L epitope. Such fully human antibodies will be advantageous over whole mouse (or other fully or partially non-human species antibodies), humanized antibodies, or chimeric antibodies in minimizing harmful or unwanted side effects, such as immune responses to non-fully human antibodies (e.g., anti-hOX40L antibodies derived from other species), when administered to a subject.

[0560] The antibodies of this invention can be monospecific, bispecific, trispecific, or even more multispecific. Multispecific antibodies can be specific to different epitopes of the hOX40L peptide or to both the hOX40L peptide and heterologous epitopes, such as heterologous peptides or solid support materials. In a preferred embodiment, the antibody provided herein is monospecific to a given epitope of the hOX40L peptide and does not specifically bind to other epitopes.

[0561] This article also provides B cells (e.g., immortalized B cells) or hybridomas that produce the anti-hOX40L antibody or fragment described herein.

[0562] In some embodiments, this document provides an isolated antibody that specifically binds to the hOX40L epitope, wherein the binding of the antibody to the hOX40L epitope is competitively blocked by the antibody or fragment of the present invention (e.g., in a dose-dependent manner). The antibody may or may not be a fully human antibody. In a preferred embodiment, the antibody is a fully human monoclonal anti-hOX40L antibody, and even more preferably a fully human, monoclonal, antagonistic anti-hOX40L antibody. Exemplary competitive blocking assays are provided in the embodiments herein.

[0563] In some embodiments, the antibody or fragment of the present invention competitively (e.g., in a dose-dependent manner) binds to hOX40L expressed on the cell surface against the OX40 receptor (or its fusion protein). In other embodiments, the antibody or fragment of the present invention competitively (e.g., in a dose-dependent manner) binds to soluble hOX40L against the OX40 receptor (or its fusion protein). Exemplary competitive binding assays available are provided in the embodiments herein. In one embodiment, the antibody or fragment partially or completely inhibits the binding of hOX40 to OX40L expressed on the cell surface, such as hOX40L. In another embodiment, the antibody partially or completely inhibits the binding of hOX40 to soluble hOX40L. In some embodiments, the antibody or fragment partially or completely inhibits the secretion of CCL20, IL-8 and / or RANTES or INF-γ, TNF-α or IL-2, especially INF-γ, from cells having OX40 expressed on their cell surface. In some embodiments, the cells expressing OX40 are colonic epithelial cells.

[0564] Preferably, the antibody of the present invention is a fully human, monoclonal antibody, such as a fully human, monoclonal antagonistic antibody that specifically binds to hOX40L.

[0565] In some embodiments, the antibody or fragment provided herein binds to an hOX40L epitope, which is a three-dimensional surface feature of an hOX40L polypeptide (in the form of a trimer of the hOX40L polypeptide). The hOX40L polypeptide region contributing to the epitope may be a continuous amino acid of the polypeptide, or the epitope may originate together from two or more discontinuous regions of the polypeptide. The hOX40L epitope may be present in (a) a trimer of hOX40L (“trimeric hOX40L epitope”), (b) a monomer of hOX40L (“monomer hOX40L epitope”), (c) a trimer and monomer of hOX40L, (d) a trimer of hOX40L but not a monomer, or (e) a monomer of hOX40L but not a trimer.

[0566] For example, in some embodiments, the epitope exists or is available for binding only in a trimer (natural) form, but not in a monomer (denatured) form or for binding to an anti-hOX40L antibody. In other embodiments, the hOX40L epitope is a linear characteristic of the hOX40L polypeptide (e.g., in a trimer or monomeric form). The antibodies provided herein can specifically bind to (a) the hOX40L monomeric (denatured) epitope, (b) the hOX40L trimeric epitope, (c) the hOX40L monomeric but not trimeric epitope, (d) the hOX40L trimeric but not monomeric epitope, or (e) both the hOX40L monomeric and trimeric forms. In a preferred embodiment, the antibodies provided herein specifically bind to the hOX40L trimeric epitope, but not specifically bind to the hOX40L monomeric epitope.

[0567] This invention also provides antibodies that specifically bind to the hOX40L epitope, comprising derivatives of the VH domain, VH CDR, VL domain, and VL CDR of the hOX40L antigen as described herein. This invention also provides antibodies comprising the antibody derivatives disclosed in the examples, wherein said antibodies specifically bind to the hOX40L epitope. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecule of this invention, including, for example, site-directed mutagenesis producing amino acid substitutions and PCR-mediated mutagenesis. Preferably, the derivative comprises fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In another embodiment, the derivative has conserved amino acid substitutions. In a preferred embodiment, the derivative has conserved amino acid substitutions produced at one or more predicted non-essential amino acid residues. Optionally, mutations may be introduced randomly along the entire or partial coding sequence, for example, by saturation mutagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and its activity can be measured.

[0568] In another embodiment, the antibody that specifically binds to the hOX40L epitope comprises a variable domain amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the variable domain amino acid sequence in the sequence listing.

[0569] In specific embodiments, the antibody is a fully human anti-human antibody, such as a fully human monoclonal antibody. Fully human antibodies can be generated by any method known in the art. Exemplary methods include immunizing a transgenic animal (e.g., a mouse) with an hOX40L antigen (any hOX40L polypeptide capable of evoking an immune response and optionally conjugated to a vector) that is capable of generating a human antibody library in the absence of endogenous immunoglobulin production; see, for example, Jakobovits et al., (1993) Proc. Natl. Acad. Sci., 90: 2551; Jakobovits et al., (1993) Nature, 362: 255-258 (1993); Bruggermann et al., (1993) Year in Immunol., 7: 33. Other methods for generating fully human anti-hOX40L antibodies can be found in the embodiments provided herein.

[0570] Optionally, fully human antibodies can be generated by screening phage display antibody libraries in vitro; see, for example, Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991), which are incorporated herein by reference. Various antibody-containing phage display libraries have been described and are readily prepared by those skilled in the art. Libraries can contain a wide variety of human antibody sequences that can be screened for suitable targets, such as human Fab, Fv, and scFv fragments.

[0571] The antibodies and fragments of the present invention include chemically modified antibodies and fragments, i.e., modified by covalent linking of any type of molecule to the antibody. For example, but not in a limiting manner, antibody derivatives include antibodies that have been chemically modified (e.g., by known glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / capping groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. Additionally, the antibodies may contain one or more non-classical amino acids.

[0572] The present invention also provides antibodies that specifically bind to the hOX40L antigen, comprising a backbone region (e.g., human or non-human backbone) known to those skilled in the art. The backbone region may be, for example, a naturally occurring or shared backbone region. More preferably, the backbone region of the antibodies of the present invention is human (for a list of human backbone regions, see, for example, Chothia et al., 1998, J. Mol. Biol. 278: 457-479, which is incorporated herein by reference in its entirety). See also Kabat et al. (1991) Sequences of Proteins of Immunological Interest (USD Department of Health and Human Services, Washington, DC), 5th edition.

[0573] In a specific embodiment, the present invention provides an antibody that specifically binds to the hOX40L antigen, said antibody comprising the amino acid sequence of one or more CDRs in the sequence listing (i.e., for HCDR1, Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96 or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42; for HCDR2, Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98 or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44; for HCDR3, Seq ID No: 8, Seq ID No: 14, Seq ID No: 40, Seq ID No: 4 ...42, Seq ID No: 4 Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46; for LCDR1, Seq ID No: 18, Seq ID No: 24, Seq ID No: 50, Seq ID No: 56, Seq ID No: 82, Seq ID No: 88, Seq ID No: 110, or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56; for LCDR2, Seq ID No: 20, Seq ID No: 26, Seq ID No: 52, Seq ID No: 58, Seq ID No: 84, Seq ID No: 90, Seq ID No: 112, or Seq ID No: 118, especially Seq ID No: 52 or Seq ID No: 58; and for LCDR3, Seq ID No: 22, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46; Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114 or Seq ID No: 120, especially Seq ID No: 54 or Seq ID No: 60) and human backbone regions having one or more amino acid substitutions at 1, 2, 3 or more of the following: (a) rare backbone residues that differ between the mouse antibody backbone (i.e., the donor antibody backbone) and the human antibody backbone (i.e., the recipient antibody backbone);(b) Vernier zone residues at different times between the donor and recipient antibody backbones; (c) different interchain packaging residues at the VH / VL interface between the donor and recipient antibody backbones; (d) different canonical residues between the donor and recipient antibody backbone sequences, particularly backbone regions crucial for defining the canonical class of the mouse antibody CDR ring; (e) residues adjacent to the CDR; (g) residues capable of interacting with the antigen; (h) residues capable of interacting with the CDR; and (i) contact residues between the VH and VL domains. In some embodiments, antibodies that specifically bind to the hOX40L antigen containing one or more amino acid substitutions at one, two, three, or more of the above-identified residues are antagonistic hOX40L antibodies.

