Anti-CD40 antibodies and methods of use

Abstract

To provide high affinity anti-CD40 monoclonal antibodies and related compositions, which may be used in any of a variety of therapeutic methods for treatment of cancer and other diseases.SOLUTION: The present invention provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a CD40 epitope set forth in any one or more of SEQ ID NOs: 196, 197, 199 and 202, for example. Also provided is an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Citation of Related Applications This application claims priority to U.S. Provisional Application No. 61 / 720,289, filed October 30, 2012, the entire disclosure of which is incorporated herein by reference.

[0002] Description of the Sequence Listing A sequence listing related to this application is provided in text format instead of a hard copy and is incorporated herein by reference. The text file name containing the sequence listing is APEX-016_01WO_ST25.txt. The text file is 92 KB, was created on October 30, 2013, and is being electronically submitted via EFS-Web.

[0003] Background Technical Field The present invention generally relates to anti-CD40 antibodies, compositions, and methods of using them. Such antibodies are useful, for example, in methods for treating various oncological diseases.

Background Art

[0004] Description of Related Art Most leukemias and lymphomas result from the malignant transformation of B-lineage cells. The expression of cell surface B-lineage-restricted antigens such as CD20 makes them attractive targets for antibody therapy. Antibody therapy has dramatically changed the management of patients with non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Since the approval of rituximab, the antibody alone or in combination with chemotherapy has significantly improved response rates, long-term outcomes, and quality of life (Chinn P, Braslawsky G, White C et al., Antibody therapy of non-Hodgkin’s B-cell lymphoma, Cancer Immunol Immunother 2003;52:257-280; Rastetter W, Molina A, White CA, Rituximab: Expanding role in therapy for lymphomas and autoimmune diseases, Annu Rev Med 2004;55:477-503). However, a significant number of patients exhibit either primary or acquired resistance to rituximab, indicating that the current approach targeting CD20 has limitations in clinical outcomes and the need to develop new immunotherapies for B-cell lymphomas and leukemias with different mechanisms of action such as anti-CD40 mAb, APX005 (Stolz C, Schuler M, Molecular mechanisms of resistance to Rituximab and pharmacologic strategies for its circumvention, Leukemia and lymphoma, 2009;50(6):873-885; Bello C, Sotomayor EM, Monoclonal antibodies for B-cell lymphomas: Rituximab and beyond, Hematology Am Soc Hematol Educ Program 2007;233-242; Dupire S, Coiffier B, Targeted treatment and new agents in suggests diffuse large B cell lymphoma, Int J Hematol 2010; June 18 (online).

[0005] The role of CD40 in the regulation of immune responses Full activation of T cells requires two distinct but synergistic signals. The first signal delivered by the T cell antigen receptor is provided by the antigen and MHC complex on the APC and is responsible for the specificity of the immune response. The second, co-stimulatory signal is provided by the interaction of CD28 with B7-1 (CD80) / B7-2 (CD86) and the interaction of CD40 with CD40L, and these interactions are required to initiate a full-scale T cell response. In the absence of co-stimulatory signals, T cells may become unresponsive (anergic) to antigen stimulation or may undergo programmed cell death (apoptosis) upon antigen stimulation.

[0006] CD40 is a member of the TNF receptor (TNFR) superfamily and is expressed mainly on B cells as well as on other antigen-presenting cells (APCs), such as dendritic cells and macrophages. CD40 ligand (CD40L) is expressed mainly by activated T cells.

[0007] The interaction between CD40 and CD40L serves as a costimulatory signal for T cell activation. CD40-CD40L engagement on resting B cells induces proliferation, immunoglobulin class switch, antibody secretion, and also plays a role in germinal center development and memory B cell survival, all of which are essential for the humoral immune response (Kehry MR, J Immunol 1996;156:2345-2348). Ligation of CD40L to CD40 on dendritic cells induces DC maturation as manifested by increased expression of costimulatory molecules such as the B7 family (CD80, CD86) and production of proinflammatory cytokines such as interleukin 12. These result in a potent T cell response (Stout, R.D., J. Suttles, 1996, Immunol. Today 17:487-492; Brendan O’Sullivan, Ranjeny Thomas, Critical Reviews in Immunology, 2003;23:83-107; Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber, J. Exp. Med., 1996;184:747-452).

[0008] CD40 signaling activates a number of pathways, including NF-kappaB (nuclear factor-kappaB), MAPK (mitogen-activated protein kinase), and STAT3 (signal transducer and activator of transcription-3) (Pype S et al., J Biol Chem, June 16, 2000; 275(24):18586-93), which regulate gene expression through the activation of activating proteins, c-Jun, ATF2 (activating transcription factor-2), and Rel transcription factor (Dadgostar H et al., Proc Natl Acad Sci U S A, February 5, 2002; 99(3):1497-502). TNFR receptor-associated factor adapter proteins (e.g., TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6) interact with this receptor and serve as mediators of signaling. Depending on the specific cell type, CD40 engagement results in a specific gene expression pattern. Genes activated in response to CD40 signaling include a very large number of cytokines and chemokines (IL-1, IL-6, IL-8, IL-10, IL-12, TNF-alpha, and macrophage inflammatory protein-1alpha (MIP1alpha)). In certain cell types, activation of CD40 can also result in the production of cytotoxic radicals (Dadgostar et al., supra), COX2 (cyclooxygenase-2), and NO (nitric oxide).

[0009] Role of CD40 in tumors CD40 is expressed not only by normal immune cells but also by many malignant cells. Specifically, CD40 is expressed in B-cell NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease (Uckun FM, Gajl-Peczalska K, Myers DE et al., Blood 1990;76:2449-2456; O’Grady JT, Stewart S, Lowrey J et al., Am J Pathol 1994;144:21-26), multiple myeloma (Pellat-Deceunynck C, Bataille R, Robillard N, Harousseau JL, Rapp MJ, Juge-Morineau N, Wijdenes J, Amiot M, Blood, 1994;84(8):2597-603), as well as carcinomas and malignant melanomas of the bladder, kidney, ovary, cervix, breast, lung, oropharynx (Young LS, Eliopoulos AG, Gallagher NJ et al., Immunol Today 1998;19:502-6; Ziebold JL, Hixon J, Boyd A et al., Arch Immunol Ther Exp(Warsz)2000;48:225-33; Gladue R, Cole S, Donovan C et al., J Clin Oncol 2006;24(18S):103s).

[0010] Ligation of CD40 on the surface of tumor cells often mediates a direct cytotoxic effect, resulting in tumor regression by apoptosis and necrosis (Grewal IS, Flavell RA, Annu Rev Immunol 1998;16:111-35; van Kooten C, Banchereau J, J Leukoc Biol 2000;67(1):2-17). The exact function of CD40 in tumor cells is unknown (Tong AW, Stone MJ, Cancer Gene Ther, 2003 10(1):1-13), but in vitro engagement of CD40 inhibits the growth of solid tumor cells and high-grade B-cell lymphoma cells (Magi Khalil and Robert H. Vonderheide, Update Cancer Ther 2007; 2(2):61-65; Young LS, Eliopoulos AG, Gallagher NJ, Dawson CW, Immunol Today 1998;19(11):502-6; Funakoshi S, Longo DL, Beckwith M et al., Blood 1994;83(10):2787-94; Hess S, Engelmann H, J Exp Med 1996;183(1):159-67; Eliopoulos AG, Dawson CW, Mosialos G et al., Oncogene 1996;13(10):2243-54; von Leoprechting A, van der Bruggen P, Pahl HL, Aruffo A, Simon JC, Cancer Res 1999;59(6):1287-94). These effects are in contrast to the proliferation induced after engagement of CD40 on non-neoplastic B cells and dendritic cells.

[0011] In addition to direct tumor inhibition, activation of CD40 signaling rescues the function of antigen-presenting cells in tumor-bearing hosts and elicits or restores an active immune response against tumor-associated antigens. CD40 agonists have been reported to overcome T cell tolerance in tumor-bearing mice, induce an effective cytotoxic T cell response against tumor-associated antigens, and enhance the efficacy of anti-tumor vaccines (Eliopoulos AG, Davies C, Knox PG et al., Mol Cell Biol 2000;20(15):5503-15; Tong AW, Papayoti MH, Netto G et al., Clin Cancer Res 2001;7(3):691-703).

[0012] CD40 as a Molecular Target CD40 is overexpressed on a wide range of malignant cells. The role of CD40 in tumor inhibition and immune system stimulation makes CD40 an attractive target for antibody-based immunotherapy (van Mierlo GJ, den Boer AT, Medema JP et al., Proc Natl Acad Sci U S A, 2002;99(8):5561-5566; French RR, Chan HT, Tutt AL, Glennie MJ, Nat Med, 1999;5(5):548-553). Anti-CD40 antibodies can act on cancer cells by a number of mechanisms: (i) antibody effector functions, such as ADCC, (ii) a direct cytotoxic effect on tumor cells, and (iii) activation of an anti-tumor immune response.

[0013] Antibodies Against CD40 for Therapeutic Development Several anti-CD40 antibodies have been reported to have potential as anti-tumor therapeutic agents. CP-870,893 is a fully human IgG2 CD40 agonist antibody developed by Pfizer. It has a K -10 of 3.48×10 DIt binds to CD40 but does not block the binding of CD40L (see, e.g., U.S. Patent No. 7,338,660). CP-870893 has not been shown to exhibit an ADCC effect, which is attributed to its IgG2 isotype, inter alia. Thus, this antibody acts as a CD40 agonist (i.e., it does not affect CD40L binding), induces apoptosis-promoting signaling, and activates DC and immune surveillance mechanisms. However, this antibody does not mediate ADCC.

[0014] HCD122 is a fully human IgG1 CD40 antagonist antibody developed by Novartis. It binds to CD40 with a K -10 of 5.1×10 D M and blocks the binding of CD40 to CD40L, inhibiting CD40 ligand-induced signaling and biological effects on B cells as well as certain primary CLL and MM cells (Tai YT et al., Cancer Res, July 1, 2005; 65(13):5898-906; Luqman M, Klabunde S et al.: Blood 112:711-720, 2008). The major mechanism of action for its antitumor effect in vivo is ADCC (Long L et al., 2005 IMF Oral Presentation and Abstract No.3; Blood 2004, 104(11, Part 1): Abst 3281). Due to its antagonist characteristics, this antibody cannot directly induce a CD40-mediated antitumor immune response.

[0015] SGN-40 is a humanized IgG1 antibody developed by Seattle Genetics from the mouse antibody clone S2C6, produced using a human bladder cancer cell line as an immunogen. It binds to CD40 with a K -9 of 1.0×10 DIt binds to CD40 and acts by enhancing the interaction between CD40 and CD40L, thus exhibiting a partial agonist effect (Francisco JA et al., Cancer Res, 60:3225 - 31, 2000). SGN - 40 delivers growth inhibitory and apoptotic signals to a panel of B - lymphoma lines of high - grade non - Hodgkin lymphoma and MM cell origin (Tai YT, Catley LP, Mitsiades CS et al., Cancer Res 2004;64(8):2846 - 2852). In vitro and in vivo studies suggest that both apoptotic signaling by ADCC and antibody effector functions contribute to the antitumor activity of SGN - 40 (Law CL, Gordon KA, Collier J et al.: Cancer Res 2005;65:8331 - 8338). Recent studies suggest that the antitumor activity of SGN - 40 significantly depends on the Fc interaction with effector cells and that macrophages are the major effectors contributing to its therapeutic activity (Oflazoglu E et al., Br J Cancer, January 13, 2009;100(1):113 - 7, Epub December 9, 2008). Since SGN - 40 is a partial agonist and requires CD40L expressed on T cells, there may be a limit to the ability of SGN - 40 to fully boost the antitumor immune response.

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Summary of the Invention

Problems to be Solved by the Invention

[0018] Therefore, there is still a need in the art for a novel immunotherapeutic agent that targets CD40, acts as an agonist against this target, activates dendritic cells and the immune surveillance mechanism, activates ADCC, and thereby provides improved anti-cancer properties.

Means for Solving the Problems

[0019] Brief Summary One aspect of the present disclosure provides an isolated antibody that binds to human CD40, or an antigen-binding fragment thereof, comprising (i) a heavy-chain variable region comprising a VHCDR1 region shown in SEQ ID NO: 3, a VHCDR2 region shown in SEQ ID NO: 4, and a VHCDR3 region shown in SEQ ID NO: 5, and (ii) a light-chain variable region comprising a VLCDR1 region shown in SEQ ID NO: 6, a VLCDR2 region shown in SEQ ID NO: 7, and a VLCDR3 region shown in SEQ ID NO: 8; or a variant of the antibody, or an antigen-binding fragment thereof, comprising a heavy-chain variable region and a light-chain variable region that are identical to the heavy-chain variable region and the light-chain variable region of (i) and (ii) except for 8 or fewer amino acid substitutions in the CDR regions. In one embodiment of the antibody disclosed herein, the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1. In a further embodiment, the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2.

[0020] Another aspect of the present disclosure provides an isolated antibody that binds to human CD40, or an antigen-binding fragment thereof, comprising a heavy-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1. In one embodiment of this aspect, the isolated antibody, or an antigen-binding fragment thereof, comprises a light-chain variable region having an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2. In a further embodiment of this aspect, the isolated antibody, or an antigen-binding fragment thereof, comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.

[0021] A further aspect of the present disclosure provides an isolated antibody that binds to human CD40 and includes a light chain variable region that includes the amino acid sequence set forth in SEQ ID NO: 2, or an antigen-binding fragment thereof. In one embodiment of this aspect, the isolated antibody, or antigen-binding fragment thereof, includes a heavy chain variable region that includes an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0022] In certain embodiments, the isolated antibodies disclosed herein are humanized. Illustrative humanized antibody variable regions are shown in the VH region amino acid sequence of SEQ ID NO: 9 and the VL region amino acid sequence of SEQ ID NO: 10.

[0023] In one embodiment, the isolated antibodies disclosed herein can be single-chain antibodies, ScFv, monovalent antibodies lacking a hinge region, minibodies, Fab, Fab’ fragments, or F(ab’)2 fragments. In certain embodiments, the antibodies herein are whole antibodies.

[0024] In another embodiment, the isolated antibodies described herein include human IgG constant domains, such as, but not limited to, the IgG1 CH1 domain or the IgG1 Fc region.

[0025] A further embodiment of the present disclosure provides an isolated antibody, or antigen-binding fragment thereof, that competes with the anti-CD40 antibodies described herein for binding to human CD40.

[0026] In one aspect of the present disclosure, the isolated antibody or antigen-binding fragment thereof that binds CD40 binds with a KD of 0.96 nM or less. In a further embodiment, the isolated antibody or antigen-binding fragment thereof that binds CD40 binds with a Kd between 1.1 nM and 0.90 nM. In a further embodiment, the isolated antibody or antigen-binding fragment thereof that binds CD40 binds with a Kd of about 1.2, 1.1, 1.0, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0.85, or about 0.80 nM. In another embodiment, the antibody binds CD40 with a Kd of about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, or 1.3 nM.

[0027] In a further aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof as described herein that blocks the binding of CD40 to CD40L; is a CD40 agonist; activates antigen-presenting cells; stimulates cytokine release from antigen-presenting cells; induces tumor cell apoptosis; inhibits tumor cell proliferation; kills tumor cells by inducing an effector function selected from the group consisting of antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis; stimulates an anti-tumor T cell response; reduces established tumors; inhibits rituximab-resistant tumors; or is a combination of any one or more of the foregoing.

[0028] Another aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to CD40 and comprises (i) a heavy chain variable region comprising any one of the VH CDR1, VH CDR2, and VH CDR3 of the VH region shown in FIG. 16, and (ii) a light chain variable region comprising the corresponding VL CDR1, VL CDR2, and VL CDR3 regions of any one of the VL regions shown in FIG. 16, or a variant of said antibody or antigen-binding fragment thereof that comprises heavy chain and light chain variable regions that are identical to the heavy chain and light chain variable regions of (i) and (ii) except for 8 or fewer amino acid substitutions in said CDR regions.

[0029] Another aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to CD40 and comprises a heavy chain variable region comprising any one of the VH regions shown in FIG. 16. In one embodiment, such an antibody further comprises a light chain variable region comprising an amino acid sequence having at least 90% identity to the corresponding VL region as shown in FIG. 16. In another embodiment, such an antibody or antigen-binding fragment thereof further comprises the corresponding light chain variable region as shown in FIG. 16.

[0030] Another aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to CD40 and comprises a light chain variable region comprising any one of the VL regions shown in FIG. 16. In one embodiment, such an antibody further comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the corresponding VH region as shown in FIG. 16. In another embodiment, such an antibody or antigen-binding fragment thereof further comprises the corresponding heavy chain variable region as shown in FIG. 16.

[0031] The present disclosure also provides an isolated polynucleotide encoding the isolated antibody or antigen-binding fragment thereof disclosed herein.

[0032] The present disclosure also provides a composition comprising a physiologically acceptable carrier and a therapeutically effective amount of the anti-CD40 antibody or antigen-binding fragment thereof described herein.

[0033] Another aspect of the present disclosure is a method for treating a patient having cancer, the method comprising administering to the patient a composition comprising a physiologically acceptable carrier and a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof described herein, thereby treating the cancer. In certain embodiments, the cancer is associated with abnormal CD40 expression. In a further aspect, the cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, and rituximab-resistant NHL and leukemia.

[0034] Another aspect of the present disclosure is a method for treating a patient having cancer and / or an autoimmune disease and / or an inflammatory disease, the method comprising administering to the patient a composition comprising a physiologically acceptable carrier and a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof described herein, thereby treating the patient having the autoimmune disease and the inflammatory disease.

[0035] Another aspect of the present disclosure is a method for improving symptoms in a patient having cancer and / or an autoimmune disease and / or an inflammatory disease, the method comprising administering to the patient a composition comprising a physiologically acceptable carrier and a therapeutically effective amount of an anti-CD40 antibody or antigen-binding fragment thereof described herein, thereby improving the symptoms in the patient having cancer and / or the autoimmune disease and / or the inflammatory disease.

