MXPA06006153A - Cd40 antibody formulation and methods - Google Patents

Abstract

The present invention provides a method of treating tumor in a patient comprising administering to said patient a CD40 agonist antibody according to an intermittent dosing schedule. The present invention also provides a method of treating tumor in a patient comprising administering a combination of a CD40 agonist antibody and a DNA replication inhibitor. Also provided is a formulation for use in the treatment.

Description

FORMULATION AND METHODS OF THE CD40 ANTIBODY BACKGROUND OF THE INVENTION CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, is expressed in antigen presenting cells (B cells, cells dendritic cells, monocytes), hematopoietic precursors, endothelial cells, soft muscle cells, epithelial cells, as well as in most human tumors (Grewal and Flavell, Ann. Rev. Immunol., 1996, 16: 111-35; and Schoenberger, Seminars in immunology, 1998, 10 {6): 443-8.) Studies using CD40 agonist agents have reported that stimulation of the CD40 receptor causes a cascade of associated effects - with antitumor activity. , it has been seen that the stimulation of the CD40 receptor in antigen-presenting cells improves its maturation, the function that the antigen presents, the co-stimulatory potential and its release of immunoregulatory cytokines (Lee et al., PNAS USA, 1999, 96 (4): 1421-6; Celia et al., J. Exp. Med., 1996, 184 (2): 747-52). It has also been reported that CD40 agonists promote apoptosis of CD40 + tumors and improve their ability to be processed by dendritic cells (von Leoprechting et al., Cancer Res., 1999, 59: 1287-94; Sotomayo et al., Nature Medicine, 1999, 5 (7): 780-87; Elíopoulos et al., Mol. Ceil Biol., 2000, 29 (15): 5,503-15; Ziebold et al., Aren. Immunol. Therapiae Experimenta / te, 2000, 48 (4): 225-33; Hoffmann et al., J. Immunol., 2001, 24 (2): 162-71). The importance of these direct immune and antitumor stimulatory effects has been illustrated in animal models in which CD40 agonist antibodies have been shown to prevent tumor growth and reverse tumor tolerance (Diehl et al., Nature Med., 1999 , 5 (7): 774-9; Francisco et al., Cancer Res., 2000, 60 (12): 32,225-31). CD40 antibodies are mentioned in the following patent publications: U.S. 5,786,456; U.S. 5,674,492; WO 02/088186; U.S. 2003059427; U.S. 20020142358; WO 01/56603; U.S. 5,801,227; EP 806963; WO 88/06891 and WO 94/04570. However, methods of administration and formulations of highly effective CD40 antibodies have not been described. A stable formulation suitable for use in such treatment would also be useful.