[0574] This invention covers antibodies that specifically bind to the hOX40L antigen, said antibodies comprising the amino acid sequences of the VH domain and / or VL domain in the sequence listing (i.e., Seq ID No: 2, Seq ID No: 34, Seq ID No: 66, or Seq ID No: 94 for the VH domain, especially Seq ID No: 34; and Seq ID No: 16, Seq ID No: 48, Seq ID No: 80, or Seq ID No: 108 for the VL domain, especially Seq ID No: 48), but with mutations (e.g., one or more amino acid substitutions) in the backbone region. In some embodiments, the antibody that specifically binds to the hOX40L antigen comprises the amino acid sequences of the VH domain and / or VL domain of the antibody disclosed in the examples or an antigen-binding fragment thereof, having one or more amino acid residue substitutions in the backbone region of the VH domain and / or VL domain.

[0575] In some embodiments, the antibodies provided herein reduce or inhibit the binding of hOX40L to hOX40, and / or reduce or inhibit the secretion of hOX40L bioactivity, such as CCL20, IL8, and / or RANTES or INF-γ, TNF-α, or IL-2, especially INF-γ, in a subject (e.g., a human subject). In some embodiments, the antibodies provided herein, such as human monoclonal anti-hOX40L antibodies, reduce or inhibit the binding of soluble or cell surface-expressed hOX40L to hOX40, and / or reduce or inhibit the secretion of CCL20 and / or RANTES or INF-γ, TNF-α, or IL-2, especially INF-γ, in a subject after contact with soluble or cell surface-expressed hOX40L. The blocking activity of the antibodies provided herein against the binding of hOX40L to hOX40 can be detected using the assays described in the examples. The inhibitory effect of the hOX40L antibodies provided herein on the bioactivity of OX40-expressing cells can be detected using the assays described in the examples.

[0576] This invention also provides fusion proteins comprising an antibody that specifically binds to the hOX40L antigen, as provided herein, and a heterologous peptide. In some embodiments, the heterologous peptide fused with the antibody is useful for targeting the antibody to cells expressing hOX40L on their cell surface.

[0577] Antibody conjugates and fusion proteins

[0578] The following discussion of conjugates and fusion proteins also applies to fragments, so that the disclosure of antibodies, with necessary modifications, can also be applied to fragments of this invention.

[0579] In some embodiments, the antibodies of the present invention are conjugated or recombinantly fused to diagnostic agents, detectable reagents, therapeutic agents, or any other molecules. The conjugated or recombinantly fused antibodies can be used as part of clinical trial procedures, for example, to monitor or predict the onset, development, progression, and / or severity of hOX40L-mediated diseases.

[0580] Such diagnostics and detections can be achieved, for example, by coupling antibodies with detectable substances, including but not limited to various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances such as, but not limited to, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin, and jellyfish luminescent protein; and radioactive substances such as, but not limited to, iodine (…). 131 I, 125I, 123 I and 121 I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 115 In、 113 In、 112 In and 111 In), Technetium ( 99 Tc), thallium 201 Ti, gallium 68 Ga、 67 Ga), Palladium ( 103 pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F) 153 Sm、 177 Lu、 159 Gd, 149 pm、 140 La、 175 Yb、 166 Ho、 90 Y、 47 Sc、 186 Re、 188 Re、 142 pr、 105 Rh、 97 Ru、 68 Ge 57 Co、 65 Zn, 85 Sr、 32 p、 153 Gd, 169 Yb、 51 Cr 54 Mn, 75 Se、 113 Sn and 117 Sn; and positron-emitting metals and non-radioactive paramagnetic metal ions obtained using various positron emission tomography (PET) scans.

[0581] This invention also covers the use of the antibodies of the invention conjugated or recombinantly fused to a therapeutic portion (or one or more therapeutic portions). The antibodies may be conjugated or recombinantly fused to a therapeutic portion, such as a cytotoxic agent (e.g., a cell inhibitor or cytotoxic agent), a therapeutic agent, or a radioactive metal ion (e.g., an alpha emitter). Cytotoxic agents or cytotoxic agents include any agents that are harmful to cells. Treatment components include, but are not limited to, antimetabolites (e.g., methotrexate, 6-ceratopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine); alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, and mitomycin C). C) and cis-dichlorodiamineplatin(II) (DDP) and cisplatin; anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (e.g., d-actinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); auristatin molecules (e.g., auristatin PHE, bryostatin 1, and solatatin 10); see Woyke et al., Antimicrob. Agents Chemother. 46: 3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother. 45: 3580-4 (2001), Mohammad et al., Anticancer Drugs 12: 735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun 266: 76-80 (1999), Mohammad et al., Int. J. Oncol. 15: 367-72 (1999), all of which are incorporated herein by reference.Hormones (e.g., glucocorticoids, progestin, androgens, and estrogens), DNA repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7): 2167-76 (2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide). bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues, and U.S. Patents 6,245,759, 6,399,633, 6,383,790, and 6,335,150. 6, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, and 5,587,459 (the compounds disclosed in these documents).Farnesyltransferase inhibitors (e.g., R115777, BMS-214662, and, for example, U.S. Patent Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, etc.). 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, ​​6,232,338, 6,228,865, 6,228,856, 6,225,322, 6 218,406、6,211,193、6,187,786、6,169,096、6,159,984、6,143,766、6,133,303、6,127,366、6,124,465、6,124,295、6,103,723、6,093,737、6,090,948、6,080,870、6,077,853、6, Those disclosed in patents 071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574 and 6,040,305); topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamycin; bulgarein; DNA minor groove binding agents such as Hoescht dye 33342 and Hoescht dye 33258; nitidine; fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1;Bisphosphonates (e.g., alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate), HMG-CoA reductase inhibitors (e.g., lovastatin) Atorvastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lescol, lupitor, rosuvastatin, and atorvastatin; antisense oligonucleotides (e.g., those disclosed in U.S. Patents 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminase inhibitors (such as fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan; Tositumomab (tositumomab) ) and its pharmaceutically acceptable salts, solvates, inclusion compounds and prodrugs.

[0582] Furthermore, the antibodies of the present invention can be coupled or recombinantly fused to a therapeutic or pharmaceutical portion that alters a given biological response. The therapeutic or pharmaceutical portion should not be construed as limited to classical chemotherapeutic agents. For example, the pharmaceutical portion can be a protein, peptide, or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis agents such as TNF-γ, AIM I (see International Publication No. WO 97 / 33899), AIM II (see International Publication No. WO 97 / 34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6: 1567-1574), and VEGF (see International Publication No. WO). 99 / 23105), anti-angiogenic agents, such as angiostatin, endostatin, or components of the coagulation pathway (e.g., tissue factor); or biological response modulators, such as lymphokines (e.g., interferon-γ, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”), interleukin-7 (“IL-7”), interleukin-9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12 (“IL-12”), interleukin- 15 (“IL-15”), interleukin-23 (“IL-23”), granulocyte-macrophage colony-stimulating factor (“GM-CSF” and granulocyte colony-stimulating factor (“G-CSF”)) or growth factors (e.g., growth hormone (“GH”)) or coagulants (e.g., calcium, vitamin K, tissue factors, such as but not limited to Hageman factor (factor XII), high molecular weight kininogen (HMWK), prokalopeptidase (PK), coagulant factor II (prothrombin), factors V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipids and fibrin monomers).

[0583] This invention encompasses antibodies of the present invention that are recombinantly fused or chemically conjugated to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce a fusion protein. Specifically, the present invention provides fusion proteins comprising an antigen-binding fragment (e.g., Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain, or VL CDR) of the antibody of the present invention and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide fused with the antibody is useful for targeting the antibody to a specific cell type, such as cells expressing hOX40L or the hOX40L receptor. For example, antibodies that specifically bind to cell surface receptors expressed by a specific cell type (e.g., immune cells) may be fused or conjugated to the modified antibody of the present invention.

[0584] The conjugated or fusion proteins of this invention comprise the antibodies and heterologous peptides described herein. In one embodiment, the conjugated or fusion protein of this invention comprises the variable domains of the antibody disclosed in the examples and the heterologous peptide.

[0585] Furthermore, the antibodies of this invention can be coupled to the therapeutic site, such as radioactive metal ions like alpha emitters (e.g., 213 Bi), or may be coupled to a device for containing radioactive metal ions, including but not limited to 131 In、 131 Lu、 131 Y、 131 Ho、 131 Sm is a macrocyclic chelator that conjugates to peptides. In some embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA), which can be linked to the antibody via a linker molecule. Such linker molecules are well known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4(10): 2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4): 553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8): 943-50, each of which is incorporated herein by reference in its entirety.

[0586] Furthermore, the antibodies of the present invention can be fused with labeled sequences, such as peptides that facilitate purification. In a preferred embodiment, the labeled amino acid sequence is a hexahistine peptide, such as the tags provided in pQE vectors (QIAGEN, Inc.), many of which are commercially available. As described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistine provides convenient purification of the fusion protein. Other peptide tags for purification include, but are not limited to, hemagglutinin (“HA”) tags corresponding to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37:767), and “FLAG” tags.

[0587] Methods for fusing or conjugating therapeutic portions (including peptides) to antibodies are well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), pp. 623–53 (Marcel Dekker, Inc., 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475–506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of "Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62: 119-58; U.S. Patents 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; Ashkenazi et al., Proc. Natl. Acad. Sci.USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331: 84-86, 1988; Zheng et al., J. Immunol., 154: 5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89: 11337-11341, 1992, which are incorporated herein by reference in their entirety.