[0036] Another aspect of the present disclosure provides an isolated antibody that binds to human CD40 and includes a heavy chain variable region that includes the amino acid sequence shown in SEQ ID NO: 11, or an antigen-binding fragment thereof. In one embodiment, the isolated antibody that binds to human CD40, or an antigen-binding fragment thereof, includes a heavy chain variable region that includes the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region that includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 22, or includes a light chain that includes the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the isolated antibody described herein includes a light chain as set forth in SEQ ID NO: 22 and a heavy chain variable region that includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 11.

[0037] A further aspect of the present disclosure provides an isolated antibody that binds to human CD40 and includes a heavy chain variable region that includes the amino acid sequence shown in SEQ ID NO: 13, or an antigen-binding fragment thereof. In one embodiment, the antibody includes a heavy chain variable region that includes the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region that includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 24. In one embodiment, the light chain includes the amino acid sequence shown in SEQ ID NO: 24.

[0038] A further aspect of the present disclosure provides an isolated antibody that binds to human CD40 and includes a light chain variable region that includes the amino acid sequence shown in SEQ ID NO: 24, or an antigen-binding fragment thereof. In one embodiment, the antibody includes a light chain variable region that includes the amino acid sequence shown in SEQ ID NO: 24 and a heavy chain variable region that includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 13.

[0039] In certain embodiments, the isolated antibody that binds CD40, or an antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, the isolated antibody that binds CD40 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 28. In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0040] Another aspect of the disclosure provides an isolated antibody that binds human CD40, or an antigen-binding fragment thereof, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28. In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds human CD40 comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0041] Another aspect of the disclosure provides an isolated antibody that binds human CD40, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19. In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds human CD40 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 30. In one particular embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0042] Yet another aspect of the disclosure provides an isolated antibody that binds human CD40, or an antigen-binding fragment thereof, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30. In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds human CD40 comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain variable region comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19.

[0043] Another aspect of the present disclosure provides an isolated antibody that binds to human CD40, or an antigen-binding fragment thereof, comprising a heavy-chain variable region comprising CDRs of the heavy-chain variable region and a light-chain variable region comprising CDRs of the corresponding light-chain variable region, wherein the CDRs are as shown in FIG. 16.

[0044] Another aspect of the invention provides an isolated antibody, or an antigen-binding fragment thereof, that binds to a CD40 epitope shown in any one or more of SEQ ID NOs: 196, 197, 199, and 202. In one particular embodiment, the isolated antibody, or an antigen-binding fragment thereof, binds to the CD40 epitope shown in SEQ ID NO: 202. In another embodiment, the isolated antibody, or an antigen-binding fragment thereof, that binds to the CD40 epitope shown in SEQ ID NO: 196, 197, 199, or 202 does not comprise the CDRs shown in SEQ ID NOs: 3-8. In one embodiment, the isolated antibody that binds to a CD40 epitope shown in any one or more of SEQ ID NOs: 196, 197, 199, and 202 comprises: (i) a heavy-chain variable region comprising a VHCDR1 region shown in SEQ ID NO: 3, a VHCDR2 region shown in SEQ ID NO: 4, and a VHCDR3 region shown in SEQ ID NO: 5; and (ii) a light-chain variable region comprising a VLCDR1 region shown in SEQ ID NO: 6, a VLCDR2 region shown in SEQ ID NO: 7, and a VLCDR3 region shown in SEQ ID NO: 8; or a variant of the antibody, or an antigen-binding fragment thereof, comprises a heavy-chain variable region and a light-chain variable region that are identical to the heavy-chain variable region and the light-chain variable region of (i) and (ii) except for 8 or fewer amino acid substitutions in the CDR regions; the isolated antibody, or an antigen-binding fragment thereof, further comprises an Fc region modified such that the isolated antibody, or an antigen-binding fragment thereof, has an increased FcγRIIB binding affinity, increased ADCC, or increased anti-CD40 agonist activity, or a combination thereof, compared to the isolated antibody, or an antigen-binding fragment thereof, that comprises the unmodified version of the Fc region. In this regard, the Fc region may comprise an S267E mutation.

[0045] The present disclosure also provides an isolated polynucleotide encoding an isolated antibody or an antigen-binding fragment thereof that binds to a CD40 epitope shown by any one or more of SEQ ID NOs: 196, 197, 199, and 202; an expression vector (expection vector) comprising the isolated polynucleotide; and an isolated host cell comprising such a vector.

[0046] The present disclosure also provides a composition comprising a physiologically acceptable carrier and a therapeutically effective amount of the isolated antibody or an antigen-binding fragment thereof that binds to a CD40 epitope shown by any one or more of SEQ ID NOs: 196, 197, 199, and 202.

[0047] The present disclosure further provides a method for treating or ameliorating cancer in a patient, the method comprising administering to the patient a composition comprising the antibody that binds to a CD40 epitope shown by any one or more of SEQ ID NOs: 196, 197, 199, and 202 as described herein. In this regard, the cancer can be non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinoma, malignant melanoma, or rituximab-resistant NHL or leukemia.

[0048] The present disclosure also provides a method for ameliorating the symptoms of an autoimmune disease or an inflammatory disease in a patient by administering to the patient a composition comprising the antibody that binds to a CD40 epitope shown by any one or more of SEQ ID NOs: 196, 197, 199, and 202. In certain embodiments, for example, the following are provided: (Item 1) An isolated antibody or an antigen-binding fragment thereof that binds to a CD40 epitope shown by any one or more of SEQ ID NOs: 196, 197, 199, and 202. (Item 2) The isolated antibody, or antigen-binding fragment thereof, according to item 1, wherein the isolated antibody, or antigen-binding fragment thereof, binds to the CD40 epitope shown in SEQ ID NO: 202. (Item 3) The isolated antibody, or antigen-binding fragment thereof, according to item 1 or item 2, wherein the antibody, or antigen-binding fragment thereof, does not contain the CDRs shown in SEQ ID NOs: 3-8. (Item 4) The isolated antibody, or antigen-binding fragment thereof, comprises: (i) a heavy chain variable region comprising a VHCDR1 region shown in SEQ ID NO: 3, a VHCDR2 region shown in SEQ ID NO: 4, and a VHCDR3 region shown in SEQ ID NO: 5; and (ii) a light chain variable region comprising a VLCDR1 region shown in SEQ ID NO: 6, a VLCDR2 region shown in SEQ ID NO: 7, and a VLCDR3 region shown in SEQ ID NO: 8; or except for 8 or fewer amino acid substitutions in the CDR regions, comprises a heavy chain variable region and a light chain variable region that are identical to the heavy chain variable region and the light chain variable region of (i) and (ii), which is a variant of the antibody, or an antigen-binding fragment thereof. The isolated antibody, or antigen-binding fragment thereof, further comprises an Fc region modified such that the isolated antibody, or antigen-binding fragment thereof, has increased FcγRIIB binding affinity, increased ADCC, or increased anti-CD40 agonist activity, or a combination thereof, compared to the isolated antibody, or antigen-binding fragment thereof, that contains an unmodified version of the Fc region. The isolated antibody according to item 1. (Item 5) The isolated antibody, or antigen-binding fragment thereof, according to item 4, wherein the Fc region is modified by an S267E mutation. (Item 6) The isolated antibody according to item 1, which is humanized. (Item 7) The isolated antibody according to item 1, which is selected from the group consisting of a single-chain antibody, ScFv, a monovalent antibody lacking a hinge region, and a minibody. (Item 8) The isolated antibody according to item 1, which is a Fab fragment or a Fab' fragment. (Item 9) The isolated antibody according to item 1, which is an F(ab’)2 fragment. (Item 10) The isolated antibody according to item 1, which is the whole antibody. (Item 11) The isolated antibody according to item 1, which contains a human IgG constant domain. (Item 12) The isolated antibody according to item 11, wherein the IgG constant domain contains an IgG1 CH1 domain. (Item 13) The isolated antibody according to item 11, wherein the IgG constant domain contains an IgG1 Fc region. (Item 14) The isolated antibody according to item 13, wherein the Fc region of the IgG1 has an S267E substitution. (Item 15) An isolated antibody, or an antigen-binding fragment thereof, that competes with the antibody according to item 1 for binding to CD40. (Item 16) An isolated polynucleotide encoding the isolated antibody or an antigen-binding fragment thereof according to any one of the foregoing items. (Item 17) An expression vector containing the isolated polynucleotide according to item 16. (Item 18) An isolated host cell containing the vector according to item 17. (Item 19) A composition comprising a physiologically acceptable carrier and a therapeutically effective amount of the isolated antibody or an antigen-binding fragment thereof according to any one of the foregoing items. (Item 20) A method for treating or ameliorating the symptoms of cancer in a patient, comprising administering to the patient the composition according to item 19, thereby treating or ameliorating the symptoms of the cancer. (Item 21) The method according to item 20, wherein the cancer is selected from the group consisting of non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, bladder carcinoma, kidney carcinoma, ovarian carcinoma, cervical carcinoma, breast carcinoma, lung carcinoma, nasopharyngeal carcinoma, malignant melanoma, and rituximab-resistant NHL and leukemia. (Item 22) A method for improving the symptoms of an autoimmune disease in a patient, comprising administering to the patient the composition according to item 20, thereby improving the symptoms of the autoimmune disease. (Item 23) A method for improving the symptoms of an inflammatory disease in a patient, comprising administering to the patient the composition according to item 20, thereby improving the symptoms of the inflammatory disease.

[0049] Brief Description of the Sequences SEQ ID NO: 1 is the amino acid sequence of the VH region of the R-8 rabbit anti-CD40 antibody.

[0050] SEQ ID NO: 2 is the amino acid sequence of the VL region of the R-8 rabbit anti-CD40 antibody.

[0051] SEQ ID NO: 3 is the amino acid sequence of the VHCDR1 region of the R-8 rabbit anti-CD40 antibody.

[0052] SEQ ID NO: 4 is the amino acid sequence of the VHCDR2 region of the R-8 rabbit anti-CD40 antibody.

[0053] SEQ ID NO: 5 is the amino acid sequence of the VHCDR3 region of the R-8 rabbit anti-CD40 antibody.

[0054] SEQ ID NO: 6 is the amino acid sequence of the VLCDR1 region of the R-8 rabbit anti-CD40 antibody.

[0055] SEQ ID NO: 7 is the amino acid sequence of the VLCDR2 region of the R-8 rabbit anti-CD40 antibody.

[0056] SEQ ID NO: 8 is the amino acid sequence of the VLCDR3 region of the R-8 rabbit anti-CD40 antibody.

[0057] SEQ ID NO: 9 is the amino acid sequence of the VH region of APX005, the humanized version of the R-8 rabbit anti-CD40 antibody, without a signal peptide.

[0058] SEQ ID NO: 10 is the amino acid sequence of the VL region of APX005, the humanized version of the R-8 rabbit anti-CD40 antibody, without a signal peptide.

[0059] SEQ ID NOs: 11-21 and 33-44 are the heavy chain amino acid sequences of rabbit anti-CD40 antibody candidates that showed functional activity (see Figure 16).

[0060] SEQ ID NOs: 22-32 and 45-56 are the light chain amino acid sequences of rabbit anti-CD40 antibody candidates that showed functional activity (see Figure 16).

[0061] SEQ ID NOs: 57-79 are the VHCDR1 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0062] SEQ ID NOs: 80-102 are the VHCDR2 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0063] SEQ ID NOs: 103-125 are the VHCDR3 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0064] SEQ ID NOs: 126-148 are the VLCDR1 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0065] SEQ ID NOs: 149-171 are the VLCDR2 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0066] SEQ ID NOs: 172-194 are the VLCDR3 amino acid sequences for the anti-CD40 antibody shown in Figure 16.

[0067] SEQ ID NO: 195 is the amino acid sequence of the human IgG1 heavy chain constant region containing the Fc region with an S267E substitution.

[0068] SEQ ID NO: 196 is amino acids 92 - 107 of human CD40, identified as an epitope bound by the APX005 antibody.

[0069] SEQ ID NO: 197 is amino acids 125 - 144 of human CD40, identified as an epitope bound by the APX005 antibody.

[0070] SEQ ID NO: 198 is the amino acid sequence of human CD40.

[0071] SEQ ID NO: 199 is the amino acid sequence of the CD40 CLIPS peptide from residues 84 - 102 of CD40.

[0072] SEQ ID NO: 200 is the amino acid sequence of residues 84 - 102 of CD40 with serine substitutions in place of the cysteine residues at positions 91 and 96.

[0073] SEQ ID NO: 201 is amino acid residues 122 - 125 of human CD40.

[0074] SEQ ID NO: 202 is amino acid residues 92 - 102 of human CD40, bound by the APX005 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0075]

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Mode for Carrying Out the Invention

[0076] Detailed Description The present disclosure relates to antibodies that specifically bind to CD40 and antigen-binding fragments thereof, in particular, antibodies having specific epitope specificities and functional properties. One embodiment of the invention encompasses specific humanized antibodies and fragments thereof that are capable of binding to CD40 and function as CD40 agonists by inducing / augmenting CD40-mediated downstream cell signaling and biological effects. In more specific embodiments of the invention, the antibodies described herein bind specifically to CD40 with a very high affinity, e.g., at least between 980 picomolar and 950 picomolar, at least between 970 picomolar and 950 picomolar, and in certain embodiments with an affinity of 960 picomolar. The antibodies described herein, among other several properties, induce CD40 signaling in tumor cells; activate dendritic cells and the immune surveillance mechanism; activate antibody-dependent cell cytotoxicity (ADCC) against tumor cells; block the binding of CD40 to CD40L; have CD40 agonist activity; activate antigen-presenting cells; stimulate cytokine release from antigen-presenting cells; induce tumor cell apoptosis; inhibit tumor cell proliferation; kill tumor cells by inducing effector functions including, but not limited to, ADCC, CDC, and ADCP; stimulate anti-tumor T cell responses; reduce established tumors; and inhibit rituximab-resistant tumors. The antibodies described herein can have or induce any one or more combinations of these properties or activities.

[0077] Embodiments of the present invention relate to the use of anti-CD40 antibodies or antigen-binding fragments thereof for the diagnosis, assessment, and treatment of diseases and injuries associated with CD40 or its aberrant expression. The subject antibodies are used in the treatment or prevention of cancer (including, but not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, and rituximab-resistant NHL and leukemia), autoimmune diseases, and inflammatory diseases, among a number of other diseases.

[0078] The practice of the present invention will, unless otherwise indicated to the contrary, utilize conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art, and many of these methods are described below for purposes of illustration. Such techniques are well explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vols. I and II (D. Glover ed.); Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid Hybridization (B. Hames and S. Higgins eds., 1985); Transcription and Translation (B. Hames and S. Higgins eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0080] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps and not the exclusion of any other integer or step or group of integers or steps.

[0081] Each embodiment described in this specification is applicable to every other embodiment described herein unless specifically stated otherwise.

[0082] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques can be performed according to the manufacturer's specifications, or as commonly practiced in the art, or as described herein. Generally, these techniques and procedures, as well as related techniques and procedures, can be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. Unless otherwise defined, nomenclature used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, as well as experimental procedures and techniques of the above chemistry, are those well known and commonly used in the art. Standard techniques can be used for recombinant techniques, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0083] Embodiments of the invention relate to antibodies that bind to CD40. Specifically, the antibodies described herein specifically bind to CD40 with unexpectedly high affinity, enhance CD40 signaling activity, activate the immune system, activate ADCC, and have therapeutic utility in the treatment of diseases associated with abnormal expression of CD40.

[0084] The sequences of the exemplary antibodies, or their antigen-binding fragments, or complementarity-determining regions (CDRs) are shown in SEQ ID NOs: 1 to 194.

[0085] As is well known in the art, an antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., by at least one epitope recognition site, and the epitope recognition site is located within the variable region of the immunoglobulin molecule. As used herein, this term includes not only intact polyclonal or monoclonal antibodies, but also their fragments (e.g., dAb, Fab, Fab’, F(ab’)2, Fv), single-chain (ScFv), their synthetic variants, naturally occurring variants, fusion proteins containing antibody portions having antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified form of immunoglobulin molecule containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. “Diabodies,” i.e., multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993) are also a special form of antibodies contemplated herein. Minibodies containing scFv linked to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, E.S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0086] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that binds to a target antigen, particularly CD40. In this context, the antigen-binding fragments of the antibodies described herein may include one, two, three, four, five, or all six CDRs of the VH and VL sequences shown herein from an antibody that binds CD40. The antigen-binding fragments of the CD40-specific antibodies described herein are capable of binding to CD40. In certain embodiments, the antigen-binding fragment, or an antibody containing the antigen-binding fragment, prevents or inhibits the binding of CD40L to CD40. In certain embodiments, the antigen-binding fragment specifically binds to human CD40 and / or enhances or modulates the biological activity of human CD40. Such biological activities include, but are not limited to, cell signaling and activation of dendritic cells.

[0087] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antibody and, in addition, can be used in an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen can have one or more epitopes.

[0088] The term "epitope" includes any determinant capable of specific binding to an immunoglobulin or T cell receptor, preferably a polypeptide determinant. An epitope is the region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface atomic groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind to an antigen when the antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. The antibody is said to specifically bind to the antigen when the equilibrium dissociation constant is ≤ 10 -7 or 10 -8 M. In some embodiments, the equilibrium dissociation constant is ≤ 10 -9 M or ≤ 10 -10It may be M.

[0089] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise sets of heavy and light chain complementarity-determining regions (CDRs) inserted between sets of heavy and light chain framework regions (FRs), wherein said FR sets support said CDRs and define the spatial relationship of said CDRs to one another. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site comprises six CDRs, including a CDR set from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystal structures of numerous antigen-antibody complexes have demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, and that the most extensive antigen contacts are with the heavy chain CDR3. Thus, said molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0090] As used herein, the term "FR set" refers to four contiguous amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues can contact the bound antigen; however, the FRs, particularly the FR residues immediately adjacent to the CDRs, are primarily responsible for the folding of the V region into the antigen-binding site. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. As the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FRs that influence the CDR loop shape folded into certain "canonical" structures, regardless of the exact CDR amino acid sequence. Further, it is known that certain FR residues are involved in non-covalent domain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0091] The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, 1987, and the current version thereof now available on the Internet (immuno.bme.nwu.edu).