SUMMARY OF THE INVENTION The present invention relates to a method for treating cancer in a patient in need of such treatment, which comprises administering to said patient a CD40 agonist antibody or a fragment thereof, wherein said antibody is administered in accordance with a intermittent dosing regimen of at least two cycles, each cycle comprising (a) a period of dosing during which a therapeutically effective amount of said CD40 agonist antibody is administered to said patient and then, (b) a rest period . In one embodiment, the administration produces a plasma concentration of the antibody from 0.01 μg / ml to 10 μg / ml for at least three hours and the rest period is for at least 1 week. In other embodiments, the dosing period is for at least one day, 1-5 days or 1-3 days. In other embodiments, the rest period is from 1-8, 1-6 weeks, 2-5 weeks or 3-4 weeks. In certain embodiments, the therapeutically effective amount of the CD40 agonist antibody results in the plasma concentration of said antibody from about 0.03 μg / ml to 10 μg / ml, about 0.03 μg / ml at 1 μg / ml, about 0. , 03 μg / ml to 0.3 μg / ml or approximately 0.1 μg / ml to 0.3 μg / ml for 3 to 120 hours. In some embodiments, the specified plasma concentration is maintained for at least one day, 24 to 30 hours, 24 to 36 hours, 24 to 48 hours, 24 to 72 hours, 24 to 96 hours or 24 to 120 hours. In some embodiments, the plasma concentration is maintained for 3 to 96 or 12 to 72 hours. In certain embodiments, the therapeutically effective amount of the CD40 agonist antibody administered during the dosing period is approximately 0, 03 to 3.0 mg / kg / day, 0.1 to 3.0 mg / kg / day, 0.1 to 1.0 mg / kg / day or approximately 0.1 to 0.3 mg / kg / day. In one embodiment, the dosage is administered for 1-5 days or 1-3 days, either consecutively or every other day. The intermittent dosing regimen of CD40 agonist antibodies, as described above in connection with tumor treatment, is also useful in the improvement of immune responses in patients and, therefore, the present invention also provides such use. In certain embodiments, the improvement of a patient's immune response results in increased expression of CD23 or MHC-II in B cells of the patient's whole blood, which, for example, can be measured at the end of a dosing period. . In some embodiments, the anti-CD40 antibody is administered to a patient suffering from combined and / or primary immunodeficiencies, including: CD40-dependent immunodeficiency with Hyper IgM syndrome, Common Variable Immunodeficiency, Bruton's Agammaglobulinemia, IgG subclass deficiencies, and SCID linked to X (mutations in the common gamma chain). In some embodiments, the anti-CD40 antibody is administered to treat a patient who is immunosuppressed, for example, due to chemotherapy, or has an immunodeficient disease, including any acquired immunodeficiency disease, such as HIV. In some embodiments, anti-CD40 antibody is administered to improve the immunity of an elderly patient. In some embodiments, the anti-CD40 antibody is administered to treat a patient having a bacterial, viral, fungal or parasitic infection. In some embodiments, an anti-CD40 human agonist antibody can be administered prophylactically to a patient who, due to age, disease or poor general health, is susceptible to infection to prevent or reduce the number or severity of infections. The present invention also provides a method for treating a tumor in a patient comprising administering a CD40 agonist antibody and an inhibitor of DNA replication, preferably a platinum derivative, especially cisplatin. In certain embodiments, cisplatin is administered intravenously. In some embodiments, cisplatin is administered in an amount of about 25 to 300 mg per m2, about 50 to 150 mg per m2 or about 75 to 100 mg per m2 of the patient's body area. In one embodiment, cisplatin is administered in a dose (eg, a single intravenous infusion). In another embodiment, it is administered for 2-5 days. In certain embodiments, the amount of the CD40 antibody, which is administered in combination with cisplatin, is administered in a dosage of about 0.1 to 3.0 mg / kg, or about 0.1 to 1.0 mg / kg, or about 0.1 to 0.3 mg / kg. In another aspect, the administration of cisplatin is combined with the intermittent dosing regimen of the CD40 antibody, administered with cisplatin during one or more of the dosing periods or rest periods. In another aspect, the invention relates to a method for treating a tumor in a patient in need of such treatment, administering to the patient a CD40 agonist antibody or a fragment thereof in a dosage of less than 1 mg / kg / day. , wherein the Cmax serum concentration in the patient resulting from the administration of the antibody is less than 50 μg / ml. In one embodiment, the dosage is between 0.1 and 0.3 mg / kg and the Cmax serum concentration of the antibody in the patient is between 0.5 and 10 μg / ml. In another aspect, the invention relates to a liquid pharmaceutical formulation, stable and suitable for parenteral administration comprising an anti CD40 antibody at a pH of 5.0 to 6.0 and a pharmaceutically acceptable carrier, the formulation being stable for a period of time. period of at least three months. Preferably, the formulation has a CD40 antibody concentration of at least about 5 mg / ml. In one embodiment, the formulation comprises an anti-CD40 antibody, sodium acetate, sodium chloride and polysorbate 80. Preferably, it comprises 20 mM sodium acetate, 140 mM sodium chloride and 0.2 mg / ml polysorbate. Preferably, the anti CD40 antibody has the amino acid sequence of an antibody selected from the group consisting of antibody 21.4.1 or 3.1.1 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibition of growth of Tumor CD40 (-) K562 by a CD40 agonist antibody in the presence of immune cells. The animals received a single injection (IP, intraparenteral) of 21.4.1 or KLH at the same time as the tumor challenge. The tumor size for each individual animal is reported on Day 21 in mm2 (10 animals per group). The study is representative of at least 5 separate studies. Figure 2 shows the inhibition of the growth of the BT 474 cell line of the human breast tumor by a CD40 agonist antibody. The values represent individual measurements of the tumor taken on Day 53 after injection using 6 animals per group. The study is representative of two separate experiments. The mean for each treatment group is indicated by the horizontal line.