[0588] Fusion proteins can be generated, for example, through techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively, “DNA shuffling”). DNA shuffling can be used to alter the activity of the antibodies of the present invention (e.g., to give the antibody higher affinity and lower dissociation rate). See typically U.S. Patents 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8: 724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson et al., 1999, J. Mol. Biol. 287: 265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313 (each of these patents and publications is incorporated herein by reference in its entirety). Antibodies or encoded antibodies can be altered prior to recombination by error-prone PCR, random nucleotide insertion, or other methods of random mutagenesis. The polynucleotide encoding the antibody of the present invention can be recombinated with one or more components, motifs, segments, parts, domains, fragments, etc. of one or more heterologous molecules.

[0589] As described in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety, the antibodies of the present invention can also be coupled to a second antibody to form antibody heteroconjugates.

[0590] The therapeutic portion or drug substance of the antibody of the present invention, which specifically binds to the hOX40L antigen, should be selected for conjugation or recombinant fusion to achieve the desired preventive or therapeutic effect. In some embodiments, the antibody is a modified antibody. Clinicians or other medical personnel should consider the following factors when deciding which therapeutic portion or drug substance to conjugate or recombinantly fuse to the antibody of the present invention: disease characteristics, disease severity, and the subject's condition.

[0591] The antibodies of this invention can also be attached to solid supports, which is particularly useful for the immunoassay or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0592] Pharmaceutical Composition

[0593] The following discussion of the composition also applies to the fragments, so that the disclosure of antibodies, with necessary modifications, can also be applied to the fragments of this invention.

[0594] Therapeutic formulations containing one or more antibodies of the present invention provided herein can be prepared for storage in lyophilized or aqueous forms by mixing antibodies of the desired purity with optionally physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroquinone quaternary ammonium chloride; benzalkonium chloride, benzyl ethoxymmonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than The following substances are considered as a whole: polypeptides (approximately 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0595] The antibodies of the present invention provided herein can also be formulated, for example, into liposomes. Liposomes containing the target molecule are prepared by methods known in the art, such as Epstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended cycle times are disclosed in U.S. Patent No. 5,013,556.

[0596] Particularly useful immunoliposomes can be generated from lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE) via reverse-phase evaporation. The liposomes are extruded through a filter with a specified pore size to produce liposomes with the desired diameter. The Fab' fragment of the antibody described herein can be coupled to liposomes as described by Martin et al. (1982) J. Biol. Chem. 257: 286-288 via a disulfide exchange reaction. Chemotherapeutic agents (such as doxorubicin) are optionally contained within the liposomes; see Gabizon et al. (1989) J. National Cancer Inst. 81(19): 1484.

[0597] Formulations, such as those described herein, may also contain more than one active compound when necessary for a specific indication of treatment. In some embodiments, the formulation comprises the antibody of the present invention and one or more active compounds having complementary activities that do not adversely affect each other. Such molecules are appropriately combined in amounts effective for the intended purpose. For example, the antibody of the present invention may be combined with one or more other therapeutic agents. Such combination therapies may be administered to the patient sequentially, simultaneously, or in sequence.

[0598] In one embodiment, the combination comprises the anti-OX40L antibody of the present invention and another therapeutic agent, said other therapeutic agent being independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, anti-IL12 / IL-23 antibody (e.g., uterotumab), anti-CD20 antibody (e.g., rituximab), anti-CD30 antibody (e.g., butuximab), CTLA4-Fc molecule (e.g., abatacept), CCR5 receptor antagonist (e.g., malaviro), anti-CD40L antibody, anti-V LA4 antibodies (e.g., nastatin), anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies (e.g., alenzab), anti-CD45 antibodies, cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibodies (e.g., eculizumab), anti-a4b7 integrin antibodies (e.g., vedolizumab), anti-IL6 antibodies (e.g., tocilizumab), anti-IL2R antibodies (e.g., baliximab), anti-CD25 antibodies (e.g., daliximab), anti-TNFa / TNFa-Fc molecules (e.g., etanercept, adalimumab, infliximab, golimumab, or sertozumab), and vorinostat. In another embodiment, the combination comprises the anti-OX40L antibody of the present invention and another therapeutic agent independently selected from rapamycin (sirolimus), tacrolimus, cyclosporine, corticosteroids (e.g., methylprednisolone), methotrexate, mycophenolate mofetil, anti-CD28 antibody, CTLA4-Fc molecule (e.g., abatacept), anti-CD40L antibody, anti-LFA1 antibody, anti-CD52 antibody (e.g., alemtuzumab), cyclophosphamide, and anti-thymocyte globulin.

[0599] The antibodies of the present invention can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by, for example, coagulation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pa.

[0600] Formulations that can be administered in vivo can be sterile. This can be easily achieved through filtration, for example, using a sterile filtration membrane.

[0601] Sustained-release formulations can also be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antagonists, said matrices being in the form of molded articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxy-methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid-ethyl ester, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic-glycolic acid copolymers and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic-glycolic acid allow molecules to be released for more than 100 days, some hydrogels release proteins for a shorter period. When encapsulated antibodies are held in vivo for extended periods, they can denature or aggregate due to exposure to moisture at 37°C, leading to loss of biological activity and possible changes in immunogenicity. Stabilization strategies can be designed based on the mechanisms involved. For example, if aggregation is found to occur via intermolecular SS bonds formed through thio-disulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0602] The pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more antibodies of the present invention in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents. Such pharmaceutical compositions are useful in the prevention, treatment, management, or improvement of hOX40L-mediated diseases, such as inflammatory bowel disease, transplant rejection, GvHD, or one or more of their symptoms.

[0603] Drug carriers suitable for administering the compounds provided herein include any such carriers known to those skilled in the art as being suitable for a particular mode of administration.

[0604] In addition, the antibody of the present invention can be formulated as the sole pharmaceutical active ingredient in a composition or can be combined with other active ingredients (such as one or more other preventive or therapeutic agents).

[0605] The composition may contain one or more antibodies of the present invention. In one embodiment, the antibody is formulated into a suitable pharmaceutical preparation for oral administration, such as a solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained-release formulation or elixir, or formulated into a sterile solution or suspension for parenteral administration, as well as transdermal patch formulations and dry powder inhalers. In one embodiment, the above-described antibody is formulated into a pharmaceutical composition using techniques and procedures well known in the art (see, for example, Ansel (1985) Introduction to Pharmaceutical Dosage Forms, 4th edition, page 126).

[0606] In the composition, an effective concentration of one or more antibodies or their derivatives is mixed with a suitable drug carrier. The concentration of the compound in the composition is effective for delivering an amount that can treat, prevent, or improve hOX40L-mediated diseases or their symptoms after administration.

[0607] In one embodiment, the composition is formulated for single-dose administration. To formulate the composition, a certain weight fraction of the compound is dissolved, suspended, dispersed, or otherwise mixed with a selected carrier at an effective concentration to relieve, prevent, or improve one or more symptoms.

[0608] The antibodies of the present invention are contained in a pharmaceutically acceptable carrier in an effective amount sufficient to produce a therapeutically useful effect without adverse side effects on the treated patient. Therapeuticly effective concentrations can be empirically determined by testing the compounds in vitro and in vivo systems using conventional methods, from which the dosage for humans can be deduced.

[0609] The concentration of antibodies in a pharmaceutical composition will depend on factors such as the physicochemical characteristics of the antibodies, the dosing regimen and the amount administered, as well as other factors known to those skilled in the art.

[0610] In one embodiment, the therapeutically effective dose produces an antibody serum concentration of about 0.1 ng / ml to about 50-100 μg / ml. In another embodiment, the pharmaceutical composition provides a dose of about 0.001 mg to about 2000 mg antibody / kg body weight per day. The pharmaceutical dosage units can be prepared to provide each dosage unit of about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, 1000 mg, or 2000 mg, and in one embodiment, a combination of about 10 mg to about 500 mg antibody and / or other optional essential components.

[0611] Antibodies can be administered in a single dose or divided into multiple smaller doses administered at different time intervals. It should be understood that the precise dosage and duration of treatment are functions of the disease being treated and can be determined empirically using known experimental protocols or inferred from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​may also vary depending on the severity of the symptoms to be alleviated. It should also be understood that for any given subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the composition, and the concentration ranges presented herein are merely illustrative and not intended to limit the scope or practice of the claimed compositions.

[0612] After antibodies are mixed or added, the resulting mixture can be a solution, suspension, emulsion, etc. The form of the resulting mixture depends on many factors, including the intended administration method and the solubility of the compound in the chosen carrier or medium. The effective concentration is sufficient to improve the symptoms of the treated disease, condition, or symptom and can be determined empirically.