[0092] "Monoclonal antibody" refers to a population of antibodies of the same kind, and the monoclonal antibody is composed of amino acids (naturally occurring or not) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific and are directed against a single epitope. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (e.g., Fab, Fab’, F(ab’)2, Fv), single-chain (ScFv), their variants, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified form of immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind to an epitope. It is not intended to be limited with respect to the antibody source or the method of making the antibody (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). This term includes whole immunoglobulins as well as fragments such as those described under the definition of "antibody" above.

[0093] The proteolytic enzyme papain preferentially cleaves the IgG molecule to yield several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer that includes an intact antigen-binding site. The enzyme pepsin can cleave the IgG molecule to yield several fragments including an F(ab’)2 fragment that contains both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be generated by the preferential proteolytic cleavage of IgM and rarely IgG or IgA immunoglobulin molecules. However, more commonly, Fv fragments are obtained using recombinant techniques known in the art. The Fv fragment contains a non-covalently linked V H ::V L heterodimer that retains most of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0094] In certain embodiments, single-chain Fv or scFV antibodies may be contemplated. For example, kappa bodies (Ill et al., Prot. Eng. 10:949-57 (1997)); minibodies (Martin et al., EMBO J 13:5305-9 (1994)); diabodies (Holliger et al., PNAS 90:6444-8 (1993)); or Janusins (Traunecker et al., EMBO J 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques according to the teachings of this application with respect to the selection of antibodies having the desired specificity. In still other embodiments, bispecific or chimeric antibodies comprising the ligands of the present disclosure can be made. For example, a chimeric antibody can include CDRs and frameworks from different antibodies, while a bispecific antibody that specifically binds to CD40 with one binding domain and to a second molecule with a second binding domain can be generated. These antibodies can be produced by recombinant molecular biology techniques or can be physically conjugated to each other.

[0095] A single-chain Fv (sFv) polypeptide is a covalently linked V H -encoding gene and a V L -encoding gene expressed from a gene fusion that includes a linker encoding a peptide, which is a covalently linked V H ::V L heterodimer. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods for identifying chemical structures for converting naturally aggregated - but chemically separated - light and heavy polypeptide chains from antibody V regions into sFv molecules that will fold into a three-dimensional structure substantially similar to that of the antigen-binding site have been described. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al. and U.S. Patent No. 4,946,778 to Ladner et al.

[0096] In certain embodiments, the CD40-binding antibodies described herein are in the form of diabodies. A diabody is a multimer of polypeptides, each polypeptide comprising a first domain that includes a binding region of an immunoglobulin light chain and a second domain that includes a binding region of an immunoglobulin heavy chain, wherein the two domains are linked (e.g., by a peptide linker) but are multimers that cannot associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of a first domain of one polypeptide within the multimer and a second domain of another polypeptide within the multimer (WO 94 / 13804 pamphlet).

[0097] The dAb fragment of an antibody consists of the VH domain (Ward, E.S. et al., Nature 341, 544-546 (1989)).

[0098] When using bispecific antibodies, these can be produced in various ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), for example, they can be conventional bispecific antibodies that can be prepared chemically or from hybrid hybridomas, or they can be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed using only the variable domains without the Fc region to potentially reduce the influence of anti-idiotypic reactions.

[0099] Bispecific diabodies are also particularly useful compared to whole bispecific antibodies, because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities can be readily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody, for example the arm with specificity for antigen X, is to be kept constant, libraries can be created in which the other arm is varied and antibodies with the appropriate specificity are selected. Whole bispecific antibodies can be produced by knobs-into-holes engineering (J.B.B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

[0100] In certain embodiments, the antibodies described herein can be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, for example, U.S. Patent Application Publication No. 20090226421). This unique antibody technology creates a stable, smaller antibody format that has a therapeutic window that is expected to be longer than current, smaller antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. A full human IgG4 antibody can be modified by deletion of the hinge region of the antibody to obtain a half-molecule fragment with different stability compared to the corresponding intact IgG4 (GenMab, Utrecht). By splitting the IgG4 molecule in half, only one region on the UniBody® that can bind to a cognate antigen (e.g., a disease target) remains, and thus the UniBody® binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding cannot stimulate and grow cancer cells as can be seen by using a bivalent antibody with the same antigen specificity, and thus the UniBody® technology can provide treatment options for some types of cancer that can be resistant to treatment with conventional antibodies. The small size of the UniBody® can be highly beneficial when treating some forms of cancer, allowing for better molecular distribution on larger solid tumors and, according to some, increasing efficacy.

[0101] In certain embodiments, the antibodies of the present disclosure may take the form of nanobodies. Nanobodies are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see, e.g., U.S. Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeast (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Patent No. 6,838,254)). This production process is scalable, and nanobodies have been produced in amounts of several kilograms. Nanobodies can be formulated as ready-to-use solutions with long shelf lives. The nanoclone method (see, e.g., WO 06 / 079372) is a unique method for generating nanobodies against a desired target based on the automated high-throughput selection of B cells.

[0102] In certain embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein are humanized. This refers to chimeric molecules generally prepared using recombinant techniques that have antigen-binding sites derived from immunoglobulins from non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of human immunoglobulins. The antigen-binding site may include full variable domains fused to constant domains or may include only CDRs grafted into appropriate framework regions within the variable domains. The epitope-binding site may be wild-type or may be modified by one or more amino acid substitutions. This removes the constant region as an immunogen in human individuals, but the potential for an immune response to the foreign variable region remains (LoBuglio, A.F. et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). The exemplary humanization methods of anti-CD40 antibodies disclosed herein include the methods described in U.S. Patent No. 7,462,697. Exemplary humanized antibodies according to certain embodiments of the invention include the humanized sequences provided in SEQ ID NOs: 9 and 10.

[0103] Another approach focuses not only on providing human-derived constant regions but also on modifying the variable regions to make them as close as possible to the human form. The variable regions of both the heavy and light chains are diverse depending on the epitope in question and contain three complementarity-determining regions (CDRs) that determine binding ability, with four framework regions (FRs) adjacent to the CDRs, the FRs being relatively conserved in a given species and putatively providing a scaffold for the CDRs, which is known. When preparing a non-human antibody against a particular epitope, the variable region can be "remodeled" or "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported by Sato, K. et al., (1993) Cancer Res 53:851-856; Riechmann, L. et al., (1988) Nature 332:323-327; Verhoeyen, M. et al., (1988) Science 239:1534-1536; Kettleborough, C.A. et al., (1991) Protein Engineering 4:773-3783; Maeda, H. et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, P.R. et al., (1991) Bio / Technology 9:266-271; Co, M.S. et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P. et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, M.S. et al., (1992) J Immunol 148:1149-1154. In some embodiments, the humanized antibody preserves all of the CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more (1, 2, 3, 4, 5, 6) CDRs that have been changed relative to the original antibody, and the CDRs are also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0104] In certain embodiments, the antibodies of the disclosure may be chimeric antibodies. In this context, a chimeric antibody consists of an antigen-binding fragment of an anti-CD40 antibody that is operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class from the parental antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may consist of CH2 and CH3 domains from one or more of the different Ig classes. As described above with respect to humanized antibodies, the anti-CD40 antigen-binding fragment of a chimeric antibody may contain only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may contain the entire variable domain (VL, VH, or both).

[0105] In certain embodiments, the CD40-binding antibody contains one or more of the CDRs of the antibodies described herein. In this context, it has been demonstrated in some cases that it is possible to perform grafting of only the VH CDR3 of the antibody while retaining the desired specific binding (Barbas et al., PNAS (1995) 92:2529-2533). See also McLane et al., PNAS (1995) 92:5214-5218, and Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0106] Marks et al. (Bio / Technology, 1992, 10:779-783) described a method for generating a repertoire of antibody variable domains that provides a repertoire of VH variable domains lacking CDR3 by using consensus primers directed to or adjacent to the 5' end of the variable domain region in combination with a consensus primer for the third framework region of the human VH gene. Marks et al. further described ways in which this repertoire can be combined with the CDR3 of a specific antibody. Using similar techniques, the CDR3-derived sequences of the disclosed antibodies can be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled full VH or VL domains can be combined with cognate VL or VH domains to provide antibodies or antigen-binding fragments thereof that bind CD40. The repertoire can then be presented in a suitable host system, such as the phage display system of WO 92 / 01047 pamphlet, and as a result, suitable antibodies or their antigen-binding fragments can be selected. The repertoire consists of at least about 10 4 individual members, and has an upper digit width, for example, about 10 6 to 10 8 or 10 10 or more members. Similar shuffling or combinatorial techniques have also been disclosed by Stemmer (Nature, 1994, 370:389-391), who described techniques related to the β-lactamase gene and stated that the approach can be used for antibody production.

[0107] A further alternative is to generate a novel VH or VL region having one or more CDR-derived sequences of the embodiments of the invention described herein by introducing mutations throughout the entire variable domain using random mutagenesis of one or more selected VH and / or VL genes. Such techniques have been described by Gram et al. (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR. Another method that can be used is to induce mutagenesis to the CDR regions of the VH or VL genes. Such techniques have been disclosed by Barbas et al. (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al. (1996, J. Mol. Biol. 263:551-567).

[0108] In certain embodiments, libraries of complementary variable domains can be screened using specific VH and / or VL of the antibodies described herein to identify antibodies having desirable properties such as increased affinity for CD40. Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

[0109] Antibodies having desired binding activities such as binding to CD40 can also be identified by using other methods to mix and match CDRs. For example, Klimka et al., British The Journal of Cancer (2000) 83:252-260 describes a screening process using mouse VL and human VH libraries that retain CDR3 and FR4 from mouse VH. After obtaining an antibody, its VH was screened against a human VL library to obtain an antibody that binds the antigen. Beiboer et al., J. Mol. Biol. (2000) 296:833-849 describes a screening process using a fully mouse heavy chain and human light chain library. After obtaining an antibody, one VL was combined with a human VH library that retains the mouse CDR3. An antibody capable of binding the antigen was obtained. Rader et al., PNAS (1998) 95:8910-8915 describes a process similar to that of Beiboer et al.

[0110] These techniques just described are, in themselves, known in the art as such. However, one of ordinary skill in the art could use such techniques, using conventional methodologies in the art, to obtain antibodies or antigen-binding fragments thereof according to several embodiments of the invention described herein.

[0111] A method for obtaining an antibody antigen-binding domain specific for the CD40 antigen is also disclosed herein. This method comprises providing a VH domain that is an amino acid sequence variant of the VH domain by addition, deletion, substitution, or insertion of one or more amino acids in the amino acid sequence of the VH domain shown herein; optionally, combining the thus-provided VH domain with one or more VL domains; and testing the VH domain or VH / VL combination(s) to identify a specific binding member or antibody antigen-binding domain that is specific for CD40 and optionally has one or more desired properties. The VL domain may have an amino acid sequence that is substantially as shown herein. A similar method of combining one or more sequence variants of the VL domain disclosed herein with one or more VH domains may also be used.

[0112] An epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or polypeptide is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule exhibits "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance with a higher frequency, more rapidly, for a longer duration, and / or with greater affinity than it does with an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, binding strength, more rapidly, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CD40 epitope is an antibody that binds to one CD40 epitope with greater affinity, binding strength, more rapidly, and / or for a longer duration than it binds to other CD40 epitopes or non-CD40 epitopes. It is understood from this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it may include it). Generally, although not necessarily, a reference to binding means preferential binding.

[0113] An immunological binding refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific, for example, as a result of electrostatic, ionic, hydrophilic and / or hydrophobic attractive or repulsive forces, steric hindrance forces, hydrogen bonds, van der Waals forces, and other interactions, by way of example and not limitation. The strength or affinity of an immunological binding interaction can be expressed by the dissociation constant (K d ) of that interaction, with a smaller K dexhibits a greater affinity than that. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method necessarily involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (K on ) and the "off-rate constant" (K off ) can be determined by calculation of the concentrations as well as the actual association and dissociation rates. The ratio of K off / K on allows cancellation of all parameters not related to affinity and thus is equal to the dissociation constant K d . See generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0114] In certain embodiments, the anti-CD40 antibodies described herein have an affinity of about 100, 150, 155, 160, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198 or 199 picomolar, and in some embodiments, the antibody may have an even higher affinity for CD40.

[0115] The terms "immunologically active" or "still immunologically active" with respect to an epitope present refer to the ability of an antibody (e.g., an anti-CD40 antibody) to bind to the epitope under different conditions, for example, after subjecting the epitope to reducing and denaturing conditions.

[0116] An antibody or antigen-binding fragment thereof according to certain preferred embodiments of the present application specifically binds to the antigen and (ii) contains the VH and / or VL domains disclosed herein, or the VH CDR3 disclosed herein, or any variant thereof, and may compete with any of the antibodies described herein for binding to CD40. Competition between antibodies can be assayed in vitro, for example, using ELISA and / or by tagging one antibody with a specific reporter molecule to enable identification of specific antibodies that bind to the same epitope or overlapping epitopes (this antibody can be detected in the presence of the other untagged antibody). Accordingly, provided herein are specific antibodies or antigen-binding fragments thereof that contain a human antibody antigen-binding site that competes with the antibodies described herein that bind to CD40.

[0117] In this context, as used herein, the terms "competes with", "inhibits binding", and "blocks binding" (e.g., referring to inhibition / blocking of CD40L binding to CD40 or inhibition / blocking of anti-CD40 antibody binding to CD40) are used synonymously and encompass both partial and complete inhibition / blocking. Inhibition and blocking are intended to mean any measurable decrease in the binding of CD40L to CD40 when in contact with the anti-CD40 antibody disclosed herein, compared to the ligand not in contact with the anti-CD40 antibody, e.g., including at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% blocking of CD40L binding to CD40.

[0118] The constant regions of immunoglobulins exhibit less sequence diversity than the variable regions and are responsible for binding to a number of natural proteins to elicit important biochemical events. In humans, there are five different classes of antibodies, including IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3 and IgG4) and IgM. Although more subtle differences may exist in the V regions, the distinguishing feature between these antibody classes is their constant regions.

[0119] The Fc region of an antibody interacts with a number of Fc receptors and ligands, thereby conferring a series of important functional capabilities called effector functions. The Fc region for IgG contains the Ig domains CH2 and CH3, and an N-terminal hinge connecting to CH2. An important family of Fc receptors for the IgG class is the Fc gamma receptor (FcγR). These receptors mediate the exchange of information between the antibody and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans, this protein family includes FcγRI (CD64), which contains the isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that can mediate some intracellular signaling events. These receptors are expressed in a variety of immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells and T cells. The formation of the Fc / FcγR complex recruits these effector cells to the bound antigen site and generally results in intracellular signaling events and important subsequent immune responses such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis and cytotoxic attack.

[0120] The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on the target cell and subsequently cause lysis of that target cell is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on the target cell and subsequently cause phagocytosis of that target cell is called antibody-dependent cell-mediated phagocytosis (ADCP). All FcγRs bind the same region of Fc at the N-terminus of the Cg2 (CH2) domain and the preceding hinge. This interaction has been structurally well characterized (Sondermann et al., 2001, J Mol Biol 309:737-749), and several structures of human Fc bound to the extracellular domain of human FcγRIIIb have been elucidated (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477).

[0121] Different IgG subclasses have different affinities for FcγR, and IgG1 and IgG3 typically bind their receptors substantially better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). All FcγRs bind the same region on the IgG Fc, but with different affinities; the high-affinity binder FcγRI has a Kd of 10 -8 M -1 for IgG1, while the low-affinity receptors FcγRII and FcγRIII generally have Kds of 10 -6 and 10 -5It binds with. The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical, but FcγRIIIb does not have an intracellular signaling domain. Furthermore, FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complex-induced activation, characterized by having an intracellular domain with an immunoreceptor activation tyrosine motif (ITAM), while FcγRIIb has an immunoreceptor inhibitory tyrosine motif (ITIM) and is thus inhibitory. Therefore, the former are called activating receptors and FcγRIIb is called an inhibitory receptor. These receptors also have different expression patterns and levels on different immune cells. Another level of complexity is the presence of multiple FcγRs in the human proteome. A particularly significant polymorphism with clinical significance is V158 / F158 FcγRIIIa. Human IgG1 binds to the V158 allotype with a greater affinity than the F158 allotype. This difference in terms of affinity and perhaps its effect on ADCC and / or ADCP has been shown to be a significant determinant of the efficacy of the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Patients with the V158 allotype respond favorably to rituximab treatment, while patients with the low-affinity F158 allotype do not respond well (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158 / V158 homozygous, 45% are V158 / F158 heterozygous, and 35-45% of humans are F158 / F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Therefore, 80-90% of humans are poor responders, i.e., they have at least one allele of F158 of FcγRIIIa.

[0122] The Fc region is also involved in the activation of the complement cascade. In the classical complement pathway, C1 binds, using its C1q subunit, to the Fc fragment of IgG or IgM that is complexed with antigen(s). In certain embodiments of the invention, the modification to the Fc region includes a modification that alters (enhances or reduces) the ability of the CD40-specific antibody described herein to activate the complement system (see, e.g., U.S. Patent No. 7,740,847). To assess complement activation, a complement-dependent cytotoxicity (CDC) assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996)).

[0123] Accordingly, in certain embodiments, the invention provides an anti-CD40 antibody having a modified Fc region with altered functional properties, such as reduced or enhanced CDC, ADCC, or ADCP activity, or enhanced binding affinity for specific FcγRs, or an increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in issued U.S. Patent Nos. 7,317,091, 7,657,380, 7,662,925, 6,538,124, 6,528,624, 7,297,775, and 7,364,731, U.S. Patent Application Publication Nos. 2009092599, 20080131435, and 20080138344, and International Publication Nos. 2006 / 105338, 2004 / 063351, 2006 / 088494, and 2007 / 024249 brochures.