Figure 3 shows the inhibition of CD40 (+) tumor growth by a CD40 agonist antibody, alone or in the presence of immune cells. The animals received a single injection of 21.4.1 at the same time as the tumor challenge. (a) The tumors were injected alone or (b) together with T cells and DC from human peripheral blood. The data points represent the tumor size (mm2) for each individual animal. The mean for each treatment group (N = 10) is indicated by the horizontal line. The study is representative of at least 3 separate experiments. Figure 4 shows the effects of a CD40 agonist antibody on the delay of mortality induced by a B cell lymphoma (Daudi). The data points refer to the average number of surviving animals, N = 10 per group. Figure 5 shows the tumor regression caused by a combination therapy with a CD40 agonist antibody and cisplatin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein, the term "CD40 agonist antibody" or "anti-CD40 agonist antibody" means an antibody that specifically binds to a human CD40 molecule and increases one or more CD40 activities by at least about 20% if it is added to a cell, tissue or organism that expresses CD40. In some embodiments, the antibody activates the CD40 activity by at least 40%, 50%, 60%, 70%, 80% or 85%. In some embodiments, activation occurs in the presence of CD40L. In some embodiments, the activity of the activating antibody is measured using an up-regulation assay of whole blood surface molecules. In another embodiment, the activity of the activating antibody is measured using a dendritic cell assay to measure the release of IL-12. In another embodiment, the activity of the activating antibody is measured using a tumor model in vivo. The term "antibody" as used herein, refers to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by chemical or enzymatic cleavage of intact antibodies. Binding fragments include Fab, Fab ', F (ab'), Fv and single chain antibodies. It is understood that reference to an intact (eg, complete, notoriously sized, etc.) antibody in the present invention includes an antibody having a terminal lysine deletion in the heavy chain, which is commonly encountered during recombinant expression. . Preferably, the agonist CD40 antibody is a human antibody. As used herein, the term "human antibody" means an antibody in which the variable and constant domain sequences are derived from human sequences. The human CD40 antibodies are described in detail in the provisional U.S. No. 60 / 348,980, filed November 9, 2001 and PCT International Application No. PCT / US02 / 36107 (already published as document WO 03/040170) filed November 8, 2002, the complete description of which is incorporated herein by reference. Human antibodies provide a substantial advantage in the methods of treatment of the present invention, since they are expected to minimize the immunogenic and allergic responses that are associated with the use of non-human antibodies in human patients. Illustrative and useful human anti-CD40 antibodies for the present invention include antibodies having the antibody amino acid sequences designated: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8. 3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.29.1, 24.2.1, 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V and 23.28.1L-C92A, as well as an antibody comprising a variable region or CDR of any of the illustrative antibodies. Antibodies that recognize the same or similar epitopes, or a portion thereof, as any of the illustrative antibodies may also be useful for the present invention. That is, as would be understood by one skilled in the art based on the description provided herein, an antibody that competes with an antibody of the invention (eg, 3.1.1, 3.1.1H-A78T, 3.1. 1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23. 28.1 H-D16E, 23.29.1, 24.2.1, 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V and 23. 28.1 L-C92A, and the like) may be useful as described elsewhere in this specification. An antibody of interest that competes with an antibody screened herein can be readily identified using methods well known in the art for the characterization of antibodies. More specifically, assays for evaluating the binding characteristics of an antibody, as well as for comparing those binding characteristics with those of another antibody, are well known in the art. Such methods include, but are not limited to, assays based on the ELISA test, the use of BIAcore binding studies, as well as those detailed in US Patent Application Publication No. 2003 / 0157730A1 of Walker et al. By the term "competes", as used herein with respect to an antibody, it is meant that a first antibody competes for binding with a second antibody, if the binding of the first antibody with its affinity epitope in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope in the presence of the first antibody is also perceptibly decreased, may, but not necessarily, be the cause. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody perceptibly inhibits the binding of the other antibody with its affinity epitope or ligand, both in it, to a greater or lesser degree, it is said that the antibodies "cross-compete" with each other by the union of your respective epitope (s). For example, antibodies that cross-compete may bind to the epitope, or portion of the epitope, to which the antibodies of the invention bind (eg, 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A -V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1 H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1 H -D16E, 23.29.1, 24.2.1, 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V and 23.28.1L-C92A). The present invention encompasses both competing and cross-competing antibodies. Regardless of the mechanisms by which said competition or cross competition occurs (for example, steric hindrance, conformational change or binding to a common epitope or portion thereof, and the like), the skilled person will appreciate, based on the techniques provided herein, which encompass said competing or cross-competing antibodies and may be useful for the methods described herein. In addition, illustrative antibodies can be further modified by substitution, addition or deletion of one or more amino acid residues without eliminating the ability of the antibody to bind the antigen and employ its agonist function. In fact, an antibody termed "3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V", comprises three substitutions of amino acids in the variable region of the heavy chain, that is, a substitution of alanine to threonine in amino acid residue number 78, a substitution of valine with alanine at amino acid residue number 88, and a substitution of valine with alanine at amino acid residue number 97 < SEC. ID NO: 9), all with respect to the amino acid sequence of the variable region of the heavy chain of antibody 3.1.1 (SEQ ID NO: 1). In addition, the antibody 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V further comprises an amino acid substitution of leucine to methionine at amino acid residue number 4 and a substitution of leucine to valine at the amino acid residue number 83 in the variable region of the light chain (SEQ ID NO: 10) compared to the amino acid sequence of the variable region of the light chain of antibody 3.1.1 (SEQ ID NO: 3). The amino acid sequences of the constant regions of the heavy chains (SEQ ID NO: 2) and the light chains (SEQ ID NO: 4) of antibodies 3.1.1 and 3.1.1 H- A78T-V88A / 97A / 3.1.1L-L4M-L83 are the same. The antibody 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83 is also referred to as "3.1.1H3L2" to reflect that the antibody comprises three amino acid substitutions in the heavy chain and two amino acid substitutions in the light chain concerning the antibody 3.1.1. A) Yes, in some embodiments, the illustrative antibodies can be modified by substitution, addition or deletion of one to ten, one to five or one to three amino acid residues, for example, in a framework region ("framework") or CDR. These antibodies and methods for illustrative production are described in detail in the provisional application U.S.