[0613] Pharmaceutical compositions are provided in unit dose forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, as well as oil-water emulsions containing an appropriate amount of the compound or a pharmaceutically acceptable derivative thereof, for administration to humans and animals. In one embodiment, the antibody is formulated and administered in unit dose or multiple dose forms. As used herein, a unit dose form refers to a physically discrete unit suitable for human and animal subjects and individually packaged as known in the art. Each unit dose contains a predetermined amount of antibody sufficient to produce the desired therapeutic effect when combined with the desired drug carrier, mediator, or diluent. Examples of unit dose forms include ampoules and syringes, and individually packaged tablets or capsules. Unit dose forms can be administered in multiple doses or multiple times. Multiple dose forms are multiple identical unit dose forms packaged in a single container and administered in separate unit dose forms. Examples of multiple dose forms include vials, bottles, or pints or gallon bottles of tablets or capsules. Thus, a multiple dose form is multiple unit doses not separated on the packaging.

[0614] In a preferred embodiment, one or more anti-hOX40L antibodies of the present invention are in the form of a liquid pharmaceutical formulation. Pharmaceutically applicable liquid compositions can be prepared, for example, by dissolving, dispersing, or otherwise mixing the above-defined active compound and optional pharmaceutical excipients in a carrier such as water, saline, dextran aqueous solution, glycerol, ethylene glycol, ethanol, etc., to form a solution or suspension. If desired, the pharmaceutical composition to be applied may also contain trace amounts of non-toxic excipients such as wetting agents, emulsifiers, solubilizers, pH buffers, etc., for example, acetates, sodium citrate, cyclodextrin derivatives, sorbitol monolaurate, sodium triethanolamine acetate, triethanolamine oleate, and other such reagents.

[0615] The actual methods for preparing such dosage forms are known or will be obvious to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pa.

[0616] Dosage forms or compositions containing 0.005% to 100% antibody, with the remainder consisting of a non-toxic carrier, can be prepared. Methods for preparing these compositions are known to those skilled in the art.

[0617] Oral medications are available in solid, gel, or liquid dosage forms. Solid dosage forms include tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges, and tablets, which may be enteric-coated, sugar-coated, or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent forms in combination with other ingredients known to those skilled in the art.

[0618] In some embodiments, the formulation is a solid dosage form. In some embodiments, the formulation is a capsule or tablet. Tablets, pills, capsules, lozenges, etc., may contain one or more of the following components or compounds with similar properties: binders; lubricants; diluents; glidants; disintegrants; colorants; sweeteners; flavorings; wetting agents; emetic coatings; and film coatings. Examples of binders include microcrystalline cellulose, tragacanth gum, glucose solution, gum arabic paste, gelatin solution, molasses, polyvinylpyrrolidone, polyvinylpyrrolidone, crosspovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silica. Disintegrants include croscarmellose sodium, glycolic acid starch sodium, alginate, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Colorants include, for example, any one or mixtures of approved water-soluble FD and C dyes; and water-soluble FD and C dyes suspended on alumina hydrate. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and many spray-dried flavorings. Flavorings include natural flavorings extracted from plants such as fruits and synthetic blends of pleasure-producing compounds, such as, but not limited to, peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauroyl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

[0619] The antibodies of the present invention can be provided in compositions that protect them from the acidic environment of the stomach. For example, the composition is formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with antacids or other such ingredients.

[0620] When the dosage unit is in capsule form, it may contain a liquid carrier, such as fatty oil, in addition to the materials of the types mentioned above. Furthermore, the dosage unit form may contain various other materials that alter the physical form of the dosage unit, such as sugars and other enteric coatings. The compound may also be applied as a component of elixirs, suspensions, syrups, rice paper capsules, sprays, chewing gum, etc. Syrups may contain sucrose as a sweetener, as well as certain preservatives, dyes and colorants, and flavorings, in addition to the active compound.

[0621] The antibody may also be mixed with other active materials that do not impair the desired effect or with other materials that complement the desired effect, such as antacids, H2 blockers, and diuretics. The active ingredient is an antibody or a pharmaceutically acceptable derivative thereof as described herein. Higher concentrations of the active ingredient, up to about 98% by weight, may be included.

[0622] In all embodiments, the tablet and capsule formulations may be coated, as is known to those skilled in the art, to alter or maintain the solubility of the active ingredient. Thus, for example, they may be coated with conventional enterically digestible coatings such as phenyl salicylate, waxes, and cellulose acetate phthalate.

[0623] In a preferred embodiment, the formulation is a liquid dosage form. Liquid dosage forms include aqueous solutions, emulsions, suspensions, solutions, and / or suspensions rehydrated from non-effervescent granules and effervescent formulations rehydrated from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are oil-in-water or water-in-oil emulsions.

[0624] Emulsions are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in another liquid in the form of small spheres. Pharmaceutically acceptable carriers used in emulsions include non-aqueous liquids, emulsifiers, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.

[0625] Pharmaceutically acceptable substances for rehydration into liquid oral dosage forms in non-effervescent granules include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances for rehydration into liquid oral dosage forms in effervescent granules include organic acids and carbon dioxide sources. Colorants and flavoring agents are used in all of the above dosage forms.

[0626] Solvents include glycerol, sorbitol, ethylene glycol, and syrups. Examples of preservatives include glycerol, methylparaben and propylparaben, benzoic acid, sodium benzoate, and alcohols. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, gum arabic, astragalus gum, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethyl cellulose, pectin, astragalus gum, Veegum, and gum arabic. Sweeteners include sucrose, syrups, glycerol, and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauroyl ether. Organic acids include citric acid and tartaric acid. Carbon dioxide sources include sodium bicarbonate and sodium carbonate. Colorants include any one or mixtures of approved water-soluble FD and C dyes. Flavorings include natural flavorings extracted from plants such as fruits and synthetic blends of compounds that produce a pleasant taste.

[0627] For solid dosage forms, solutions or suspensions in, for example, propylene carbonate, vegetable oils, or triglycerides, are, in one embodiment, encapsulated in gelatin capsules. Such solutions, their preparation, and encapsulation are disclosed in U.S. Patent Nos. 4,328,245, 4,409,239, and 4,410,545. For liquid dosage forms, solutions, such as those in polyethylene glycol, may be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, for easy measurement and administration.

[0628] Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those proposed in U.S. Patents RE28,819 and 4,358,603. In short, such formulations include, but are not limited to, those containing the compounds provided herein, dialkylated mono- or polyalkylene glycols (including, but not limited to, 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether), and one or more antioxidants (such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates), wherein 350, 550, and 750 refer to the approximate average molecular weight of polyethylene glycol.

[0629] Other formulations include, but are not limited to, aqueous alcoholic solutions, including pharmaceutically acceptable acetals. The alcohols used in these formulations are any pharmaceutically acceptable water-soluble solvents having one or more hydroxyl groups, including but not limited to propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diacetal.

[0630] In one embodiment, this document also considers parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection. The injection can be prepared in conventional forms, such as as a liquid solution or suspension, a solid form of a solution or suspension suitable for use in a liquid prior to injection, or as an emulsion. The injection, solution, and emulsion also contain one or more excipients. Suitable excipients are, for example, water, saline, dextran, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered may also contain trace amounts of non-toxic excipients such as wetting agents, emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, and cyclodextrin.

[0631] This document also considers implantation of sustained-release or continuous-release systems that maintain a constant dose level (see, for example, U.S. Patent No. 3,710,795). In short, the compounds described herein are dispersed in an internal solid matrix, such as polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethicone, silica-carbonate copolymer, hydrophilic polymers (such as hydrogels of acrylic acid and methacrylate), collagen, and an external polymeric membrane (e.g., polyethylene, polypropylene, ethylene / ...). Crosslinked polyvinyl alcohol (CVA) surrounded by propylene copolymers, ethylene / ethyl acrylate copolymers, and ethylene / vinyl acetate copolymers, and partially hydrolyzed crosslinked polyvinyl acetate, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride and vinyl acetate, vinylidene chloride, copolymers of ethylene and propylene, ionomers of polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers, and ethylene / vinyloxyethanol insoluble in body fluids. In a controlled release rate step, the antibody diffuses through an external polymer membrane. The amount of antibody contained in such parenteral compositions is highly dependent on their specific properties, the activity of the compound, and the needs of the subject.

[0632] Preparations intended for parenteral administration include readily injectable sterile solutions, sterile dried soluble products (such as lyophilized powders, including subcutaneous tablets) readily recombinable with solvents immediately before use, readily injectable sterile suspensions, sterile dried insoluble products readily recombinable with media immediately before use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0633] If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS) and solutions and mixtures thereof containing thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol.

[0634] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, masking or chelating agents, and other pharmaceutically acceptable substances.

[0635] Examples of aqueous parenteral media include sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, and dextran and lactated Ringer's injection. Non-aqueous parenteral media include non-volatile plant-derived oils, cottonseed oil, sesame oil, and peanut oil. Antimicrobial agents, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl ethoxymonochloride, can be added to parenteral preparations packaged in multi-dose containers. Isotonic agents include sodium chloride and dextran. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensors and dispersants include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (…). 80). Masking or chelating agents for metal ions include EDTA. Drug carriers also include ethanol, polyethylene glycol, and polypropylene glycol for water-soluble media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0636] The concentration of the active pharmaceutical compound is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. As is known in the art, the precise dosage depends on the patient's or animal's age, weight, and condition.