[0124] In one embodiment, one or more substitutions in the Fc can increase the binding affinity for FcγRIIB, enhance the cross-linking of CD40 molecules, and result in stronger CD40 activation by the anti-CD40 antibody. In one embodiment, the invention provides an anti-CD40 antibody having an Fc region modified at position 267 (EU numbering; see, e.g., Edelman, G.M. et al., 1969 Proc. Natl. Acad. USA, 63, 78-85; also see the ImMunoGeneTics (IMGT) database website at imgt.org / IMGTScientificChart / Numbering). In one embodiment, the anti-CD40 antibodies herein include a modified Fc containing the S267E substitution (Li Fu, Ravetch JV. 2011 Science 333:1030; J. Immunol. 2011, 187:1754-1763; mAbs 2010, 2:181-189 are also referred to). The amino acid sequence of the heavy chain constant region containing the exemplary modified Fc is shown in SEQ ID NO: 195.

[0125] Thus, in certain embodiments, an antibody variable domain having a desired binding specificity is fused to an immunoglobulin constant domain sequence. In certain embodiments, the fusion is with an Ig heavy chain constant domain comprising at least a portion of the hinge, C H 2 and C H 3 regions. A first heavy chain constant region (C HIt is preferred to have (1). DNA encoding an immunoglobulin heavy chain fusion and, optionally, an immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host cell. Thereby, in embodiments where the unequal ratios of the three polypeptides used in its construction result in an optimal yield of the desired bispecific antibody, a greater degree of freedom in adjusting the mutual ratio of these three polypeptide fragments is obtained. However, when expression in equimolar amounts of at least two polypeptide chains results in a high yield, or when their ratio does not significantly affect the yield of the desired chain combination, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0126] The antibodies of the present invention (as well as their antigen-binding fragments and variants) can also be modified to include an epitope tag or label, for example for use in purification or diagnostic applications. Many linking groups for making antibody conjugates are known in the art, including, for example, those disclosed in U.S. Patent No. 5,208,020 or European Patent No. 0 425 235 B1, as well as Chari et al., Cancer Research 52:127-131 (1992). Examples of such linking groups include disulfide groups, thioether groups, acid-labile groups, photosensitive groups, peptidase-labile groups, or esterase-labile groups as disclosed in the patents specified above, with disulfide and thioether groups being preferred.

[0127] In another possible embodiment, the CD40-specific antibodies described herein can be conjugated or operably linked to another therapeutic compound, referred to herein as a conjugate. The conjugate can be a cytotoxic agent, a chemotherapeutic agent, a cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, a radioisotope, or other therapeutically active agent. Chemotherapeutic agents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors and other therapeutic agents are described above, and all of these above-described therapeutic agents can find use as antibody conjugates.

[0128] In an alternative embodiment, the antibody (including its fragments and / or variants) is conjugated or operably linked to toxins including (but not limited to) small molecule toxins and enzymatically active toxins derived from bacteria, fungi, plants or animals. Small molecule toxins include, but are not limited to, saporin (Kuroda K et al., The Prostate 70:1286-1294 (2010); Lip, WL et al., 2007 Molecular Pharmaceutics 4:241-251; Quadros EV et al., 2010 Mol Cancer Ther;9(11);3033-40; Polito L et al., 2009 British Journal of Haematology, 147, 710-718), calicheamicin, maytansine (U.S. Patent No. 5,208,020), trichothene, and CC1065. Toxins include, but are not limited to, RNase, gelonin, enjine, ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin (PE40), Shigella toxin, Clostridium perfringens toxin, and yamabushitoxin.

[0129] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure is conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. Immunoconjugates containing maytansinoids and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064 and European Patent No. 0 425 235 B1. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describes an immunoconjugate containing a maytansinoid designated DM1 conjugated to the monoclonal antibody C242 against human colorectal cancer. The conjugate has been found to be highly cytotoxic to cultured colon cancer cells and to exhibit high tumor activity in in vivo tumor growth assays.

[0130] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. On average, 3 to 4 maytansinoid molecules per antibody molecule have shown the potency to enhance the cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, and even one molecule of toxin / antibody was expected to enhance cytotoxicity over the use of the naked antibody. Maytansinoids are well known in the art and maytansinoids can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020, as well as in other patents and non-patent publications referred to above herein. Preferred maytansinoids are maytansinol, and maytansinol analogs in which the aromatic ring or other moiety of the maytansinol molecule is modified, such as various maytansinol esters.

[0131] Another conjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibodies is capable of producing double-stranded DNA breaks at subpicomolar concentrations. Structural analogs of calicheamicin that can also be used (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928) (U.S. Patent Nos. 5,714,586, 5,712,374, 5,264,586, and 5,773,001). Dolastatin 10 analogs, such as auristatin E (AE) and monomethyl auristatin E (MMAE), can be found to be utilized as conjugates for the disclosed antibodies or their variants (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65). Useful enzyme-active toxins include, but are not limited to, diphtheria A chain, the non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, PCT International Publication No. 93 / 21232 pamphlet. The present disclosure further contemplates embodiments that form conjugates or fusions between the CD40-specific antibodies described herein and compounds having nuclease activity, such as ribonucleases or DNA endonucleases, such as deoxyribonuclease (DNase).

[0132] In an alternative embodiment, the disclosed antibodies herein can be conjugated or operably linked to a radioisotope to form a radio conjugate. A variety of radioactive isotopes can be utilized in the production of radio conjugate antibodies. Examples include 90 Y, 123 I, 125 I, 131 I, 186 Re, 188 Re, 211 At and 212 Bi, but are not limited thereto.

[0133] In certain other embodiments, the antibodies described herein can be conjugated to a therapeutic moiety, such as a cytotoxin (e.g., a cell growth inhibitor or a cell destroying agent), a therapeutic agent or a radioisotope (e.g., an α-emitter, a γ-emitter, etc.). Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxel / paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin as well as analogs or homologs thereof. One preferred exemplary cytotoxin is saporin (available from Advanced Targeting Systems, San Diego, CA). Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine and vinblastine), but are not limited thereto.

[0134] Furthermore, in certain embodiments, a CD40-specific antibody (including its functional fragments as provided herein, such as antigen-binding fragments) can be conjugated to a therapeutic moiety useful for conjugation of a radioactive metal ion, such as a radioactive material or a macrocyclic chelator. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), which can be attached to the antibody by a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50.

[0135] In yet another embodiment, an antibody can be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretreatment, in which the antibody-receptor conjugate is administered to a patient, followed by using a chelating agent to remove unbound conjugate from the circulation, and then administering a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radioactive nucleotide). In an alternative embodiment, the antibody is conjugated or operably linked to an enzyme for use in antibody-dependent enzyme-mediated prodrug therapy (ADEPT). ADEPT can be used by conjugating or operably linking the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see PCT International Publication No. 81 / 01145 pamphlet) to an active anticancer drug. See, for example, PCT International Publication No. 88 / 07378 pamphlet and U.S. Patent No. 4,975,278 specification. Enzyme components of immunoconjugates useful for ADEPT include any enzyme capable of acting on a prodrug to convert it to its more active cytotoxic form.Enzymes useful in these and related embodiments of the method include alkaline phosphatase useful for the conversion of phosphate-containing prodrugs to free drugs; arylsulfatase useful for the conversion of sulfate-containing prodrugs to free drugs; cytosine deaminase useful for the conversion of non-toxic 5-fluorocytosine to the anticancer drug, 5-fluorouracil; proteases useful for the conversion of peptide-containing prodrugs to free drugs, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsin (e.g., cathepsin B and L); D-alanyl carboxypeptidase useful for the conversion of prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes useful for the conversion of glycosylated prodrugs to free drugs, such as β-galactosidase and neuraminidase; β-lactamase useful for the conversion of drugs derivatized with β-lactam to free drugs; and penicillin amidase, such as penicillin V amidase or penicillin G amidase, useful for the conversion of drugs in which the amine nitrogen is derivatized with a phenoxyacetyl or phenylacetyl group, respectively, to free drugs, but not limited thereto. Alternatively, antibodies having enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs to active drugs (see, e.g., Massey, 1987, Nature 328:457-458). Antibody-abzyme conjugates can be prepared for the delivery of abzymes to tumor cell populations.

[0136] A variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutareldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene) can be used to prepare immunoconjugates. Specific coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737

[1978] ) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) for providing disulfide linkages. The linker may be a "cleavable linker" that promotes the release of one or more cleavable components. For example, an acid-labile linker may be used (Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0137] Other modifications of the antibodies (and polypeptides) of the invention are also contemplated herein. For example, the antibody can be conjugated to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. The antibody can also be entrapped in microcapsules (e.g., hydroxy methylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th Edition, Oslo, A. Ed. (1980).

[0138] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to the cells or mammals exposed thereto at the dosages and concentrations employed. In many cases, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™), polyethylene glycol (PEG) and poloxamer (PLURONICS™), and the like.

[0139] As described elsewhere in this specification, the antibodies of the present disclosure induce CD40 signaling in tumor cells, activate dendritic cells and immune surveillance mechanisms, activate antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, block the binding of CD40 to CD40L; have CD40 agonist activity; activate antigen-presenting cells; stimulate cytokine release from antigen-presenting cells; induce tumor cell apoptosis; inhibit tumor cell proliferation; kill tumor cells by inducing effector functions including (but not limited to) ADCC, CDC and ADCP; stimulate anti-tumor T cell responses; reduce established tumors; and inhibit rituximab-resistant tumors. The antibodies described herein can have or induce any one or more combinations of these properties or activities. Various methods known to those of skill in the art, such as affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation, activation or differentiation assays, ADCC and CDC assays, other cellular activities resulting from CD40 cell signaling events (e.g., production of cytokines including STAT3 phosphorylation, IL-1, IL-6, IL-8, IL-10, IL-12, TNF-α and MIP1α), and cancer cell and / or tumor growth inhibition using in vitro or in vivo models can be used to evaluate the desired functional properties of the anti-CD40 antibodies. Other assays can test the ability of the antibodies described herein to block normal CD40L binding to CD40 or CD40-mediated responses, such as cell signaling, cell activation (e.g., immune cell activation, proliferation; antigen-presenting cell activation (e.g., dendritic cells, B cells, macrophages) and maturation assays), immune responses (including cell-mediated and humoral responses), etc. The antibodies described herein can also be tested for effects on CD40 internalization, in vitro and in vivo efficacy, etc. Such assays are well-established protocols known to those of skill in the art (e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current It can be carried out using Current Protocols in Immunology (edited by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, see John Wiley & Sons, NY, NY, 2001) or commercially available kits.

[0140] In certain embodiments, the invention further provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, e.g., a nucleic acid encoding a CDR or VH or VL domain described herein. Nucleic acids include DNA and RNA. These and related embodiments can include polynucleotides encoding antibodies that bind CD40 described herein. As used herein, the term "isolated polynucleotide" means a polynucleotide of genomic, cDNA or synthetic origin that, by virtue of its origin, the isolated polynucleotide is (1) not associated with all or part of the polynucleotide in which the isolated polynucleotide is found in nature, (2) linked to a polynucleotide to which it is not linked in nature, or (3) not present in nature as part of a larger sequence, or some combination thereof.

[0141] The term "operably linked" means that the components to which the term applies are in a relationship such that they can perform their inherent functions under suitable conditions. For example, a transcriptional control sequence "operably linked" to a protein coding sequence is ligated to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0142] As used herein, the term "control sequence" refers to a polynucleotide sequence that can affect the expression, processing, or intracellular localization of a coding sequence to which it is ligated or operably linked. The nature of such control sequences may depend on the host organism. In certain embodiments, transcriptional control sequences for prokaryotes may include a promoter, ribosome binding site, and transcription termination sequence. In other certain embodiments, transcriptional control sequences for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, a transcription termination sequence, and a polyadenylation sequence. In certain embodiments, "control sequence" may include a leader sequence and / or a fusion partner sequence.

[0143] As used herein, the term "polynucleotide" means a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides that make up the polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications, such as bromouridine, ribose modifications, such as arabinose and 2',3'-dideoxyribose, and internucleotide linkage modifications, such as phosphorothioate, phosphorodithioate, phosphorothioate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoramidate. The term "polynucleotide" includes, in particular, DNA in both single-stranded and double-stranded forms.

[0144] The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar groups and the like. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranirothioate, phosphoralanidate, phosphoramidate and the like. For example, see LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (edited by F. Eckstein), Oxford University Press, Oxford England; Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543 (the disclosures of which are incorporated herein by reference for any purpose). An oligonucleotide may include a detectable label to enable detection of the oligonucleotide or their hybridization.

[0145] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing when introns are present.

[0146] As will be understood by those skilled in the art, a polynucleotide can include smaller modified gene segments that express, or can be engineered to express, genomic sequences, extra-genomic and plasmid coding sequences, as well as proteins, polypeptides, peptides and the like. Such segments may be naturally isolated or may be synthetically modified by those skilled in the art.

[0147] As will also be understood by those skilled in the art, a polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be a DNA (genomic, cDNA or synthetic) or an RNA molecule. Examples of RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one fashion, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may or may not be present within the polynucleotides according to the present disclosure, and the polynucleotides may or may not be linked to other molecules and / or support materials. The polynucleotides can include natural sequences or can include sequences encoding variants or derivatives of such sequences.

[0148] Accordingly, according to these and related embodiments, the present disclosure also provides polynucleotides encoding the anti-CD40 antibodies described herein. In certain embodiments, polynucleotides are provided that include some or all of the polynucleotide sequences encoding the antibodies described herein, and complements of such polynucleotides.

[0149] In other related embodiments, the polynucleotide variant may have substantial identity with the polynucleotide sequence encoding the anti-CD40 antibody described herein. For example, the polynucleotide may be compared to a reference polynucleotide sequence, such as the sequence encoding the antibody described herein, using the methods described herein (e.g., BLAST analysis using standard parameters as described below), and may be a polynucleotide comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity. Those skilled in the art will appreciate that these values can be appropriately adjusted by considering codon degeneracy, amino acid similarity, reading frame positioning and the like to determine the corresponding identity for the proteins encoded by the two nucleotide sequences.

[0150] Typically, the polynucleotide variant will preferably contain one or more substitutions, additions, deletions and / or insertions such that the binding affinity of the antibody encoded by the variant polynucleotide is not substantially reduced compared to the sequence encoded by the polynucleotide sequence specifically shown herein.

[0151] In certain other related embodiments, the polynucleotide fragment can comprise or consist essentially of contiguous stretches of various lengths of a sequence that is identical or complementary to the sequence encoding the antibodies described herein. For example, provided are polynucleotides comprising or consisting essentially of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of the sequence encoding the antibodies disclosed herein, or antigen-binding fragments thereof, and all intermediate lengths of nucleotides therebetween. By "intermediate length" in this context is meant any length between the recited values, e.g., 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc., and including any length that comprises all integers through 200 - 500, 500 - 1000, and the like. The polynucleotide sequences described herein may be extended at one or both ends by additional nucleotides not found in the native sequence. This additional sequence can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides at either or both ends of the polynucleotide encoding the antibodies described herein.

[0152] In another embodiment, provided herein is a polynucleotide that can hybridize under high stringency conditions to a polynucleotide sequence encoding an antibody provided herein, or an antigen-binding fragment thereof, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology techniques. For purposes of illustration, moderate stringency conditions suitable for testing the hybridization of a polynucleotide provided herein with other polynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50° C. to 60° C. in 5×SSC; followed by two washes at 65° C. for 20 minutes each in 2×, 0.5×, and 0.2×SSC containing 0.1% SDS. It will be understood by those skilled in the art that the stringency of hybridization can be readily manipulated by changing, for example, the salt content of the hybridization solution and / or the temperature at which hybridization is performed. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the temperature of hybridization is raised to, for example, 60° C. to 65° C. or 65° C. to 70° C.

[0153] In certain embodiments, the polynucleotides described above, such as polynucleotide variants, fragments, and hybridization sequences, encode an antibody that binds CD40, or an antigen-binding fragment thereof. In other embodiments, such polynucleotides encode not only the antibody sequences specifically shown herein, but also antibodies or antigen-binding fragments that bind CD40 with at least about 50%, at least about 70%, and in certain embodiments at least about 90% binding, or their CDRs. In further embodiments, such polynucleotides encode not only the antibody sequences specifically shown herein, but also antibodies or antigen-binding fragments that bind CD40 with a greater affinity than the antibodies described herein, for example, quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% binding, or their CDRs.

[0154] As described elsewhere in this specification, since the three-dimensional structure of a representative polypeptide (e.g., a variant CD40-specific antibody as provided herein, e.g., an antibody protein having an antigen-binding fragment as provided herein) can be determined by conventional methodologies, one or more amino acid substitutions, additions, deletions or insertions with selected natural or non-natural amino acids can be virtually modeled to determine whether the thus-derived structural variant retains the space-filling properties of the disclosed species. For example, various computer programs are known to those skilled in the art for determining appropriate amino acid substitutions (or appropriate polynucleotides encoding such amino acid sequences) within an antibody such that affinity is maintained or better affinity is achieved.

[0155] The polynucleotides described herein, or fragments thereof regardless of their own coding sequence length, can be combined with other DNA sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments and the like, so their full lengths can be quite diverse. Thus, it is contemplated that nucleic acid fragments of almost any length can be utilized, and their full lengths are preferably restricted by the ease of preparation and use in the desired recombinant DNA protocol. For example, illustrative polynucleotide segments having a total length of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length and lengths similar thereto (including all intermediate lengths) are considered useful.

[0156] When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequences of nucleotides in the two sequences are the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify local regions of sequence similarity. A "comparison window" as used herein refers to a segment of at least about 20, usually 30 to about 75, 40 to about 50 consecutive positions, which can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned.

[0157] Optimal alignment of the arrays for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wisconsin) with default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Volume 5, Supplement 3, pp. 345 - 358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626 - 645 (1990); Methods in Enzymology Volume 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5:151 - 153 (1989); Myers, E.W. and Muller W., CABIOS 4:11 - 17 (1988); Robinson, E.D., Comb.Theor 11:105 (1971); Santou, N. Nes, M., Mol.Biol.Evol. 4:406 - 425 (1987); Sneath, P.H.A. and Sokal, R.R., Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA (1973); Wilbur, W.J. and Lipman, D.J., Proc.Natl.Acad.,Sci.USA 80:726 - 730 (1983).

[0158] Alternatively, the optimal alignment of arrays for comparison can be performed by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin)), or by inspection.