[0637] Unit-dose parenteral preparations may be packaged in ampoules, vials, or syringes with needles. As is known and practiced in the art, all preparations intended for parenteral administration are sterile.

[0638] Illustratively, intravenous or arterial infusion of a sterile aqueous solution containing the active compound is an effective mode of administration. Another implementation is, if necessary, injection of a sterile aqueous or oil solution or suspension containing the active substance to produce the desired pharmacological effect.

[0639] Injectable formulations designed for both local and systemic application. In one embodiment, a therapeutically effective dose is formulated to contain an active compound at a concentration of at least about 0.1% w / w to about 90% w / w or higher, and in some embodiments, more than 1% w / w, for use on the treated tissue.

[0640] The antibody is suspended in a micronized or other suitable form. The form of the resulting mixture depends on many factors, including the intended administration method and the solubility of the compound in the chosen carrier or medium. An effective concentration is sufficient to improve the symptoms of the condition and can be determined empirically.

[0641] In other embodiments, the pharmaceutical preparation is a lyophilized powder that can be rehydrated into solutions, emulsions, and other mixtures for application. It can also be rehydrated and formulated into solids or gels.

[0642] Lyophilized powders are prepared by dissolving the antibodies or pharmaceutically acceptable derivatives thereof provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients or other pharmacological components of a powder or a reconstituted solution prepared from the powder to improve stability. Available excipients include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable reagents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, approximately neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the selected formulation. In one embodiment, the resulting solution is aliquoted into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0643] This lyophilized powder is rehydrated with water for injection to provide a formulation for parenteral administration. For rehydration, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends on the compound selected. This amount can be determined via [method / method / etc.].

[0644] As described for both topical and systemic application, prepare a topical mixture. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated into creams, gels, ointments, lotions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories, bandages, skin patches, or any other formulation suitable for surface application.

[0645] The antibodies of the present invention can be formulated into aerosols for application, for example, by inhalation of a surface (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids useful for treating inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract, alone or in combination with an inert carrier such as lactose, can be in aerosol or solution form for nebulizers, or in fine powder form for inhalation. In such cases, the particles of the formulation will have a diameter of less than 50 micrometers in one embodiment and less than 10 micrometers in another embodiment.

[0646] The compounds can be formulated for topical or surface application, such as in gels, creams, and lotions for application to the skin and mucous membranes (e.g., the eyes) and for application to the eyes, or for application to the intracranial or spinal cord. Surface application is considered for transdermal delivery and also for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of the active compounds, alone or in combination with other pharmaceutically acceptable excipients, can also be administered.

[0647] These solutions, especially those intended for ophthalmic use, can be prepared as 0.01%–10% isotonic solutions with a pH of approximately 5–7 using appropriate salts.

[0648] This article also considers other routes of administration, such as transdermal patches, including iontophoresis and electrophoresis devices, and rectal administration.

[0649] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art. Such patches are disclosed, for example, in U.S. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

[0650] For example, pharmaceutical dosage forms for rectal administration include systemic suppositories, capsules, and tablets. As used herein, a rectal suppository is a solid that is inserted into the rectum, melts or softens at body temperature, and releases one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances in rectal suppositories are a matrix or carrier and a melting point-enhancing agent. Examples of matrices include cocoa butter (cocoa soybean oil), glycerol-gelatin, carbon wax (polyethylene glycol), and suitable mixtures of fatty acid monoglycerides, diglycerides, and triglycerides. Combinations of various matrices can be used. Agents that enhance the melting point of suppositories include cetylene and waxes. Rectal suppositories can be prepared by compression or molding. In one embodiment, the weight of the rectal suppository is about 2 to 3 gm.

[0651] Tablets and capsules for rectal administration can be prepared using the same pharmaceutically acceptable substances and by the same methods as those used for oral administration.

[0652] The antibodies and other compositions described herein can also be formulated to target specific tissues, receptors, or other areas of the body of a subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods for use in ready-to-use compositions are considered herein. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In some embodiments, the anti-hOX40L antibody of the present invention is targeted (or otherwise administered) to the colon, for example, in a patient with IBD or at risk of developing IBD. In some embodiments, the anti-hOX40L antibody of the present invention is targeted (or otherwise administered) to the eye, for example, in a patient with uveitis or at risk of uveitis.

[0653] In one embodiment, liposomal suppositories, including tissue-targeting liposomes such as tumor-targeting liposomes, may also be suitable as pharmaceutically acceptable carriers. These can all be prepared according to methods known to those skilled in the art. For example, liposomal formulations are prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilayer vesicles (MLVs) can be formed by drying lecithin and cephalinositol serine (7:3 molar ratio) inside a flask. A solution of the compounds provided herein in phosphate-buffered saline (PBS) lacking divalent cations is added and the flask is shaken until the lipid membrane is dispersed. The resulting vesicles are washed to remove unencapsulated compounds, granulated by centrifugation, and then resuspended in PBS.

[0654] Administration and Dosage Methods

[0655] The present invention also provides compositions comprising one or more antibodies or fragments of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases (or symptoms thereof). Discussions regarding antibodies, with necessary modifications, also apply to fragments of the present invention. In an alternative embodiment, the present invention also provides compositions comprising one or more antibodies or fragments of the present invention for the prevention, management, treatment, and / or improvement of OX40L-mediated diseases (or symptoms thereof) in subjects, wherein OX40L is non-human (e.g., dog, cat, horse, cow, sheep, or pig) and the subjects are dogs, cats, horses, cows, sheep, or pigs.

[0656] In some embodiments, this document provides compositions comprising one or more antibodies of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases such as IBD (e.g., ulcerative colitis or Crohn's disease) or their symptoms. IBD symptoms can range from mild to severe and are generally dependent on the part of the intestine involved. Exemplary symptoms of IBD include abdominal cramps and pain, hemorrhagic diarrhea, severe defecation urgency, fever, loss of appetite, weight loss, anemia, fatigue, and / or sores on the lower extremities, ankles, calves, thighs, and arms. Exemplary intestinal complications of IBD include massive bleeding from ulcers, intestinal perforation or rupture, strictures and obstructions, fistulas (abnormal pathways) and perianal diseases, toxic megacolon (e.g., acute obstructive dilatation of the colon), and / or malignancies (e.g., colonic or small bowel cancer). Exemplary extraintestinal complications of IBD include arthritis, dermatitis, ocular inflammation, liver and kidney disease, and / or bone loss. Any combination of these symptoms can be prevented, managed, treated, and / or improved using the compositions and methods provided herein.

[0657] In some embodiments, this document provides compositions comprising one or more antibodies of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases such as GVHD or their symptoms. GVHD typically occurs following allogeneic or matched unrelated bone marrow transplantation (BMT).

[0658] In some implementations, GVHD is referred to as acute GVHD. Symptoms of GVHD can develop rapidly and can be mild or severe. In some cases, acute GVHD occurs within approximately 3 months post-transplantation, such as when blood cell counts recover. In some cases, acute GVHD affects the skin, gastrointestinal (GI) tract, and / or liver. For example, in some patients, acute cutaneous GVHD begins with a rash, for example, on the palms, soles, or shoulders. However, the rash can become widespread and can be itchy and painful and / or may blister and peel. Acute hepatic GVHD can affect normal liver function, such as liver enzymes, and can also cause jaundice. If the liver is enlarged, acute hepatic GVHD can also cause the abdomen to become swollen and painful. Finally, symptoms of acute intestinal GVHD (or digestive system GVHD) may include diarrhea, mucus or blood in the stool, cramps or abdominal pain, indigestion, nausea, and / or loss of appetite. Other systemic symptoms of acute GVHD may include anemia, low-grade fever, and / or increased susceptibility to infections. Any combination of these symptoms of acute GVHD can be prevented, managed, treated, and / or improved using the compositions and methods provided herein.

[0659] In other implementations, GVHD is chronic GVHD. Chronic GVHD can occur from approximately 3 months to approximately 1 year or longer after transplantation. Chronic GVHD can be mild or severe and typically includes symptoms similar to those of acute GVHD. Chronic GVHD can affect the skin and digestive system, including the liver, but can also involve other organs and the immune system (e.g., making the patient more susceptible to infection) and / or connective tissue. Symptoms of chronic skin GVHD include rash, dry skin, tight skin, itchy skin, darkening of skin color, thickening of the skin, and / or may affect hair (e.g., hair loss, turning gray) or nails (e.g., hard or brittle nails). Chronic intestinal GVHD can affect the digestive system, oral cavity, esophagus, stomach lining, and / or intestinal lining, and symptoms may include diarrhea, dry mouth or mouth ulcers, painful swallowing, poor gastric nutrient absorption, flatulence, and stomach cramps. Chronic liver GVHD can cause liver damage and scarring (cirrhosis). Chronic GVHD of the eyes can affect the lacrimal glands, causing dryness, burning, pain, or intolerance to bright light. Chronic GVHD of the lungs can cause shortness of breath, wheezing, persistent cough, and / or increased susceptibility to chest infections. Chronic GVHD can affect tendons that connect muscles to bones (e.g., inflammation), making it difficult to straighten or bend your arms and legs. Any combination of these symptoms of chronic GVHD can be prevented, managed, treated, and / or improved using the compositions and methods provided in this article.

[0660] In some embodiments, this document provides compositions comprising one or more antibodies of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases such as uveitis or their symptoms.

[0661] In some embodiments, this document provides compositions comprising one or more antibodies of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases such as pyoderma gangrenosa, giant cell arteritis, Schnitzler syndrome, or non-infectious scleritis.

[0662] In some embodiments, this document provides compositions comprising one or more antibodies of the present invention for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases or conditions selected from autoimmune diseases or conditions, systemic inflammatory diseases or conditions, or transplant rejection; such as inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, transplant rejection, allogeneic transplant rejection, graft-versus-host disease (GvHD), ulcerative colitis, systemic lupus erythematosus (SLE), diabetes, uveitis, ankylosing spondylitis, contact hypersensitivity, multiple sclerosis, and atherosclerosis, especially GvHD.

[0663] In one specific embodiment, a composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises an antibody of the present invention, such as the OX40L binding site of the antibody disclosed in the examples.

[0664] In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VH domains having an amino acid sequence having any VH domain in the sequence listing (i.e., Seq ID No: 2, Seq ID No: 34, Seq ID No: 66, or Seq ID No: 94, especially Seq ID No: 34). In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VHCDR1s having an amino acid sequence having any VH CDR1 in the sequence listing (i.e., Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96, or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42). In another embodiment, the composition for the prevention, management, treatment and / or improvement of hOX40L-mediated disease comprises one or more antibodies, said antibodies comprising one or more VH CDR2 having the amino acid sequence of any VH CDR2 in the sequence listing (i.e., Seq ID No: 6, Seq ID No: 12, Seq ID No: 38, Seq ID No: 44, Seq ID No: 70, Seq ID No: 76, Seq ID No: 98 or Seq ID No: 104, especially Seq ID No: 38 or Seq ID No: 44). In a preferred embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VH CDR3 having the amino acid sequence of any VH CDR3 in the sequence listing (i.e., Seq ID No: 8, Seq ID No: 14, Seq ID No: 40, Seq ID No: 46, Seq ID No: 72, Seq ID No: 78, Seq ID No: 100, or Seq ID No: 106, especially Seq ID No: 40 or Seq ID No: 46).

[0665] In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VL domains having an amino acid sequence having any VL domain in the sequence listing (i.e., Seq ID No: 16, Seq ID No: 48, Seq ID No: 80, or Seq ID No: 108, especially Seq ID No: 48), (optionally also comprising homologous VH domains listed in the sequence listing (i.e., Seq ID No: 2 / 16, Seq ID No: 34 / 48, Seq ID No: 66 / 80, or Seq ID No: 94 / 108, especially Seq ID No: 34 / 48)). In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VL domains having an amino acid sequence having any VL domain in the sequence listing (i.e., Seq ID No: 16, Seq ID No: 48, Seq ID No: 80, or Seq ID No: 108, especially Seq ID No: 48)). In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated disease comprises one or more antibodies having an amino acid sequence of any VL CDR1 in the sequence listing (i.e., Seq ID No: 82, Seq ID No: 88, Seq ID No: 110, or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56). In a preferred embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated disease comprises one or more antibodies having an amino acid sequence of any VL CDR2 in the sequence listing (i.e., Seq ID No: 20, Seq ID No: 26, Seq ID No: 52, Seq ID No: 58, Seq ID No: 84, Seq ID No: 90, Seq ID No: 112, or Seq ID No: 118, especially Seq ID No: 52 or Seq ID No: 58). VL CDR3 is the amino acid sequence of any of the following: No: 22, Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114 or Seq ID No: 120, especially Seq ID No: 54 or Seq ID No: 60).

[0666] In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VH domains having an amino acid sequence having any VH domain in the sequence listing (i.e., Seq ID No: 2, Seq ID No: 34, Seq ID No: 66, or Seq ID No: 94, especially Seq ID No: 34), and one or more VL domains having an amino acid sequence having any VL domain in the sequence listing (i.e., Seq ID No: 16, Seq ID No: 48, Seq ID No: 80, or Seq ID No: 108, especially Seq ID No: 48).

[0667] In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VHCDR1 having the amino acid sequence of any VH CDR1 in the sequence listing (i.e., Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96 or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42), and one or more VL CDR1 having the amino acid sequence of any VL CDR1 in the sequence listing (i.e., Seq ID No: 18, Seq ID No: 24, Seq ID No: 50, Seq ID No: 56, Seq ID No: 82, Seq ID No: 88, Seq ID No: 110 or Seq ID No: 116, especially Seq ID No: 50 or Seq ID No: 56). In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VH CDR1 having the amino acid sequence of any VH CDR1 in the sequence listing (i.e., Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96 or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42), and one or more VL CDR2 having the amino acid sequence of any VL CDR2 in the sequence listing (i.e., Seq ID No: 20, Seq ID No: 26, Seq ID No: 52, Seq ID No: 58, Seq ID No: 84, Seq ID No: 90, Seq ID No: 112 or Seq ID No: 118, especially Seq ID No: 52 or Seq ID No: 58).In another embodiment, the composition for the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases comprises one or more antibodies, said antibodies comprising one or more VH CDR1 having the amino acid sequence of any VH CDR1 in the sequence listing (i.e., Seq ID No: 4, Seq ID No: 10, Seq ID No: 36, Seq ID No: 42, Seq ID No: 68, Seq ID No: 74, Seq ID No: 96 or Seq ID No: 102, especially Seq ID No: 36 or Seq ID No: 42), and one or more VL CDR3 having the amino acid sequence of any VL CDR3 in the sequence listing (i.e., Seq ID No: 22, Seq ID No: 28, Seq ID No: 54, Seq ID No: 60, Seq ID No: 86, Seq ID No: 92, Seq ID No: 114 or Seq ID No: 120, especially Seq ID No: 54 or Seq ID No: 60).

[0668] As discussed in more detail elsewhere herein, the compositions of the present invention can be used alone or in combination with other compounds or compositions. Furthermore, the antibodies can be recombinantly fused to the N- or C-terminus of a heterologous polypeptide, or chemically coupled (including covalent and non-covalent coupling) to the polypeptide or other compositions. For example, the antibodies of the present invention can be recombinantly fused to or coupled to molecules used as markers in detection assays, and effector molecules such as heterologous polypeptides, drugs, radioactive nucleotides, or toxins. See, for example, PCT disclosures WO 92 / 08495, WO 91 / 14438, WO 89 / 12624, U.S. Patent No. 5,314,995, and EP 396,387.

[0669] In some embodiments, this document provides a method for reducing or inhibiting the binding of hOX40L to the OX40L receptor or homologous ligand (e.g., OX40) in a subject (e.g., a human subject), comprising administering to the subject an effective amount of an antibody that specifically binds to an hOX40L polypeptide (e.g., cell surface-expressed or soluble hOX40L). In some embodiments, the secretion of hOX40L biological activity in the subject, such as CCL20, IL8 and / or RANTES or INF-γ, TNF-α or IL-2, especially INF-γ or another cytokine disclosed herein, is also reduced, for example, by at least 10, 20, 30, 40, 50 or 60% or 70% or 80% or 90% or 95% or >95%.

[0670] In some embodiments, this document provides a method for reducing or inhibiting the biological activity of hOX40L in a subject (e.g., a human subject), such as interferon-γ, IL-2, CCL20, IL8 and / or RANTES or other cytokines, or the secretion of INF-γ, TNF-α or IL-2, especially INF-γ, comprising administering to the subject an effective amount of an antibody that specifically binds to an hOX40L peptide (e.g., hOX40L expressed on the cell surface), wherein the biological activity of hOX40L is reduced by said antibody.

[0671] In other embodiments, this document provides methods for reducing or inhibiting the binding of hOX40L to the OX40L receptor or homologous ligand (e.g., OX40) in cells expressing hOX40L on their cell surface, comprising contacting cells with an effective amount of specifically binding hOX40L peptides (e.g., cell surface-expressed or soluble hOX40L), such as hOX40L peptides, hOX40L peptide fragments, or antibodies against hOX40L epitopes. In some embodiments, the secretion of hOX40L biological activities in cells, such as interferon-γ, IL-2, CCL20, IL8, and / or RANTES or INF-γ, TNF-α, or IL-2, especially INF-γ or other cytokines disclosed herein, is also reduced.

[0672] In some embodiments, this document provides a method for reducing or inhibiting the secretion of hOX40L biological activity, such as interferon-γ, IL-2, CCL20, IL8 and / or RANTES or other cytokines disclosed herein, in cells having a cell surface-expressed hOX40L receptor (e.g., OX40). This method comprises contacting cells with an effective amount of an antibody that specifically binds to an hOX40L polypeptide (e.g., cell surface-expressed or soluble hOX40L), wherein the hOX40L biological activity is reduced by said antibody.

[0673] In in vitro and in vivo diagnostic and therapeutic methods, the antibodies of the present invention can be used, for example, to purify, detect, and target the hOX40L antigen. For example, the modified antibodies have the use in immunoassays to qualitatively and quantitatively determine hOX40L levels in biological samples. See, for example, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) (incorporated herein by reference in its entirety).