[0159] One preferred example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used with, for example, the parameters described herein to determine the percent sequence identity between two or more polynucleotides. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. In one illustrative example, for nucleotide sequences, cumulative scores can be calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of word hits in each direction stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score goes below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. The BLASTN program (for nucleotide sequences) uses, as defaults, a word length (W) of 11, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment, (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and both-strand comparison.

[0160] In certain embodiments, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the components of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) of less than 20 percent, usually 5 to 15 percent or 10 to 12 percent, as compared to the reference sequence for optimal alignment of the two sequences (excluding additions and deletions). The percent is calculated by determining the number of positions at which the identical nucleic acid base appears in both sequences to obtain the number of match positions, dividing the number of match positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percent sequence identity.

[0161] As a result of the degeneracy of the genetic code, it will be appreciated by those skilled in the art that there are many nucleotide sequences encoding the antibodies described herein. Some of these polynucleotides have minimal sequence identity with the nucleotide sequence of the native or original polynucleotide sequence encoding an antibody that binds CD40. Nevertheless, different polynucleotides are clearly contemplated by the present disclosure due to differences in codon usage frequency. In certain embodiments, sequences that are codon-optimized for mammalian expression are particularly contemplated.

[0162] Accordingly, in another embodiment of the invention, mutagenesis approaches such as site-directed mutagenesis can be utilized to prepare variants and / or derivatives of the antibodies described herein. This approach allows for specific modification of the polypeptide sequence by mutagenesis of the underlying polynucleotide encoding them. These techniques provide a straightforward approach for preparing and testing sequence variants, including, for example, one or more of the considerations described above, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0163] Site-directed mutagenesis is the production of mutants by use of a specific oligonucleotide sequence encoding the DNA sequence of a desired mutation and a sufficient number of adjacent nucleotides, enabling production to yield primer sequences of sufficient size and sequence complexity to form stable double strands on both sides across the deletion junction. Mutations can be used in the selected polynucleotide sequence to improve, alter, reduce, modify or otherwise change the properties of the polynucleotide itself, and / or to change the properties, activity, composition, stability or primary sequence of the encoded polypeptide.

[0164] In certain embodiments, the inventors contemplate mutagenesis of a polynucleotide sequence encoding an antibody, or antigen-binding fragment thereof, disclosed herein, for the purpose of altering one or more properties of the encoded polypeptide, such as the binding affinity of said antibody or antigen-binding fragment thereof, or the function of a particular Fc region, or the affinity of the Fc region for a particular FcγR. Techniques for site-directed mutagenesis are well known in the art and are widely used to generate variants of both polypeptides and polynucleotides. For example, site-directed mutagenesis is often used to alter specific portions of a DNA molecule. In such embodiments, primers of a length typically containing about 14 to about 25 nucleotides are used to alter about 5 to about 10 residues on either side of the junction of the sequence.

[0165] As will be appreciated by those skilled in the art, site-directed mutagenesis techniques often utilize phage vectors that exist in both single-stranded and double-stranded forms. Exemplary vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phages are readily available on the market and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely utilized in site-directed mutagenesis that eliminates the step of transferring the gene of interest from the plasmid to the phage.

[0166] Generally, site-directed mutagenesis according to the present specification is carried out by first obtaining a single-stranded vector or melting the two strands of a double-stranded vector containing a DNA sequence encoding a desired peptide separately within the sequence. An oligonucleotide primer carrying the desired mutant sequence is generally prepared synthetically. Next, this primer is annealed to the single-stranded vector and subjected to a DNA polymerase such as the Klenow fragment of Escherichia coli polymerase I to complete the synthesis of the strand carrying the mutation. Thus, a heteroduplex is formed in which one strand encodes the original unmutated sequence and the second strand carries the desired mutation. Thereafter, this heteroduplex vector is used to transform an appropriate cell such as an Escherichia coli cell, and clones containing recombinant vectors carrying the mutant sequence arrangement are selected.

[0167] The preparation of sequence variants of a DNA segment encoding a selected peptide using site-directed mutagenesis provides a means of producing potentially useful species and is not intended to be limiting as there are other methods by which peptide sequence variants and the DNA sequences encoding them can be obtained. For example, a recombinant vector encoding the desired peptide sequence can be treated with a mutagen such as hydroxylamine to obtain sequence vari ants. Specific details regarding these methods and protocols can be found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, and for the purposes of this, each of the said teachings is incorporated herein by reference.

[0168] As used herein, the term "oligonucleotide-directed mutagenesis procedure" refers to a template-dependent process and vector-mediated propagation that results in an increase in the concentration of a specific nucleic acid molecule or an increase in the concentration of a detectable signal such as amplification. As used herein, the term "oligonucleotide-directed mutagenesis procedure" is intended to refer to a process that includes template-dependent extension of primer molecules. The term template-dependent process refers to nucleic acid synthesis of RNA or DNA molecules where the sequence of the newly synthesized strand of nucleic acid is defined by well-known rules for complementary base pairing (see, e.g., Watson, 1987). Typically, vector-mediated methodologies include introduction of a nucleic acid fragment into DNA or RNA, clonal amplification of the vector, and recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided in U.S. Patent No. 4,237,224, which is hereby specifically incorporated by reference in its entirety.

[0169] In another approach for the production of polypeptide variants, recursive sequence recombination as described in U.S. Patent No. 5,837,458 can be utilized. In this approach, iterative cycles of recombination and screening or selection are performed to "generate", for example, individual polynucleotide variants with increased binding affinity. Certain embodiments also provide constructs in the form of plasmids, vectors, transcription or expression cassettes that include at least one polynucleotide described herein.

[0170] In many embodiments, a nucleic acid encoding a subject monoclonal antibody is introduced directly into a host cell and the cell is incubated under conditions sufficient to induce expression of the encoded antibody. The antibodies of the disclosure are prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein. Appropriate nucleic acid sequences encoding the specific antibodies disclosed thereby can be determined using the polypeptide sequences. The nucleic acid sequences can be optimized to reflect the individual codon "preferences" for various expression systems according to standard methods well known to those of skill in the art.

[0171] According to certain related embodiments, provided are recombinant host cells comprising one or more constructs described herein; nucleic acids encoding any antibody, its CDR, VH or VL domain, or antigen-binding fragment; and a method for producing the encoded product, said method comprising expression from the nucleic acid encoding the product. Expression can be conveniently achieved by culturing the recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, the antibody or its antigen-binding fragment can be isolated and / or purified using any suitable technique and then used as needed.

[0172] Antibodies or antigen-binding fragments, as well as encoding nucleic acid molecules and vectors, as provided herein can be isolated and / or purified, for example, in a substantially pure or homogeneous form from their natural environment, or in the case of nucleic acids, may be free or substantially free of the original nucleic acid or gene other than the sequence encoding the polypeptide having the desired function. The nucleic acid can include DNA or RNA and can be wholly or partially synthetic. References to nucleotide sequences in the context shown herein include DNA molecules having the specified sequence and, unless otherwise required by context, RNA molecules having the specified sequence with T substituted by U.

[0173] Systems for the cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous peptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A commonly preferred bacterial host is Escherichia coli.

[0174] The expression of antibodies and antigen-binding fragments in prokaryotic cells such as Escherichia coli is well established in the art. For reviews, see, e.g., Pluckthun, A. Bio / Technology 9:545-551 (1991). Expression in eukaryotic cell culture can also be utilized by those skilled in the art as an option for the production of antibodies or their antigen-binding fragments. For recent reviews, see, e.g., Ref, M.E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J.J. et al. (1995) Curr. Opinion Biotech 6:553-560.

[0175] A suitable vector can be selected or constructed that contains appropriate regulatory sequences, optionally including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. The vector can optionally be a plasmid, viral, e.g., phage, or phagemid. For further details, see, e.g., Molecular Cloning: a Laboratory Manual: 2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols regarding nucleic acid manipulation, e.g., preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression, as well as protein analysis, are described in detail in Current Protocols in Molecular Biology, 2nd Edition, Ausubel et al. eds., John Wiley & Sons, 1992 or its later updates.

[0176] The term "host cell" refers to a cell into which a nucleic acid sequence encoding one or more of the antibodies described herein has been introduced, or a cell capable of tolerating such introduction, and further capable of expressing or being capable of expressing a selected gene of interest, such as a gene encoding any of the antibodies described herein. This term includes progeny of the parent cell, whether or not the progeny are identical to the original parent in terms of morphology or the genes they contain, as long as the selected gene is present. Thus, methods involving the introduction of such nucleic acids into host cells are contemplated. Any available technique can be used for such introduction. Techniques suitable for eukaryotic cells include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus or, for insect cells, baculovirus. Techniques suitable for bacterial cells include calcium chloride transformation, electroporation, and transfection using bacteriophage. After such introduction, expression from the nucleic acid can be caused or enabled, for example, by culturing the host cell under conditions for gene expression. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences that promote recombination in the genome by standard techniques.

[0177] In certain embodiments, the invention also provides methods that include the use of the above-described constructs in an expression system for expressing certain polypeptides, such as the CD40-specific antibodies described herein. The term "transduction" is used herein to generally refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when exogenous DNA has been introduced into the interior of the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986, BASIC METHODS 1N MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0178] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it is genetically modified from its natural state. After transfection or transduction, the transforming DNA can be recombined with the cell's DNA by physical integration into the cell's chromosome, or can be transiently maintained as an episomal element without replication, or can replicate independently as a plasmid. A cell is considered to be stably transformed when the DNA is replicated with the cell's division. The terms "naturally occurring" or "natural" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells and the like, refer to materials found in nature that have not been manipulated by humans. Similarly, "not naturally occurring" or "non-natural" as used herein refers to materials not found in nature that have been structurally modified or synthesized by humans.

[0179] The terms "polypeptide", "protein", "peptide", and "glycoprotein" are used synonymously and mean a polymer of amino acids that is not limited to any particular length. This term does not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more chains of amino acids, where each chain contains amino acids covalently linked by peptide bonds, and the polypeptide or protein can include chains that are non-covalently and / or covalently linked to each other by peptide bonds, and have the sequence of a natural protein, i.e., a protein that is naturally occurring and produced in particular by non-recombinant cells, or a protein produced by genetically modified or recombinant cells, and can also include molecules having the amino acid sequence of a natural protein, or molecules having a deletion of one or more amino acids from said natural sequence, an addition to said amino acids, and / or a substitution of said amino acids. The terms "polypeptide" and "protein" specifically include antibodies that bind to CD40 of the present disclosure, or sequences having a deletion of one or more amino acids from an anti-CD40 antibody, an addition to said amino acids, and / or a substitution of said amino acids. Thus, a "polypeptide" or "protein" can include one amino acid chain (referred to as a "monomer") or multiple amino acid chains (referred to as a "multimer").

[0180] As used herein, the term "isolated protein" means that the protein of interest lacks (1) at least some other proteins typically found together in nature, (2) is substantially free of other proteins from the same source, e.g., from the same species, (3) is expressed by cells from different species, (4) is separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) does not associate (by covalent or non-covalent interactions) with the protein moieties with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or non-covalent interactions) with a polypeptide with which it is not associated in nature, or (7) does not exist in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, may be of synthetic origin, or may be any combination thereof. In certain embodiments, the isolated protein is substantially free of proteins, polypeptides, or other contaminants found in its native environment that would interfere with its use (for therapy, diagnosis, prevention, research, or other uses).

[0181] The term "polypeptide fragment" refers to a polypeptide that may be monomeric or multimeric and that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain that is at least about 5 to about 500 amino acids in length. In certain embodiments, it will be understood that the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains, such as the antigen-binding domain or fragment of an antibody. In the case of an anti-CD40 antibody, useful fragments include the CDR regions, particularly the CDR3 region of the heavy or light chain; the variable region of the heavy or light chain; a portion of an antibody chain containing two CDRs or the variable region thereof only; and the like, but are not limited thereto.

[0182] A polypeptide may contain at its N-terminus a signal (or leader) sequence that directs the import of the protein during or after translation. Any polypeptide amino acid sequence provided herein that includes a signal peptide without such a signal or leader peptide is also contemplated for any of the uses described herein. As will be appreciated by those skilled in the art, the signal peptide is typically cleaved during processing and is not included in the active antibody protein. The polypeptide may also be in-frame fused or conjugated to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

[0183] Multiple polypeptide components can also be utilized with peptide linker / spacer arrays, if necessary, to space the polypeptides apart by a sufficient distance to ensure that each polypeptide folds into its secondary and / or tertiary structure. Such peptide linker sequences can be incorporated into the fusion polypeptide using standard techniques well known in the art.

[0184] Certain peptide spacer arrays can be selected, for example, based on (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that can interact with the functional epitopes on the first and second polypeptides; and / or (3) the absence of hydrophobic or charged residues that can react with the functional epitopes of the polypeptides.

[0185] In one exemplary embodiment, the peptide spacer array contains, for example, Gly, Asn, and Ser residues. Other substantially neutral amino acids, such as Thr and Ala, can also be included in the spacer array.

[0186] Other amino acid sequences that can be usefully employed as spacers are disclosed in Maratea et al., Gene 40:39 - 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 - 8262 (1986); U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.

[0187] Other exemplary spacers include, for example, Glu - Gly - Lys - Ser - Ser - Gly - Ser - Gly - Ser - Glu - Ser - Lys - Val - Asp (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066 - 1070) and Lys - Glu - Ser - Gly - Ser - Val - Ser - Ser - Glu - Gln - Leu - Ala - Gln - Phe - Arg - Ser - Leu - Asp (Bird et al., 1988, Science 242:423 - 426).

[0188] In some embodiments, when the first and second polypeptides have a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric hindrance, a spacer sequence is not required. The two coding sequences can be fused directly without using any spacer, or by using, for example, a flexible polylinker consisting of a pentamer Gly-Gly-Gly-Gly-Ser repeated 1 to 3 times. Such spacers have been used by inserting them between VH and VL when constructing single-chain antibodies (scFv) (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).

[0189] In certain embodiments, a peptide spacer is designed to allow proper interaction between the two beta-sheets forming the variable region of the single-chain antibody.

[0190] In certain illustrative embodiments, the peptide spacer is an amino acid between 1 and 5, between 5 and 10, between 5 and 25, between 5 and 50, between 10 and 25, between 10 and 50, between 10 and 100, or any intervening range of amino acids.

[0191] In other illustrative embodiments, the peptide spacer comprises an amino acid having a length of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more.

[0192] Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of said antibody. For example, amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes into the polynucleotide encoding the antibody or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of said antibody. Combinations of any of deletion, insertion and substitution may be made to arrive at the final antibody, provided that the final construct has the desired properties (e.g., high affinity binding to CD40). The amino acid changes may also alter post-translational processes of the antibody, such as changing the number or location of glycosylation sites. Any mutations and modifications described above for the polypeptides of the invention can be included in the antibodies of the invention.

[0193] The present disclosure provides variants of the antibodies disclosed herein. In certain embodiments, not only the antibody sequences specifically shown herein, but also such variant antibodies or their antigen-binding fragments, or their CDRs bind to CD40 at least about 50%, at least 70%, and in certain embodiments, at least about 90%. In further embodiments, not only the antibody sequences specifically shown herein, but also such variant antibodies or their antigen-binding fragments, or their CDRs bind to CD40 with a greater affinity than the antibodies shown herein, e.g., quantitatively at least about 105%, 106%, 107%, 108%, 109% or 110% bind.

[0194] In certain embodiments, the subject antibody can have a heavy chain variable region that is at least 80% identical, at least 90%, at least 95%, at least 98% or 99% identical to the heavy chain variable region of the anti-CD40 antibodies described herein; and a light chain variable region that is at least 80% identical, at least 85%, at least 90%, at least 95%, at least 98% or 99% identical to the light chain variable region of the anti-CD40 antibodies described herein. Amino acid sequences of illustrative heavy and light chain regions are shown in SEQ ID NOs: 1-56.

[0195] In certain embodiments, the antibody comprises a) a heavy chain variable region comprising i. a CDR1 region that is identical to the heavy chain CDR1 region of a selected antibody described herein in terms of amino acid sequence; ii. a CDR2 region that is identical to the heavy chain CDR2 region of the selected antibody in terms of amino acid sequence; and iii. a CDR3 region that is identical to the heavy chain CDR3 region of the selected antibody in terms of amino acid sequence, and b) a light chain variable domain comprising i. a CDR1 region that is identical to the light chain CDR1 region of the selected antibody in terms of amino acid sequence; ii. a CDR2 region that is identical to the light chain CDR2 region of the selected antibody in terms of amino acid sequence; and iii. a CDR3 region that is identical to the light chain CDR3 region of the selected antibody in terms of amino acid sequence, and specifically binds to a selected target (e.g., CD40). In a further embodiment, the antibody or antigen-binding fragment thereof is a variant antibody, the variant comprising the same heavy and light chains as the selected antibody, except for 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions in the CDR regions of the VH and VL regions. In this context, the CDR regions of the selected antibody may further have 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments 9, 10, 11, 12, 13, 14, 15 amino acid substitutions. Substitutions can be in the CDRs in either the VH and / or VL regions. (See, e.g., Muller, 1998, Structure 6:1153-1167).

[0196] Since the three-dimensional structure of a representative polypeptide (e.g., a variant CD40-specific antibody as provided herein, e.g., an antibody protein having an antigen-binding fragment as provided herein) can be determined by routine methodologies, one or more amino acid substitutions, additions, deletions or insertions with selected natural or unnatural amino acids can be virtually modeled to determine whether the thus-derived structural variant retains the space-filling properties of the disclosed species. See, e.g., Donate et al., 1994 Prot.Sci. 3:2378; Bradley et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc.Nat.Acad.Sci.USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al., Science 327:1014-1018 (2010). For these and related embodiments, additional non-limiting examples of some computer algorithms that can be used, for example, for the rational design of CD40-specific antibodies and their antigen-binding domains as provided herein, include VMD, a molecular visualization program that uses 3-D graphics and built-in scripts to display, animate and analyze large biomolecular systems (see the website of the Theoretical and Computational Biophysics Group at the University of Illinois at Urbana-Champagne at ks.uiuc.edu / Research / vmd / ).Many other computer programs are known in the art and can be used by those skilled in the art to determine atomic dimensions from the space-filling model (van der Waals radius) of the energy-minimized conformation; GRID, which attempts to determine regions of high affinity for various chemical groups and thereby strengthen the bonds, Monte Carlo searches that calculate mathematical algorithms, and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al. (1981) J. Comput. Chem. 106:765) (also see Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488) that evaluate and analyze force field calculations. Various suitable computational computer programs are commercially available, for example, from Schroedinger (Munich, Germany).