[0674] The present invention also provides a method for preventing, managing, treating, and / or improving hOX40L-mediated diseases by administering to a subject an effective amount of an antibody or a pharmaceutical composition comprising the antibody of the present invention. In one aspect, the antibody is substantially purified (i.e., substantially free of substances that limit its effectiveness or produce adverse side effects). In a preferred embodiment, the antibody is a fully human monoclonal antibody, such as a fully human monoclonal antagonistic antibody. The subject administering the therapy is preferably a mammal such as a non-primate (e.g., a cow, pig, horse, cat, dog, rodent, mouse, or rat) or a primate (e.g., a monkey such as a rhesus monkey or a cynomolgus monkey or a human). In one preferred embodiment, the subject is a human. In another embodiment, the subject is a human infant or a premature human infant. In yet another embodiment, the subject is a person suffering from an hOX40L-mediated disease.

[0675] Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., the antibodies of the present invention), including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies, receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)), and nucleic acids constructed as part of retroviruses or other vectors. Methods of administering prophylactic or therapeutic agents (e.g., the antibodies of the present invention) or pharmaceutical compositions include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal administration (e.g., intranasal and oral routes). In one specific embodiment, prophylactic or therapeutic agents (e.g., the antibodies of the present invention) or pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously. Prophylactic or therapeutic agents or compositions can be administered via any conventional route, such as by infusion or rapid concentration, through epithelial or mucosal linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Application can be systemic or localized. Alternatively, pulmonary administration can be used, for example, by using an inhaler or nebulizer and formulating a nebulizer. See, for example, U.S. Patents 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0676] In one specific embodiment, it is desirable to apply a preventative or therapeutic agent, or the pharmaceutical composition of the present invention, topically to the area requiring treatment. This can be achieved, for example, but not limited to, local infusion, surface application (e.g., via nasal spray), injection, or by means of an implant, which may be a porous, non-porous, or gelatinous material, including membranes such as silicone rubber membranes or fibers. Preferably, when administering the antibodies of the present invention, care must be taken to use materials that do not absorb the antibodies.

[0677] In another embodiment, the preventive or therapeutic agent or the composition of the present invention may be delivered in vesicles, especially liposomes (see Langer, 1990, Science 249: 1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; generally see sources ibid.).

[0678] In another embodiment, the preventive or therapeutic agent or the composition of the present invention can be delivered in a controlled-release or sustained-release system. In one embodiment, controlled-release or sustained-release can be achieved using a pump (see Langer, above; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, the controlled or sustained release of a preventive or therapeutic agent (e.g., the antibody of the present invention) or a composition of the present invention can be achieved using polymeric materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985, Science). 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253. Examples of polymers used in sustained-release formulations include, but are not limited to, poly(hydroxyethyl 2-methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolic acid (PLG), polyanhydride, N-vinylpyrrolidone, poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolic acid) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, storage-stable, sterile, and biodegradable.In another embodiment, the controlled-release or sustained-release system may be placed near the therapeutic target, i.e., the nasal passage or lungs, thus requiring only a portion of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, Vol. 2 above, pp. 115-138 (1984)). Langer (1990, Science 249: 1527-1533) provides a comprehensive overview of controlled-release systems. Any techniques known to those skilled in the art can be used to prepare sustained-release formulations comprising one or more antibodies of the present invention. See, for example, U.S. Patent No. 4,526,938, PCT Publication WO 91 / 05548, PCT Publication WO 96 / 20698, Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39: 179-189; Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50: 372-397; Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery, Proc. Int'l. Symp. ControlRel. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety.

[0679] In one specific embodiment, when the composition of the present invention is a nucleic acid encoding a preventive or therapeutic agent (e.g., the antibody of the present invention), it can be administered in vivo, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286) as part of a suitable nucleic acid expression vector and administering it to make it intracellular, or by direct injection, or by using microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or by coating it with lipids or cell surface receptors or transfection agents, or by administering it in combination with a homeobox-like peptide known to enter the cell nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), to promote the expression of the preventive or therapeutic agent encoded therein. Alternatively, the nucleic acid can be introduced into cells and expressed by homologous recombination into the host cell DNA.

[0680] In one specific embodiment, the composition of the present invention comprises one, two, or more antibodies or fragments of the present invention. In another embodiment, the composition of the present invention comprises one, two, or more antibodies or fragments of the present invention and a preventive or therapeutic agent other than the antibody of the present invention. Preferably, the agent is known for, or has been or is currently used for, the prevention, management, treatment, and / or improvement of hOX40L-mediated diseases. In addition to the preventive or therapeutic agent, the composition of the present invention also comprises a carrier.

[0681] The compositions of the present invention comprise bulk pharmaceutical compositions for use in preparing unit-dose pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient). In a preferred embodiment, the compositions of the present invention are pharmaceutical compositions. Such compositions comprise a preventive or therapeutically effective amount of one or more preventive or therapeutic agents (e.g., antibodies or other preventive or therapeutic agents of the present invention) and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is formulated for administration to a subject via a route suitable for administration.

[0682] In one specific implementation, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or medium that is administered with the therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, Pa. Such compositions will contain a prophylactic or therapeutically effective amount of antibody, preferably in purified form, along with an appropriate amount of carrier, to provide a form suitable for appropriate administration to the patient. The formulation should be appropriate for the mode of administration.

[0683] In a preferred embodiment, the composition is formulated according to a standard procedure suitable for intravenous administration of pharmaceutical compositions to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic buffers. If necessary, the composition may also include a solubilizer and a local anesthetic such as lignocaine to reduce pain at the injection site. However, such compositions can be administered via routes other than intravenous administration.

[0684] Typically, the components of the compositions of this invention are provided individually or mixed together in unit doses, for example, as lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent. When the composition is to be administered by infusion, it can be dispersed in an infusion bottle containing pharmaceutical-grade sterile water or saline. When the composition is to be administered by injection, a single ampoule of sterile water or saline for injection can be provided so that the components can be mixed prior to administration.

[0685] The present invention also specifies that the antibodies of the present invention be packaged in a sealed container, such as an ampoule or sachet, indicating the amount of antibody. In one embodiment, the antibody is provided in a sealed container as a dry sterile lyophilized powder or anhydrous concentrate and can be rehydrated, for example, with water or saline to an appropriate concentration for administration to a subject. Preferably, the antibody is provided in a sealed container as a dry sterile lyophilized powder in unit doses of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, or at least 3 mg, and more preferably at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody can be stored in its original container between 2 and 8°C and the antibody can be administered within 12 hours, preferably within 6 hours, 5 hours, 3 hours, or 1 hour after rehydration. In an alternative embodiment, the antibody is provided in liquid form in a closed container indicating the amount and concentration of the antibody. Preferably, the antibody in liquid form is provided in a closed container at a concentration of at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml, and more preferably at least 5 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, at least 70 mg / ml, at least 80 mg / ml, at least 90 mg / ml, or at least 100 mg / ml.

[0686] The compositions of the present invention can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0687] The amount of a therapeutic agent (e.g., the antibody of the present invention) or a composition of the present invention that will be effective in the prevention, management, treatment and / or remission of hOX40L-mediated diseases can be determined using standard clinical techniques.

[0688] Therefore, a concentration of about 0.1 μg / ml to about 450 μg / ml can be administered to a patient, and in some embodiments at least 0.1 μg / ml, at least 0.2 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.8 μg / ml, at least 1 μg / ml, at least 1.5 μg / ml, and more preferably at least 2 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml. Antibodies or compositions at serum titers of at least 30 μg / ml, at least 35 μg / ml, at least 40 μg / ml, at least 50 μg / ml, at least 75 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 200 μg / ml, at least 250 μg / ml, at least 300 μg / ml, at least 350 μg / ml, at least 400 μg / ml, or at least 450 μg / ml are intended for the prevention, management, treatment, and / or relief of hOX40L-mediated disease. Alternatively, in vivo assays may be used to help identify the optimal dose range. The precise dose used in the formulation will also depend on the route of administration and the severity of hOX40L-mediated disease, and should be determined based on the physician's judgment and the individual patient's condition.

[0689] The effective dose can be inferred from the dose-response curves obtained from in vitro or animal model testing systems.

[0690] For the antibodies of the present invention, the dosage administered to a patient is typically from 0.1 mg / kg to 100 mg / kg of patient body weight. In some embodiments, the dosage administered to a patient is from about 1 mg / kg to 75 mg / kg of patient body weight. Preferably, the dosage administered to a patient is between 1 mg / kg and 20 mg / kg of patient body weight, more preferably between 1 mg / kg and 5 mg / kg of patient body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to exogenous peptides. Therefore, lower doses of human antibodies and less frequent administration are often possible. Furthermore, the dosage and frequency of administration of the antibodies of the present invention can be reduced by modifications such as lipolysis to enhance antibody absorption and tissue penetration.

[0691] In one embodiment, the antibody or fragment of the present invention is administered 5, 4, 3, 2 times, or preferably once, at a dose of about 100 mg / kg or less, about 75 mg / kg or less, about 50 mg / kg or less, about 25 mg / kg or less, about 10 mg / kg or less, about 5 mg / kg or less, about 1 mg / kg or less, about 0.5 mg / kg or less, or about 0.1 mg / kg or less, to manage hOX40L-mediated disease. In some embodiments, the antibody of the present invention is administered about 1-12 times, wherein the dose is determined by a physician and may be administered (e.g.) weekly, every two weeks, monthly, every two months, every three months, etc., as necessary. In some embodiments, a lower dose (e.g., 1-15 mg / kg) may be administered more frequently (e.g., 3-6 times). In other embodiments, a higher dose (e.g., 25-100 mg / kg) may be administered less frequently (e.g., 1-3 times). However, as will be apparent to those skilled in the art, other dosages and regimens are readily determined and are within the scope of the present invention.

[0692] In one specific embodiment, the antibody or fragment of the present invention in a sustained-release formulation is administered to a subject, preferably to a human, at a dose of about 100 mg / kg, about 75 mg / kg or less, about 50 mg / kg or less, about 25 mg / kg or less, about 10 mg / kg or less, about 5 mg / kg or less, about 1 mg / kg or less, about 0.5 mg / kg or less, or about 0.1 mg / kg or less, to prevent, manage, treat, and / or improve hOX40L-mediated disease. In another specific embodiment, the antibody pills of the present invention, which are not in a sustained-release formulation, are administered to a subject, preferably to a human, at a dose of about 100 mg / kg, about 75 mg / kg or less, about 50 mg / kg or less, about 25 mg / kg or less, about 10 mg / kg or less, about 5 mg / kg or less, about 1 mg / kg or less, about 0.5 mg / kg or less, or about 0.1 mg / kg or less, to prevent, manage, treat, and / or improve hOX40L-mediated disease, and after a certain period of time, the subject is administered (e.g., nasally or intramuscularly) two, three, or four times (preferably once) the antibody of the present invention at a dose of about 100 mg / kg, about 75 mg / kg or less, about 50 mg / kg or less, about 25 mg / kg or less, about 10 mg / kg or less, about 5 mg / kg or less, about 1 mg / kg or less, about 0.5 mg / kg or less, or about 5 mg / kg or less. According to this implementation plan, a certain period of time can be 1 to 5 days, one week, two weeks or one month.

[0693] In some embodiments, a single dose of th...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to hOX40L and comprises: (i) a VH domain and a VL domain, wherein the VH domain comprises the HCDR1 sequence of SEQ ID NO: 36, the HCDR2 sequence of SEQ ID NO: 38, and the HCDR3 sequence of SEQ ID NO: 40 as defined by the IMGT nomenclature, and the VL domain comprises the LCDR1 sequence of SEQ ID NO: 50, the LCDR2 sequence of SEQ ID NO: 52, and the LCDR3 sequence of SEQ ID NO: 54 as defined by the IMGT nomenclature, or (ii) a VH domain and a VL domain, wherein the VH domain comprises the HCDR1 sequence of SEQ ID NO: 42, the HCDR2 sequence of SEQ ID NO: 44, and the HCDR3 sequence of SEQ ID NO: 46 as defined by the Kabat nomenclature, and the VL domain comprises the LCDR1 sequence of SEQ ID NO: 56, the LCDR2 sequence of SEQ ID NO: 58, and the LCDR3 sequence of SEQ ID NO: 60 as defined by the Kabat nomenclature, and further comprises a human heavy chain constant region and a human light chain constant region.

2. An antibody or antigen-binding fragment thereof that specifically binds to hOX40L and comprises a VH domain containing SEQ ID NO: 34 and a VL domain containing SEQ ID NO:

48.

3. An antibody or antigen-binding fragment thereof that specifically binds to hOX40L and comprises two copies of a heavy chain and two copies of a light chain, wherein the heavy chain comprises SEQ ID NO: 62 and the light chain comprises SEQ ID NO:

64.

4. An antibody or antigen-binding fragment thereof that specifically binds to hOX40L and consists of two copies of a heavy chain and two copies of a light chain, wherein the heavy chain consists of SEQ ID NO: 62 and the light chain consists of SEQ ID NO:

64.

5. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable excipient, diluent, or carrier.

6. A composition comprising the antibody or antigen-binding fragment thereof according to claim 2 and a pharmaceutically acceptable excipient, diluent, or carrier.

7. A composition comprising the antibody or antigen-binding fragment thereof according to claim 3 and a pharmaceutically acceptable excipient, diluent, or carrier.

8. A composition comprising the antibody or antigen-binding fragment thereof according to claim 4 and a pharmaceutically acceptable excipient, diluent, or carrier.

9. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-4 or a composition as claimed in any one of claims 5-8 in the manufacture of a medicament for the treatment or prevention of hOX40L-mediated diseases or conditions in humans, wherein the hOX40L-mediated diseases or conditions are selected from atherosclerosis, allograft rejection, colitis or autoimmune diseases or conditions selected from encephalomyelitis, diabetes or rheumatoid arthritis.

10. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-4 or a composition as claimed in any one of claims 5-8 in the manufacture of a medicament for the treatment or prevention of high IgE-related asthma.

11. Use as claimed in claim 9 or 10, wherein the medicament further comprises another therapeutic agent, which is independently selected from rapamycin, tacrolimus, cyclosporine, corticosteroids, methotrexate, mycophenolate mofetil, anti-CD28 antibodies, anti-IL12 / IL-23 antibodies, anti-CD20 antibodies, anti-CD30 antibodies, CTLA4-Fc molecules, CCR5 receptor antagonists, anti-CD40L antibodies, anti-VLA4 antibodies, anti-LFA1 antibodies, fludarabine, anti-CD52 antibodies, anti-CD45 antibodies, cyclophosphamide, antithymocyte globulin, anti-complement C5 antibodies, anti-a4b7 integrin antibodies, anti-IL6 antibodies, anti-IL2R antibodies, anti-CD25 antibodies, anti-TNFa / TNFa-Fc molecules and vorinostat.

12. Use as claimed in claim 11, wherein the another therapeutic agent is selected from sirolimus, methylprednisolone, ustekinumab, rituximab, brentuximab vedotin, abatacept, maraviroc, natalizumab, alemtuzumab, eculizumab, vedolizumab, tocilizumab, basiliximab, daclizumab, etanercept, adalimumab, infliximab, golimumab and certolizumab pegol.

13. Use as claimed in claim 11, wherein the antibody and the another therapeutic agent are in different compositions and / or are administered sequentially.

14. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-4 or a composition as claimed in any one of claims 5-8 in the manufacture of a medicament for administration to humans for the treatment or prevention of hOX40L-mediated diseases or conditions, wherein the hOX40L-mediated diseases or conditions are selected from atherosclerosis, allograft rejection, colitis or autoimmune diseases or conditions selected from encephalomyelitis, diabetes or rheumatoid arthritis, wherein the human is also administered a therapeutically effective amount of another therapeutic agent. Wherein the other therapeutic agent is independently selected from rapamycin, tacrolimus, cyclosporine, corticosteroids, methotrexate, mycophenolate mofetil, anti-CD28 antibody, anti-IL12 / IL-23 antibody, anti-CD20 antibody, anti-CD30 antibody, CTLA4-Fc molecule, CCR5 receptor antagonist, anti-CD40L antibody, anti-VLA4 antibody, anti-LFA1 antibody, fludarabine, anti-CD52 antibody, anti-CD45 antibody, cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibody, anti-α4β7 integrin antibody, anti-IL6 antibody, anti-IL2R antibody, anti-CD25 antibody, anti-TNFα / TNFα-Fc molecule, and vorinostat.

15. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1-4 or a composition according to any one of claims 5-8 in the manufacture of a medicament for administration to a human for the treatment or prevention of high IgE-related asthma, wherein the human is also administered a therapeutically effective amount of another therapeutic agent, Wherein the other therapeutic agent is independently selected from rapamycin, tacrolimus, cyclosporine, corticosteroids, methotrexate, mycophenolate mofetil, anti-CD28 antibody, anti-IL12 / IL-23 antibody, anti-CD20 antibody, anti-CD30 antibody, CTLA4-Fc molecule, CCR5 receptor antagonist, anti-CD40L antibody, anti-VLA4 antibody, anti-LFA1 antibody, fludarabine, anti-CD52 antibody, anti-CD45 antibody, cyclophosphamide, anti-thymocyte globulin, anti-complement C5 antibody, anti-α4β7 integrin antibody, anti-IL6 antibody, anti-IL2R antibody, anti-CD25 antibody, anti-TNFα / TNFα-Fc molecule, and vorinostat.

16. Use according to claim 14 or 15, wherein the other therapeutic agent is selected from sirolimus, methylprednisolone, ustekinumab, rituximab, brentuximab vedotin, abatacept, maraviroc, natalizumab, alemtuzumab, eculizumab, vedolizumab, tocilizumab, basiliximab, daclizumab, etanercept, adalimumab, infliximab, golimumab, and certolizumab pegol.

17. Use according to claim 14 or 15, wherein the antibody and the other therapeutic agent are in different compositions and / or are administered sequentially.

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