[0197] In another embodiment of the invention, the anti-CD40 antibodies and their humanized versions are derived from rabbit monoclonal antibodies and, in particular, are produced using the RabMAb® technology. These antibodies require minimal sequence modification, thereby facilitating retention of functional properties after humanization using mutational lineage guided (MLG) humanization techniques (see, e.g., U.S. Patent No. 7,462,697). Thus, an exemplary method of making the anti-CD40 antibodies of the present disclosure involves the RabMab® rabbit monoclonal antibody technology as described, for example, in U.S. Patent Nos. 5,675,063 and 7,429,487. In this regard, in certain embodiments, the anti-CD40 antibodies of the present disclosure are produced in rabbits. In a particular embodiment, rabbit-derived immortalized B lymphocytes capable of fusing with rabbit splenocytes are used to produce hybrid cells that produce the antibodies. The immortalized B lymphocytes do not detectably express endogenous immunoglobulin heavy chains and, in certain embodiments, may contain a gene encoding a modified immunoglobulin heavy chain.

[0198] Compositions and Methods of Use The present disclosure provides compositions comprising CD40-specific antibodies, antigen-binding fragments thereof, and administration of such compositions in various therapeutic settings.

[0199] Administration of the CD40-specific antibodies described herein, in pure form or in a suitable pharmaceutical composition, can be effected by any mode approved for administration of agents that provide a similar utility. The pharmaceutical composition can be prepared by combining the antibody or antibody-containing composition with a suitable physiologically acceptable carrier, diluent or excipient, and formulated into a solid, semi-solid, liquid or gaseous dosage form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. In addition, other pharmaceutically active ingredients (including other anti-cancer agents described elsewhere herein) and / or suitable excipients, such as salts, buffers and stabilizers, although not necessary, may be present in the composition. Administration can be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical routes. The preferred mode of administration depends on the nature of the condition to be treated or prevented. An amount that reduces, inhibits, prevents or delays cancer progression and / or metastasis after administration is considered to be effective.

[0200] In certain embodiments, the amount administered is an amount sufficient to result in tumor regression as demonstrated by a statistically significant decrease in the amount of viable tumor, e.g., at least a 50% decrease in tumor mass or a change in scan dimensions (e.g., a statistically significant decrease). In other embodiments, the amount administered is an amount sufficient to result in a clinically significant reduction in the symptoms of a particular disease indication known to a skilled clinician.

[0201] The correct dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be conducted. The dosage can be varied as the severity of the condition is alleviated. The pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect and, moreover, to minimize undesirable side effects. The composition may be administered in a single dose or divided into a number of smaller doses and administered at intervals. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0202] The CD40-specific antibody-containing composition may be administered alone or in combination with other known cancer treatments, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition can also be administered in combination with an antibiotic.

[0203] Accordingly, exemplary routes of administration of these and related pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, intrarectal, vaginal, and intranasal routes. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intracostal injection, or infusion techniques. Pharmaceutical compositions according to certain embodiments of the invention are formulated so that upon administration to a patient, the active ingredient contained therein can be made biologically available. The composition to be administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of the CD40-specific antibody described herein in aerosol form can hold multiple dosage units. The actual methods of preparation of such dosage forms are known to, or will be apparent to, those of ordinary skill in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any event, the composition to be administered contains a therapeutically effective amount of an antibody of the present disclosure for treating the disease or condition of interest in accordance with the teachings herein.

[0204] The pharmaceutical composition may be in solid form or in liquid form. In one embodiment, the carrier(s) is / are granular because the composition is, for example, in tablet or powder form. The carrier(s) may be liquid if the composition is, for example, an oral oil, an injection solution, or an aerosol useful for inhalation administration. When oral administration is intended, the pharmaceutical composition is preferably in solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid.

[0205] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gums, wafers or the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of capsules, for example gelatin capsules, it may contain, in addition to materials of the above types, a liquid carrier such as polyethylene glycol or an oil.

[0206] The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may, by way of two examples, be for oral administration or for delivery by injection. When oral administration is intended, preferred compositions contain, in addition to the compound, one or more of a sweetening agent, a preservative, a dye / coloring agent and a flavor enhancer. In the case of compositions intended for administration by injection, they may contain one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent.

[0207] The liquid pharmaceutical composition, whether it is a solution, a suspension, or in other similar forms, may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils that can serve as solvents or suspension media, such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and agents for adjusting isotonicity, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The pharmaceutical composition for injection is preferably sterile.

[0208] The liquid pharmaceutical composition intended for either parenteral or oral administration must contain an amount of the CD40-specific antibody disclosed herein such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody in the composition. When oral administration is intended, this amount can be varied to be between 0.1% and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody. In certain embodiments, the pharmaceutical compositions and formulations according to the invention are prepared such that the parenteral dosage unit contains between 0.01% and 10% by weight of the antibody before dilution.

[0209] The pharmaceutical composition may be intended for topical administration, and in this case the carrier may suitably include a solution, an emulsion, an ointment or a gel base. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. There may also be a thickening agent present in the pharmaceutical composition for topical administration. When transdermal administration is intended, the composition may include a transdermal patch or an iontophoresis device. The pharmaceutical composition may also be intended for rectal administration, for example, in the form of a suppository that will melt in the rectum to release the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0210] The pharmaceutical composition may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may contain materials that form a coating shell around the active ingredient. The materials forming the coating shell are typically inert and can be selected, for example, from sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient can be placed in a gelatin capsule. The pharmaceutical composition in solid or liquid form may contain an agent that binds to the antibody of the present invention, thereby assisting in the delivery of the compound. Suitable agents that can act in this capacity include other monoclonal or polyclonal antibodies, one or more proteins, or liposomes. The pharmaceutical composition can consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote various systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery can be by liquefied or compressed gas, or by a suitable pump system for dosing the active ingredient. The aerosol can be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, and the like that can form a kit together. One skilled in the art can determine the preferred aerosol without undue experimentation.

[0211] The pharmaceutical composition can be prepared by methodologies well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection can be prepared by forming a solution of the composition containing the CD40-specific antibody described herein and optionally one or more of salts, buffers, and / or stabilizers together with sterile distilled water. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the antibody composition to facilitate the dissolution or homogeneous suspension of the antibody in an aqueous delivery system.

[0212] The composition can be administered in a therapeutically effective amount, which will vary depending on the specific compound utilized (e.g., a CD40-specific antibody); the metabolic stability and duration of action of the compound; the age, weight, general health, gender and diet of the patient; the mode and frequency of administration; the rate of excretion; drug combinations; the severity of the particular disorder or condition; and various factors including the subject being treated. Generally, a therapeutically effective daily dose will be from about 0.001 mg / kg (i.e., 0.07 mg) to about 100 mg / kg (i.e., 7.0 g) for a 70 kg mammal; preferably, the therapeutically effective amount will be from about 0.01 mg / kg (i.e., 0.7 mg) to about 50 mg / kg (i.e., 3.5 g) for a 70 kg mammal; more preferably, the therapeutically effective amount will be from about 1 mg / kg (i.e., 70 mg) to about 25 mg / kg (i.e., 1.75 g) for a 70 kg mammal.

[0213] The composition comprising a CD40-specific antibody of the present disclosure may also be administered simultaneously with, prior to, or subsequent to one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation containing the compound of the invention and one or more additional active agents, as well as administration of the antibody of the invention and each active agent in their own separate pharmaceutical dosage formulations. For example, the antibody and other active agents described herein can be administered together to a patient in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in separate oral dosage formulations. Similarly, the antibody and other active agents described herein can be administered together to a patient in a single parenteral dosage composition, such as a saline solution or other physiologically acceptable solution, or each agent can be administered in separate parenteral dosage formulations. When separate dosage formulations are used, the compositions comprising the antibody and one or more additional active agents can be administered essentially simultaneously, i.e., at the same time, or separately at staggered times, i.e., sequentially and in any order; combination therapy is understood to include all of these regimens.

[0214] Thus, in certain embodiments, administration of the anti-CD40 antibody compositions of the present disclosure in combination with one or more other therapeutic agents is also contemplated. Such therapeutic agents may be acceptable in the art as standard treatments for the particular medical conditions described herein, such as rheumatoid arthritis, inflammation or cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy agents, or other active and adjuvant agents.

[0215] In certain embodiments, the anti-CD40 antibodies disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbazom, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburemabine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodrubicin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine;Pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, for example, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone; antiadrenal drugs, for example, aminoglutethimide, mitotane, trilostane; folic acid supplements, for example, folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL (registered trademark), Bristol-Myers Squibb Oncology, Princeton, New Jersey) and docetaxel (TAXOTERE (registered trademark), Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, for example, cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;Difluoromethylornithine (DMFO); retinoid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine; pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition includes antihormonal agents that act to modulate or inhibit the hormonal action on tumors, such as antiestrogens (including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston)); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing are also included.;

[0216] A variety of other therapeutic agents can be used in combination with the anti-CD40 antibodies described herein. In one embodiment, the antibody is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) (including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate).

[0217] Exemplary NSAIDs are selected from the group consisting of ibuprofen, naproxen, sodium naproxen, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialates. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol or propoxyphene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone or prednisolone. Exemplary biological response modifiers include molecules directed at cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular) and minocycline.

[0218] In certain embodiments, the antibodies described herein are administered together with a cytokine. As used herein, the term "cytokine" refers to a general term for proteins released by a certain cell population that act as intercellular mediators on other cells. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Müllerian-inhibiting factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-β; platelet growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-α, β, and -γ; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors including leukemia inhibitory factor (LIF) and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cells, and bioactive equivalents of native sequence cytokines.

[0219] The composition comprising a CD40-specific antibody described herein can be administered to an individual suffering from a disease as described herein, and the diseases include, but are not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, pharyngeal carcinomas, malignant melanoma, as well as rituximab-resistant NHL and leukemia, autoimmune diseases and inflammatory diseases. Autoimmune diseases include, but are not limited to, arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), psoriasis and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, alopecia areata, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture syndrome, Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including patients with refractory aplastic anemia with frequent blood transfusions), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans syndrome, factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0220] Inflammatory disorders include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In certain embodiments, the inflammatory disorder is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndromes, and chronic obstructive pulmonary disease. In this context, one embodiment provides a method of treating an inflammation or inflammatory disorder, reducing the severity of an inflammation or inflammatory disorder, or preventing an inflammation or inflammatory disorder, by administering to a patient in need thereof a therapeutically effective amount of the compositions disclosed herein comprising an anti-CD40 antibody.

[0221] For in vivo use in the treatment of human diseases, the antibodies described herein are generally incorporated into a pharmaceutical composition prior to administration. The pharmaceutical composition comprises one or more of the antibodies described herein in combination with a physiologically acceptable carrier or excipient described elsewhere herein. To prepare the pharmaceutical composition, an effective amount of one or more of the compounds is mixed with any pharmaceutically acceptable carrier(s) or excipient(s) known to those skilled in the art to be suitable for a particular mode of administration. The pharmaceutically acceptable carrier may be liquid, semi-liquid, or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical application may contain, for example, sterile diluents such as water, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; and chelating agents such as ethylenediaminetetraacetic acid (EDTA); and buffers such as acetate, citrate, and phosphate. For intravenous administration, suitable carriers include solutions containing physiological saline or phosphate-buffered saline (PBS), as well as thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0222] The composition containing the CD40-specific antibody described in this specification can be prepared using a carrier that prevents the rapid excretion of the antibody from the body, for example, a sustained-release formulation or a coating. Such carriers include controlled-release formulations, such as, but not limited to, implants and microencapsulation delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, PEG, polyorthoesters, polylactic acid, and those known to those skilled in the art.

[0223] A treatment method using an antibody that binds to CD40 is provided in this specification. In one embodiment, the antibody of the present invention is administered to a patient having a disease associated with inappropriate expression of CD40, and the disease associated with inappropriate expression of CD40, in the context of the present disclosure, is, for example, abnormal CD40 expression or activity caused by a change in the amount of the existing protein (e.g., a statistically significant increase or decrease), or the presence of a mutant protein, or both. The excess amount can be caused by any cause, including, but not limited to, overexpression at the molecular level of CD40 relative to what can be normally detected, a persistent or accumulated aspect at the site of action, or increased (e.g., statistically significantly) activity. Such an excess amount of CD40 can be measured based on the normal expression, aspect, or activity of CD40 signaling events, and such measurement can play an important role in the development and / or clinical trials of the antibodies described in this specification.

[0224] This antibody is useful for the treatment of various cancers. In certain embodiments, the antibodies described herein exhibit antitumor activity by activating the antitumor immune response. In certain embodiments, the antibody is useful for the treatment of various cancers associated with abnormal expression of CD40. In one embodiment of the invention, a method of treating non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, and rituximab-resistant NHL and leukemia (but not limited thereto) by administering to a cancer patient a therapeutically effective amount of the CD40-specific antibody disclosed herein. After administration, an amount that statistically significantly inhibits, prevents or delays (i.e., based on an appropriate control known to those skilled in the art) cancer progression and / or metastasis is considered effective.

[0225] Another embodiment provides a method of preventing metastasis of cancer (but not limited thereto) including non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, and rituximab-resistant NHL and leukemia by administering to a cancer patient a therapeutically effective amount (e.g., an amount that statistically significantly inhibits, prevents or delays metastasis of cancer based on an appropriate control known to those skilled in the art after administration) of the CD40-specific antibody disclosed herein.

[0226] Another embodiment provides a method of preventing cancer (but not limited thereto) including non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, and rituximab-resistant NHL and leukemia by administering to a cancer patient a therapeutically effective amount of the CD40-specific antibody disclosed herein.

[0227] Another embodiment provides a method for treating non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, pancreatic, colon, gastrointestinal, prostate, bladder, kidney, ovarian, cervical, breast, lung, hypopharyngeal carcinomas, malignant melanoma, as well as rituximab-resistant NHL and leukemia, for ameliorating the symptoms of the disease, for inhibiting the progression of the disease, or for preventing the disease, the method comprising administering to a patient afflicted with one or more of these diseases a therapeutically effective amount of a CD40-specific antibody disclosed herein.

[0228] Another embodiment provides a method for treating an autoimmune disease, for ameliorating the symptoms of the disease, for inhibiting the progression of the disease, or for preventing the disease, the method comprising administering to a patient afflicted with one or more of these diseases a therapeutically effective amount of an anti-CD40 antibody disclosed herein. In this context, autoimmune diseases include, but are not limited to, arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), psoriasis and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, alopecia areata, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplastic anemia (including patients with transfusion-dependent aplastic anemia), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans syndrome, factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0229] Another embodiment provides a method for treating an inflammatory disease, ameliorating the symptoms of the disease, inhibiting the progression of the disease, or preventing the disease, the method comprising administering to a patient suffering from one or more of these diseases a therapeutically effective amount of the anti-CD40 antibody disclosed herein. Inflammatory diseases include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In certain embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndrome, and chronic obstructive pulmonary disease.

[0230] In another embodiment, the anti-CD40 antibody of the invention can be used to determine the structure of a binding antigen, such as a conformational epitope, and then the structure can be used to develop compounds having or mimicking this structure, for example, by chemical modeling and SAR methods.

[0231] Various other embodiments of the invention relate, in part, to diagnostic applications for detecting the presence of cells or tissues that express CD40. Accordingly, the present disclosure provides methods for detecting CD40 in a sample, such as detecting cells or tissues that express CD40. Such methods can utilize a variety of known detection formats including, but not limited to, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole mount in situ hybridization (WISH), fluorescence DNA in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA).

[0232] ISH is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., primary binders) to localize specific DNA or RNA sequences in a part or section of a cell or tissue (in situ) or, if the tissue is small enough, the whole tissue (whole mount ISH). It will be understood by those skilled in the art that this is different from immunohistochemistry, which uses antibodies as primary binders to localize proteins in tissue sections. DNA ISH can be used with genomic DNA to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can be used, for example, in medical diagnostics to assess the integrity of chromosomes. RNA ISH (hybridization histochemistry) is used to measure and localize mRNA and other transcripts within tissue sections or whole mounts.

[0233] In various embodiments, the antibodies described herein are conjugated to a detectable label that can be detected directly or indirectly. In this context, an antibody “conjugate” refers to an anti-CD40 antibody that is covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, their antigen-binding fragments, and their antibody derivatives, such as single-chain variable fragment antibodies or epitope-tagged antibodies, can all be covalently linked to a detectable label. In the case of “direct detection”, only one detectable antibody, i.e., a primary detectable antibody, is used. Thus, direct detection means that the antibody itself, which is conjugated to a detectable label, can be detected without the need for an additional second antibody (secondary antibody).

[0234] A "detectable label" is a molecule or material that can produce a detectable signal (e.g., visually, electronically or otherwise) indicating the presence and / or concentration of a label in a sample. When conjugated to an antibody, the detectable label can be used to locate or quantify the target to which the specific antibody is directed. Thereby, the presence and / or concentration of the target in the sample can be detected by detecting the signal generated by the detectable label. The detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific antibodies can be used in combination to detect one or more targets.

[0235] Examples of detectable labels that can be detected directly include fluorescent dyes, radioactive substances, and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after the application of the primary antibody. Thus, this detection is performed by detecting the binding of the secondary antibody or binder to the primary detectable antibody. Examples of primary detectable binders or antibodies that require the addition of a secondary binder or antibody include enzymatically detectable binders and hapten-detectable binders or antibodies.

[0236] In some embodiments, a detectable label is conjugated to a nucleic acid polymer comprising a first binder (e.g., in the case of an ISH, WISH or FISH process). In other embodiments, a detectable label is conjugated to an antibody comprising a first binder (e.g., in the case of an IHC process).

[0237] Examples of detectable labels that can be conjugated to the antibodies used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

[0238] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidocaproic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3 and Cy5, optionally substituted coumarins (including AMCA), PerCP, phycobiliproteins (including R-phycoerythrin (RPE) and allophycocyanin (APC)), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogs, and conjugates of R-phycoerythrin or allophycocyanin, inorganic fluorescent labels such as particles based on semiconductor materials such as coated CdSe nanocrystals.

[0239] Examples of polymer particle labels include microparticles or latex particles of polystyrene, PMMA or silica, or polymer micelles or capsules containing dyes, enzymes or substrates, into which fluorescent dyes can be embedded.

[0240] Examples of metal particle labels include gold particles and coated gold particles that can be converted by a silver stain. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of substrates commonly used for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker·Yates reagent (HYR), indophenol blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), α-naphthol pyronin (α-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-β-D-galactoside / ferro-ferricyanide (BCIG / FF).

[0241] Examples of substrates commonly used for alkaline phosphatase include naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / -fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuchsine (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazolium (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG).

[0242] Examples of luminescent labels include luminol, isoluminol, acridinium ester, 1,2-dioxetane, and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0243] A detectable label can be linked to the antibodies described herein, or to any other molecule that specifically binds to a target biological marker, such as an antibody, nucleic acid probe, or polymer. Furthermore, it will be understood by those skilled in the art that the detectable label can also be conjugated to a second and / or third and / or fourth and / or fifth binding agent or antibody, etc. Moreover, it will be understood by those skilled in the art that each additional binding agent or antibody used for characterizing the target biological marker can serve as a signal amplification step. When the detectable substance is, for example, a dye, colloidal gold particles, or a luminescent reagent, the biological marker can be visually detected using, for example, light microscopy, fluorescence microscopy, or electron microscopy. The visually detectable substance bound to the biological marker can also be detected using a spectrophotometer. When the detectable substance is a radioisotope, the detection can be visual detection by autoradiography or non-visual detection using a scintillation counter. See, for example, Larsson, 1988, Immunocytochemistry: Theory and Practice (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, Volume 80, 1998, John D. Pound (ed.) (Humana Press, Totowa, N.J.).

[0244] The present invention further provides a kit for detecting CD40 or cells or tissues expressing CD40 in a sample, the kit containing at least one antibody, polypeptide, polynucleotide, vector, or host cell described herein. In certain embodiments, the kit may include a buffer, an enzyme, a label, a substrate, beads or other surfaces to which the antibodies of the present invention are attached, and the like, as well as instructions for use.

Examples

[0245] Example 1: Production and Humanization of Anti-CD40 Antibodies Four New Zealand white rabbits were immunized with recombinant rabbit Fc-hCD40. Rabbits with the highest serum titers of specific binding to human CD40 were selected for cell fusion. A total of 172 hybridomas were identified as positive binders to soluble Fc-hCD40, and 44 of these clones were found to be positive binders to cell surface CD40. After epitope-clustering assay, 24 representative hybridomas were selected for recombinant expression and further characterized. A second functional screening was performed, including and as further described below: 1) induction of DC maturation as measured by CD80, CD83, CD86 upregulation (agonist activity); induction of direct tumor growth inhibition (agonist activity); and 3) ADCC antibody effector function. Candidates were selected based on a dual-functional screening that included the following two arms: 1) binding affinity, antibody internalization, antibody-dependent cell cytotoxicity (ADCC), complement-dependent cell cytotoxicity (CDC) and antibody-dependent cell phagocytosis (ADCP); and 2) agonist DC activation / maturation function, receptor-ligand interaction, mixed lymphocyte reaction (MLR), cell proliferation and apoptosis.

[0246] Screening of agonist antibodies by dendritic cell maturation To further clarify the agonist or antagonist effects of the initial panel of anti-CD40 antibodies, a DC maturation assay was used as an indicator for screening functional antibodies. Anti-CD40 or control antibodies were added to human monocyte-derived DC culture solutions over a period of two days. Upregulation of CD83, one of the best-known maturation markers of human dendritic cells, was measured to screen for agonist antibodies. The mouse monoclonal antibody 5C11, which induces dendritic cell maturation, was used as a positive control. Antibodies R-3, R-6, R-8, R-9, R-16, R-18, R-24, R-33, R-36, 19-21, 19-45, and 19-59 increased more than 50% of CD83 expression compared to the Ig control (Figure 1A). DC maturation was further determined by measuring the antibody-induced upregulation of the costimulatory molecules CD80 and CD86 for the selected antibodies. As shown in Figures 1B and 1C, antibodies R-3, R-8, R-9, R-33, and 19-21 upregulated both CD80 and CD86, while the other antibodies had only a slight effect. These results were consistent with the CD83 modulation effects of these antibodies. Interestingly, among the antibodies capable of inducing DC maturation, only clone 19-21 showed strong activity to enhance T cell proliferation in the mixed lymphocyte reaction (Figure 1D).

[0247] Screening for direct inhibition of tumor growth A panel of agonist anti-CD40 antibodies was further evaluated for its ability to induce tumor growth inhibition in CD40-expressing tumor cells. All anti-CD40 antibodies tested inhibited tumor cell proliferation. Antibody 19-21 exhibited the highest titer. (Figure 2).

[0248] Screening for ADCC activity In addition to APC activation and induction of tumor growth inhibition, antibody effector function, ADCC, was used as an important criterion for screening and ranking antibody candidates. To perform an ADCC assay using human PBMC, all selected antibodies were converted from rabbit mAbs to chimeric mAbs with rabbit Fab and human IgG1. As shown in Figure 3, all selected candidates showed significant ADCC activity compared to the IgG1 control. Based on the maximum ADCC activity, the lead mAbs were ranked as cR-8 > cR-3 > cR-33 > c19-21 > cR-9 > c19-59.

[0249] Four candidates (c19-21, cR-8, cR-3, cR-33) were selected based on in vitro functional screening. Their in vitro characterization is summarized in Table 1. Antibody c19-21 strongly enhanced DC activation and tumor growth inhibition, while antibodies cR-8 and cR-3 showed more potent ADCC activity.

Table 1

[0250] In vivo anti-tumor activity screening Since the top four candidates showed different potencies in different in vitro assays, the inventors evaluated and compared their anti-tumor activities in vivo for lead selection. vivo research was conducted. The Ramos tumor xenograft model was used. Tumor-bearing mice were intraperitoneally treated with the chimeric antibodies cR-3, cR-8, cR-33, or c19-21 at 5 mg / kg three times a week for a total of 9 doses (8 mice per group). The antitumor activity of rituximab at the same regimen was used as a reference. As shown in Figure 4A, cR-8 and cR-3 showed the strongest antitumor effects. In contrast, 19-21 exhibited lower antitumor activity and the tumor rebounded more rapidly after drug withdrawal. The antitumor effect of cR-33 was intermediate but still showed better in vivo efficacy than rituximab. The in vivo efficacy of antibodies cR-3 and cR-8 was further evaluated in a dose-response study. As shown in Figure 4B, cR-8 showed more potent antitumor efficacy than cR-3 and was thus identified as the lead anti-CD40 antibody.

[0251] The amino acid sequences of the heavy and light chain variable regions of the R-8 clone are shown in SEQ ID NOs: 1 and 2. The amino acid sequences of its VH and VL CDRs are shown in SEQ ID NOs: 3-5 and 6-8, respectively. The amino acid sequences of the heavy and light chain sequences of some of the other antibody candidates that showed functional activity are shown in SEQ ID NOs: 11-56. The VHCDR and VLCDR amino acid sequences of these antibodies are provided in SEQ ID NOs: 57-194. Figure 16 shows the alignment of these sequences including the R-8 clone, with the CDRs underlined.

[0252] R-8 was humanized using the unique mutagenesis library-guided (MLG) humanization technology (see, for example, U.S. Patent No. 7,462,697). The light and heavy chain frameworks of humanized R-8 (APX005) are 95% identical to human germline sequences. The amino acid sequences of its humanized VH and VL regions are shown in SEQ ID NOs: 9 and 10, respectively. The binding of APX005 to CD40 was found to be similar to that of its parental clone R-8.

[0253] Example 2: In Vitro Characterization of the APX005 Humanized Anti-CD40 Antibody Numerous in vitro experiments were performed to further characterize the APX005 humanized antibody.

[0254] APX005 selectively binds to CD40 The binding selectivity of APX005 was evaluated by direct ELISA against a panel of TNFR family proteins. A total of 1 μg / mL of fusion proteins of rabbit Fc and CD40, RANK TweakR, OX40, DR5, and 4-1BB were coated on ELISA plates. Bound APX005 was detected using goat anti-human HRP-conjugated IgG. As shown in Figure 5, APX005 selectively binds to human CD40 but not to the other TNFR family proteins tested.

[0255] APX005 blocks the binding of CD40L to CD40 ELISA was performed to evaluate the effect of APX005 on the binding of CD40L to CD40. Specifically, CD40L (final concentration of 4 μg / mL) was used to bind immobilized human CD40 on ELISA plates, and the immobilized CD40 was pre-incubated with APX005, after which the change in the amount of CD40L binding to CD40 was measured. The binding of CD40L to immobilized CD40 was detected with a mouse anti-CD40L monoclonal antibody. As shown in Figure 6, APX005 blocks the binding of CD40L to CD40. In contrast, SGN-40 increases the binding.

[0256] The APX005 / CD40 complex is not internalized To assess the target-mediated internalization of APX005 to evaluate its impact on ADCC activity, Ramos cells were incubated with APX005 for 4 hours at 37°C (temperature allowing internalization) or for 30 minutes at 4°C (temperature minimizing internalization). Cells were washed with staining buffer and then incubated with Alexa 488-labeled goat anti-human IgG for an additional 30 minutes at 4°C. FACS analysis was performed to examine the level of APX005 on the cell surface. As shown in Figure 7, after incubation at 37°C, there was no reduction (slight increase) in the level of APX005 on the cell surface. This data suggests that upon binding to CD40, the APX005 / CD40 complex is not internalized by tumor cells and thus optimal conditions for effector cell recruitment to ADCC are brought about.

[0257] APX005 mediates ADCC To evaluate the ADCC activity of APX005 against tumor cells expressing CD40, CD40-expressing Ramos and Daudi were used as target cells and fresh human peripheral blood mononuclear cells (PBMC) were used as effector cells. ADCC was measured by the calcein-AM release assay. Target cells were labeled with calcein-AM (15 uM / 10 6 cells), washed, and placed at 5 × 10 per well in a round-bottom 96-well plate 3Then, plating was performed in triplicate. Either APX005 or the control antibody at increasing concentrations (0.0001 - 10 μg / mL) was pre-incubated at 4°C for 30 minutes, and then PBMC effector cells from healthy human donors were added at a final effector:target cell ratio of 40:1 in a final volume of 200 μL per well. Experiments were conducted using PBMC from at least three different donors. After 4 hours of incubation, 100 μL of the culture supernatant was transferred to a Black View Plate - 96 well plate and read for arbitrary fluorescence units (AFU) using a Victor II plate reader (485 nm excitation / 535 nm emission). Percent specific lysis = (mean experimental release of AFU - mean spontaneous release of AFU) / (mean maximum release of AFU - mean spontaneous release of AFU). As shown in Figure 8, APX005 induced ADCC in a dose - dependent manner. A similar effect was observed for SGN - 40. The different sensitivities of Ramos and Daudi cells to ADCC may be due to different CD40 expression levels (Cancer Res 2005;65:8331 - 8338).

[0258] APX005 inhibits tumor cell proliferation. To assess the ability of APX005 to inhibit tumor cell proliferation, Ramos cells were seeded at 50,000 cells / well into 200 μL of RPMI 1640 supplemented with 10% FBS containing various concentrations of APX005, SGN-40 or control human IgG in 96-well flat-bottom plates. For crosslinking, APX005, SGN-40 or control IgG was pre-incubated for 30 minutes at room temperature in the medium with the F(ab’)2 fragment of a goat anti-human IgG Fc fragment-specific antibody and then added to the cells. The cells were treated for a total of 72 hours. Then, 10% AlamarBlue® (Serotec, Oxford, UK) was added to each well and incubated for an additional 24 hours. Cell viability was measured with a CytoFluor fluorescence reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. All studies were performed in duplicate and in triplicate for each sample concentration. As shown in Figure 9, monomeric APX005 inhibited the proliferation of Ramos cells (Figure 9A). When APX005 was crosslinked with a secondary antibody, it resulted in increased and dose-dependent growth inhibitory effects (Figure 9B). Crosslinking of APX005 can be achieved in vivo by Fc receptor-expressing cells.

[0259] APX005 induces DC activation To assess the ability of APX005 to stimulate DC cell maturation, PBMCs were prepared by density gradient centrifugation using lymphocyte separation solution. After incubation at 37°C for 2 hours, adherent monocytes were collected. The isolated monocytes were cultured in 24-well plates with 100 ng / mL of recombinant human GM-CSF and 100 ng / mL of recombinant human IL-4 in RPMI1640 medium supplemented with 10% FCS. Half of the medium was replaced after 3 days. On the 5th day of culture, 1.3 nM of anti-CD40 antibody, CD40L or control antibody was added to those DC cells and further cultured in 24-well plates for 48 hours. For DC activation marker staining, PE-conjugated anti-CD83, anti-CD86 antibody and anti-CD80 antibody were used. Analysis was performed using FACS. The data are from one representative study. As shown in Figure 10, APX005 induced significant DC maturation. Its effect appears to be more potent than SGN-40 and CD40L. The increased activation of DCs can lead to a more potent anti-tumor T cell response.

[0260] APX005 is cross-reactive with cynomolgus CD40 but not with mouse CD40 Cross-reactivity was evaluated by direct ELISA. A total of 1 μg / mL of human CD40, cynomolgus CD40 or mouse CD40 was coated on ELISA plates and then incubated with 1 μg / mL of APX005 or control IgG1. Antibodies bound to CD40 were detected using goat anti-human IgG conjugated to HRP. APX005 clearly cross-reacts with cynomolgus CD40 but not with mouse CD40. (Figure 11A).

[0261] The cross-reactivity of APX005 with mouse CD40 was further determined by FACS binding to the mouse A20 cell line expressing mouse CD40. 0.5×10 6An aliquot of A20 cells was added to a 96-well plate and incubated with 100 μL of diluted rat anti-mouse CD40 antibody (conjugated with PE), APX005 or IgG1 control antibody. After washing, 100 μL of goat anti-human IgG (H+L) (Southern Biotech CAT#2040-09) conjugated with R-PE was added to the samples at a 1:200 dilution in PBS and incubated. The rat anti-mouse CD40 antibody conjugated with PE was used as a positive control. The samples were resuspended in 0.5 mL PBS and analyzed by FACS. The FACS data showed that APX005 did not cross-react with mouse CD40 (Figure 11B).

[0262] In summary, the experiments in this example demonstrated that APX005 is a humanized IgG1 antibody that binds CD40. APX005 specifically binds to CD40 with a Kd of 9.6×10 -10 M and blocks CD40L binding to CD40. This is in contrast to the SGN40 anti-CD40 antibody, which enhances the CD40-CD40L interaction. This suggests that these two antibodies bind to different epitopes. In vitro, APX005 demonstrated potent ADCC activity against CD40-positive lymphoma cells (Ramos and Daudi), as well as the ability to directly inhibit tumor cell (Ramos) proliferation upon crosslinking. APX005 also stimulated dendritic cell maturation and enhanced the cellular immune response. In addition, APX005 was shown to cross-react with cynomolgus CD40.

[0263] Example 3: In Vivo Characterization of the APX005 Humanized Anti-CD40 Antibody Numerous in vivo experiments were conducted to further characterize the APX005 humanized antibody.

[0264] Inhibition of Tumor Growth by APX005 in the Ramos Model To evaluate the effect of APX005 on a xenograft model of human B-cell lymphoma, female BALB / c nu / nu mice, 6 - 8 weeks old, were used for tumor cell inoculation. 1×10 7 Xenografts were established by subcutaneous inoculation of 1×10 7 tumor cells / mouse into the dorsal and lateral flanks. When tumors reached an average volume of approximately 100 mm3 (50 - 200 mm3), the mice were randomly assigned to groups. Starting on day 13, an antibody at 3 mg / kg was administered intraperitoneally (see Figure 12). A total of 9 doses were administered three times a week (8 mice per group). The vertical dimension of the tumor was measured using a Vernier scale caliper. Tumor volume was calculated using the following formula: Volume = (length × width 2 ) / 2. As shown in Figure 12A, APX005 demonstrated potent and sustained antitumor activity. To determine in vivo drug levels by measuring human IgG concentration, serum was collected on day 34, 2 days after the final dose (see Figure 12B). The antitumor efficacy mediated by APX005 was greater than that of SGN-40 and was sustained longer after the dosing period. A single-point PK analysis showed that the superior antitumor activity of APX005 was not due to PK differences.

[0265] Inhibition of APX005 on rituximab-resistant tumors pretreated with rituximab The purpose of this experiment was to evaluate the effect of APX005 on rituximab-resistant B-cell lymphoma pretreated with rituximab. Nude mice bearing established Ramos tumors were first treated with 3 mg / kg of rituximab over 5 doses. Tumor growth was partially inhibited by rituximab (Figure 13A). When these tumors reached approximately 700 mm 3Once they reached the size, those mice were randomly assigned to 4 groups (7 mice per group), and re-treated via the intraperitoneal route for 3 weeks with APX005, rituximab, SGN40 analog 3 mg / kg or saline control (Figure 13B). As shown in Figure 13, the rituximab-pretreated tumors could not respond to rituximab re-treatment. This suggests that these tumors are rituximab-resistant (Figure 13B). APX005 demonstrated the ability to inhibit the growth of rituximab-resistant tumors.

[0266] APX005 Inhibition of Tumor Growth in the Raji Model The purpose of this experiment was to determine the relationship between the dose and efficacy of APX005 in vivo. Nude mice bearing established CD40-positive Raji tumors were treated with APX005 starting on day 15. Doses of APX005 ranging from 0.1 mg / kg to 10 mg / kg were administered intraperitoneally 3 times / week for 2 weeks (8 mice per group) (see Figure 14). Saline was used as a control treatment. Tumor volume was measured on each dosing day. Serum levels of APX005 in each group were also measured 3 days after the final dosing to determine the correlation between its in vivo efficacy and the level of APX005 in circulation. A clear dose-dependent anti-tumor activity was observed (see Figure 14). The difference in tumor volume between the control group and the antibody-treated groups at dose levels ≥1 mg / kg on days 29 to 33 was significant (P≤0.05). The minimum effective dose was determined to be 1 mg / kg, which corresponded to a median serum concentration of 0.49 μg / mL on day 36. The differences in tumor volume among the 3, 5, and 10 mg / kg dose groups were not statistically significant. Therefore, the maximum anti-tumor activity was achieved at doses ≥3 mg / kg with a median serum concentration ≥1.6 μg / mL.

[0267] Inhibition of Tumor Growth by APX005 in the Human MM IM-9 Model To evaluate the antitumor activity of APX005 in a human multiple myeloma model, nude mice bearing established CD40-positive multiple myeloma IM-9 tumors were intraperitoneally treated with APX005 or SGN40 starting on day 15. APX005 was given at 3 mg / kg three times a week for 3 weeks (5 mice per group). Tumor volume was measured on each dosing day.

[0268] APX005 demonstrated potent antitumor activity against human multiple myeloma in the IM-9 xenograft model (see Figure 15). The antitumor efficacy mediated by APX005 was significantly greater than that of SGN-40 (P < 0.05).

[0269] Inhibition of tumor growth by APX005 in the Ramos model compared to SGN-40 and rituximab The purpose of this experiment was to compare the antitumor activities of APX005, rituximab, and SGN-40 in a human B-cell lymphoma Ramos xenograft model. Female SCID C.B-17 mice were inoculated subcutaneously with Ramos cells on the dorsal and lateral flanks to establish xenografts. When the tumors reached an average volume of approximately 200 - 300 mm 3 the mice were randomly assigned to 6 groups. Antibodies were administered intraperitoneally at the doses shown in Figure 17. A total of 9 doses were administered three times a week (10 mice per group). The vertical dimension of the tumor was measured using a Vernier scale caliper. Tumor volume was calculated using the following formula: volume = (length × width 2 ) / 2. The survival period of the mice was also determined and recorded.

[0270] APX005 demonstrated dose-dependent antitumor activity. Treatment with high-dose APX005 (10 mg / kg) resulted in complete tumor regression, while rituximab only delayed tumor growth at the same dose (10 mg / kg). This suggests that APX005 is more potent than rituximab in this model. APX005 is also more potent than SGN-40 (Figure 17A). APX005 not only inhibited tumor growth but also improved the survival of tumor-bearing mice (Figure 17B).

[0271] Inhibition of tumor growth in a rituximab-resistant human Namalwa lymphoma xenograft model The purpose of this experiment was to compare the antitumor activities of APX005, rituximab, and SGN-40 in a rituximab-resistant human Namalwa lymphoma model. Xenografts were established by subcutaneous inoculation of Namalwa cells into the dorsal and flank regions of female SCID C.B-17 mice. When the tumors reached an average volume of approximately 200 - 300 mm3, the mice were randomly assigned to 6 groups. Antibodies were administered intraperitoneally at the doses shown in Figure 18. A total of 9 doses were administered three times a week (10 mice per group). The vertical dimension of the tumors was measured using a Vernier scale caliper. The tumor volume was calculated using the following formula: Volume = (length × width 2 ) / 2. The survival of the mice was also determined and recorded.

[0272] APX005 demonstrated potent antitumor activity in a rituximab-resistant Namalwa lymphoma model (Figure 18A). APX005 also improved the survival of rituximab-resistant tumor-bearing mice (Figure 18B).

[0273] In summary, the experiments in this example showed that the efficacy of APX005 was tested in a number of xenograft tumor models. APX005 significantly inhibited tumor growth in the Ramos model. Interestingly, its therapeutic effect persisted well beyond the dosing period. Treatment with APX005 resulted in inhibition of rituximab-resistant tumors pre-treated with rituximab. A study to find the dose range was conducted in the Raji model, and it was found that the minimum effective dose was determined to be 1 mg / kg, and the maximum anti-tumor activity was observed at a dose ≥ 3 mg / kg. In addition to B-cell lymphoma, APX005 also exhibited significant and potent anti-tumor activity in the human multiple myeloma IM-9 model.

[0274] Therefore, the above example demonstrates that the use of APX005 can improve the treatment of patients with NHL, CLL, multiple myeloma, and certain solid tumors expressing the CD40 target. When binding to CD40, APX005 mobilizes cytotoxic cells to kill tumor cells by ADCC. APX005 can also directly inhibit tumor cell proliferation and activate APCs through its agonist activity. In vivo, APX005 significantly inhibited the growth of a number of CD40-expressing human tumor xenografts and showed a persistent anti-tumor effect. APX005 is also capable of inhibiting human multiple myeloma and rituximab-resistant tumors pre-treated with rituximab.

[0275] (Example 4: The APX005 S267E Fc mutant increases binding to FcγRIIB and CD40 agonist activity) In an attempt to increase the binding to the FCγ receptor and the agonist activity of the APX005 antibody, serine (S) at position 267 of APX005 Fc (IgG1, EU numbering) was mutated to glutamic acid (E) to generate the APX005 S267E variant. The mutation from AGC (S) to GAG (E) at position 267 located in the heavy chain Fc region of APX005 was confirmed by sequencing. The IgG1 heavy chain constant region containing the modified Fc region sequence is provided in SEQ ID NO: 195 (this sequence includes CH1, hinge, CH2 and CH3 of IgG1).

[0276] To evaluate the CD40 binding ability of the APX005 S276E variant, the heavy chain plasmid and light chain plasmid of APX005 S267E were purified from transformed bacteria and transfected into 293-6E cells. Four days after transfection, the culture supernatant containing the antibody was collected and tested for binding to CD40 using ELISA (ELISA plate coated with CD40-Fc protein). The data of the above ELISA showed that the APX005 S267E variant had CD40 binding activity similar to that of APX005 (see Figure 19).

[0277] In another experiment, the binding ability of the above CD40 antibody to the native CD40 protein was determined by FACS analysis using live Ramos cells. Ramos cells were collected and blocked with 1.5 ml of blocking buffer (0.1% BSA / PBS) for 30 minutes. Antibodies at various concentrations were added to the cells and incubated for 1 hour. After washing, 50 μl of goat anti-human IgG (H+L) PE antibody (diluted 1:1000 in the blocking solution) was added and incubated for 30 minutes. Then the cells were washed with PBS and resuspended in 1 m PBS, and analyzed by FACS It was analyzed. The antibodies tested were SGN-40 developed by Seattle Genetics and CP870893 from Pfizer, which are 19-21 rabbit anti-CD40 agonist antibodies. As shown in Figure 20, APX005 and the APX005 S267E variant showed CD40 binding affinity similar to that of SGN40 but 6-fold higher than that of CP870893.

[0278] Experiments were conducted to compare the CD40 agonist activities of APX005, the APX005 S267E variant, and other anti-CD40 antibodies in a B cell activation assay. Specifically, in vitro, after treatment with various anti-CD40 antibodies, CD40 agonist activity was tested by measuring the upregulation of CD86, a B cell activation marker, on human B cells. Fresh human B cells were obtained by isolating B cells from peripheral blood using the negative selection method (whole cells). The above B cells were treated with different human antibodies at 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, and 0.0045 μg / ml for 48 hours. The cells were then collected and analyzed for human CD86 expression on viable B cells by flow cytometry. 19-21, SGN-40, and CP870893 (see the above description) were tested. As shown in Figure 21, the APX005 S267E variant demonstrated significantly increased potency (EC50) and efficacy (EC100) compared to APX005. Among the anti-CD40 antibodies tested in this assay, APX005 S267E was the most potent CD40 agonist antibody.

[0279] Further experiments were conducted to evaluate the ADCC activity of APX005 and the APX005 S267E variant. Specifically, the in vitro ADCC effect mediating ability of the CD40 antibody was assayed using Daudi cells as target cells and human PBMC as effector cells. Labeled target cells were seeded at 5×10 per well in a round-bottom 96-well plate. 3Cells were plated in triplicate. Antibodies at increasing concentrations (50 μl per well in complete medium) were added to the labeled cells described above. The final antibody concentrations ranged from 0 to 10 μg / ml (in complete medium). The cells were treated with the antibodies at 37 °C for 30 minutes. Effector cells (PBMC) in complete medium were seeded onto the target cells (effector cell / target cell ratio = 40 / 1). The final volume of effector cells was 100 μl / well. After incubation for 4 hours in a 37 °C incubator, 100 μl of the culture supernatant from each well was transferred to a 96-well black view plate, and the RFU was read at 485ex / 535em. Specific lysis was calculated according to the formula: Specific lysis = [(test release - natural release) / (maximum release - natural release)] × 100 and. Rituxan was used as a positive control. As shown in Figure 22, the above-mentioned APX005 S267E mutant showed slightly increased ADCC activity compared to the APX005 wild type.

[0280] Example 5 Characterization of the epitope of APX005 This example describes the characterization of the epitope of APX005, a humanized anti-CD40 antibody. As described in more detail below, a total of 5,841 linear peptides and peptides chemically linked to a scaffold (CLIPS) were synthesized and analyzed for binding. The dominant binding regions were 92 TSEACESCVLHRSCSP 107 (SEQ ID NO: 196) and 125 PCPVGFFSNVSSAFEKCHPW 144 (SEQ ID NO: 197). Among the identified binding residues, T 92 , E 97 and 100 VL 101 are known to be contact residues between CD40 and the CD40L trimer. Specifically, E 97 is considered an important residue for the binding of CD40 to the ligand.

[0281] The CD40 protein sequence is shown in SEQ ID NO: 198, and this is directly available as the single crystal structure 3QD6. The residues of CD40 that are predicted to bind to the CD40L trimer are E74, Y82, D84, N86, and E117 (see, e.g., Bajorath et al., 1995 Biochemistry 34:9884-9892. Note that the numbering in the 3QD6 structure is 20 residues larger than the numbers referred to herein. Thus, the predicted important binding residues are E54, Y62, D64, N66, and E97 are as follows.

[0282] Peptide synthesis and screening procedures

[0283] To reconstruct the discontinuous epitopes of the target molecule, a library of structured peptides was synthesized. This was done using the technology of Chemically Linked Peptides on Proprietary Supports of Pepscan (CLIPS) (Pepscan Presto, Lelystad The Netherlands) (see, for example, Timmerman et al. (2007). J. Mol. Recognit. 20:283-99; Slootstra et al. (1996). Molecular Diversity 1: 87-96). The CLIPS technology provides the ability to structure peptides into single loops, double loops, triple loops, sheet-like foldings, helix-like foldings and combinations thereof. The CLIPS template is attached to cysteine residues. For this experiment, the side chains of multiple cysteines of the above peptides were attached to one or two CLIPS templates. For example, a 0.5 mM solution of 1,3-bis(bromomethyl)benzene, a T2 CLIPS template, was dissolved in ammonium bicarbonate (20 mM, pH 7.9) / acetonitrile (1:1 (v / v)). This solution was added to the above series of peptides. The above CLIPS template binds to the side chains of two cysteines present in the peptides bound to the solid phase of the above series of peptides (455-well plate with 3 μl wells). The above series of peptides was gently shaken in the above solution for 30 - 60 minutes with the peptides completely immersed in the solution. Finally, the above series of peptides was thoroughly washed with excess H2O and sonicated in a disruption buffer containing 1 percent SDS / 0.1 percent β-mercaptoethanol in PBS (pH 7.2) at 70 °C for 30 minutes and then sonicated in H2O for an additional 45 minutes. The T3 CLIPS-bearing peptides were prepared in a similar manner except that they contained three cysteines.

[0284] The binding of antibodies to each of the synthesized peptides was tested in a PEPSCAN-based ELISA. The series of peptides were incubated with the primary antibody solution (overnight at 4 °C). After washing, the series of peptides were incubated with a 1 / 1000 dilution of the antibody peroxidase conjugate (SBA, catalog number 2010-05) for 1 hour at 25 °C. After washing, 2,2’-azino-di-3-ethylbenzthiazoline sulfonate (ABTS), a peroxidase substrate, and 2 μl / ml of 3 percent H2O2 were added. After 1 hour, the color development was measured. The color development was quantified using a charge-coupled device (CCD)-camera and an image processing system.

[0285] Data processing The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The results were quantified and stored in the Peplab database. The binding values were extracted for analysis. Occasionally, the wells contained air bubbles, generating false positive values. The cards were manually inspected and any values caused by air bubbles were scored as 0.

[0286] Epitope determination was performed by a combination of 20-mer overlapping peptides and single residue mutagenesis of 12 specific regions of the protein. These 12 regions were chosen based on the known three-dimensional structure of CD40 and the published cysteine bonds in the protein. A total of 5841 chemically synthesized CLIPS peptides were synthesized.

[0287] The evaluation of the overlapping 20-mer peptides showed two distinct binding regions ( 92 TSEACESCVLHRSCSP 107 (SEQ ID NO: 196) and 125 PCPVGFFSNVSSAFEKCHPW 144 (SEQ ID NO: 197)). Similar results were obtained by the evaluation of overlapping sequences with two alanine substitutions.

[0288] Each of the 12 selected candidate epitope "hot spots" contains at least 10 identical "control peptides" with no mutations. Evaluation of these control sequences for each of the 12 mutagenesis data sets identified two distinct but similar binding regions ( 84 TCEEGWHCTSEACESCVLH 102 (SEQ ID NO: 199) and 125 PCPVGFFSNVSSAFEKCHPW 144 (SEQ ID NO: 197)). The resulting ELISA readout information was highly statistically significant (p < 2e -10 ). 92 TSEACESCVLHRSCSP 107 (SEQ ID NO: 196) and 84 TCEEGWHCTSEACESCVLH 102 (SEQ ID NO: 199), residues 92 - 102 (SEQ ID NO: 202) are probably the residues most relevant to binding. Data from a CLIPS matrix set combining three different sequence regions to identify discontinuous epitopes provided no further insight.

[0289] Detailed analysis of two mutagenesis data sets (84TCEEGWHCTSEACESCVLH102 (SEQ ID NO: 199) and 125PCPVGFFSNVSSAFEKCHPW144 (SEQ ID NO: 197)) showed no important residues, although W144 may be an exception. However, mutagenesis of residues 84 - 102 was also performed on a linear peptide without CLIPS (sequence 84TCEEGWH S TSEA S ESCVLH102 (SEQ ID NO: 200), with the two underlined residues replaced from C to S). Binding by APX005 to this linearized peptide almost completely disappeared. This indicates that binding of this antibody specifically depends on these two cysteines or on the specific conformational state in which this peptide is placed by CLIPS assembly.

[0290] Region on the crystal structure of CD40 92 TSEACESCVLHRSCSP 107 (SEQ ID NO: 196) and 125 PCPVGFFSNVSSAFEKCHPW 144 Visualization of (SEQ ID NO: 197) showed that amino acids 92 - 107 form a loop structure facing the CD40L trimer ( 97 ESC 100 which is within 4 Å of the above ligand) (see Figure 23). Residues 125 - 144 are not resolved in structure 3QD6, but are predicted to be very proximal to residues 92 - 107 based on available data on cysteine bonding. For visualization purposes, residues 122 - 125 (SEQ ID NO: 201) are shown in Figure 23.

[0291] Therefore, this experiment describes the mapping of the binding of the humanized antibody APX005 to CD40 using linear peptides and CLIPS peptides performed by Pepscan Presto BV. This mapping identified two specific binding regions. These are 92 TSEACESCVLHRSCSP 107 (SEQ ID NO: 196) and 125 PCPVGFFSNVSSAFEKCHPW 144 (SEQ ID NO: 197). Within region 92 - 107, the residues 92 TSEACESCVLH 102 (SEQ ID NO: 202) are probably the residues most relevant to binding. Among the identified binding residues, T 92 , E 97 and 100 VL 101 are known to be contact residues between CD40 and the CD40L trimer. Specifically, E 97 is considered an important residue for the binding of CD40 to the ligand.

[0292] Additional embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified and further embodiments can be provided, utilizing, as necessary, the contents of the various patents, applications, and publications.

[0293] In view of the foregoing detailed description, these and other modifications can be made to the embodiments. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but rather the claims are to be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure of the embodiments.

Claims

1. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to CD40 and is a CD40 agonist, comprising: a. a heavy chain variable region as set forth in SEQ ID NO:9; b. a light chain variable region comprising a VLCDR1 region set forth in SEQ ID NO:6, a VLCDR2 region set forth in SEQ ID NO:7, and a VLCDR3 region set forth in SEQ ID NO:8; c. A human IgG1 Fc region modified by the S267E mutation; 2. An isolated antibody, or antigen-binding fragment thereof, comprising:

2. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody is humanized.

3. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the antibody is a monovalent antibody lacking a hinge region.

4. An isolated antibody, or an antigen-binding fragment thereof, described in claim 1, wherein the antibody is an intact antibody.

5. The isolated antibody, or antigen-binding fragment thereof, of claim 1, comprising a human IgG1 constant domain.

6. The isolated antibody, or antigen-binding fragment thereof, of claim 5, wherein the human IgG1 constant domain comprises an IgG1 CH1 domain.

7. The isolated antibody, or antigen-binding fragment thereof, of claim 5, wherein the human IgG1 constant domain comprises an amino acid sequence as set forth in SEQ ID NO:

195.

8. An isolated polynucleotide encoding an isolated antibody, or an antigen-binding fragment thereof, described in any one of claims 1 to 7.

9. An expression vector comprising the isolated polynucleotide described in claim 8.

10. An isolated host cell comprising the expression vector described in claim 9.

11. A composition comprising a physiologically acceptable carrier and a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 7.

12. Use of the composition of claim 11 in the manufacture of a medicament for treating or ameliorating symptoms of cancer in a patient, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, melanoma, rituximab-resistant leukemia, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer and lung cancer.

13. The use of claim 12, wherein the cancer is rituximab-resistant non-Hodgkin's lymphoma.

14. The composition of claim 11 for treating or ameliorating symptoms of cancer in a patient, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiple myeloma, melanoma, rituximab-resistant leukemia, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, ovarian cancer, breast cancer and lung cancer.

15. The composition described in claim 14, wherein the cancer is rituximab-resistant non-Hodgkin's lymphoma.

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