No. 60 / 348,980, filed on November 9, 2001 and the Application International PCT No. PCT / US02 / 36107 (WO 03/040170), filed November 8, 2002. However, the invention is not limited to these, nor to any other amino acid substitutions. Before the person skilled in the art, armed with the techniques provided herein, will appreciate that this invention encompasses a wide variety of amino acid substitutions.

Hybridomas 3.1.1, 7.1.2, 10.8.3, 15.1.1 and 21.4.1 were deposited, according to the Budapest Treaty, in the "American Type Culture Collection" (ATCC), 10801, University Boulevard, Manassas , VA 20110-2209, August 6, 2001. Hybridomas 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.29.1 and 24.2.1 were deposited with the ATCC on 16 July 2002. Hybridomas have been assigned to the following deposit numbers: Hybridoma Deposit No. 3.1.1 (LN 15848) PTA-3600 7.1.2 (LN 15849) PTA-3601 10.8.3 (LN 15850) PTA-3602 15.1.1 (LN 15851) PTA-3603 21.4.1 (LN 15853) PTA-3605 21.2.1 (LN 15874) PTA-4549 22.1.1 (LN 15875) PTA-4550 23.5.1 (LN 15855) PTA-4548 23.25.1 (LN 15876) PTA-4551 23.28.1 (LN 15877) PTA-4552 23.29.1 (LN 15878) PTA-4553 24.2.1 (LN 15879) PTA-4554 The sequences of these antibodies are known and are described in WO 03/040170. For convenience, the amino acid sequences of the light and heavy chains of two of these antibodies are shown below: Antibody 3.1.1: Variable (Sec I .D N °:.. L) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQA PGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNALYL QMNSLRYEDTAV YCYRRGHQLVLGYYYYNGLDVWGQGTT VTVSS 3.1.1: Sequence Constant (Sec I .D N °:.. 2) Protein of the SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCN Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN Heavy VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGl PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variable (Sec.I.D. N °: 3) DIVLTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQ 3. 1.1: KPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRLEA Sequence EDVGVYYCMQALQTPRTFGQGTKVEIK Protein of the Constant String (Sec. I .D. N °: 4) Light RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC Variable (Sec. I .D. °: 9) QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQA 3. 1.1 H-A78T- PGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNJJ-YL QMNSLRAEDTAv? YCARRGHQLVLGYYYYNGLDVWGQGTT V88A-V97A: TVSS Constant Sequence (Sec.I.D. N °: 2) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP TVSWN Protein of the SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCN VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK Chain PKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGll " Heavy PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3. 1.1 L-L4M- Variable (Sec.I.D. N °: 10) L83V: DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYL QKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE Sequence AEDVGVYYC QALQTPRTFGQGTKVEIK Protein of the Constant (Sec.I.O. N °: 4) RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK String VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC Lightweight Antibody 21.4.1: Variable (Sec I .D N °:.. 5): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA 21.4.1: Sequence MELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGT PGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAY LVTVSS Constant (Sec I .D N °:.. 6): Protein of ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN Chain Heavy SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCN VDHKPSNTKVDKTV RKCCVECPPCPAPPVAGPSVFLFPPK PKDTL ISRTPEN? -CWVDVSHEDPEVQFKIWYVDGVEVHNA KTKPREEQFNSTFRWSVL7WHQDWLNGKEYKCKVSNKGL PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 21. 4.1 Variable (Sec I .D N °:.. 7): Sequence DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPG KAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANIFPLTFGGGTKVEIK Protein Constant (Sec I .D N °:.. 8): Light Chain RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC Thus, the amino acid sequence of antibody 21.4.1 comprises the amino acid sequences shown in SEQ. ID N °: 5-8, the amino acid sequence of the antibody 3.1.1 comprises the sequences of amino acids shown in SEC. ID N °: 1-4, and the amino acid sequence of antibody 3.1.1 H-A78T-V88A-V97A / 3.1.1 L-L4M-L83 comprises the sequences shown in SEC. ID N °: 9, SEC. ID N °: 2, SEC. ID N °: 10 and SEC.

ID N °: 4. The amino acids that differ between 3.1.1 and 3.1.1 H-A78T-V88A-V97A / 3.1.1L-L4M-L83V are underlined.

It will be understood, based on the description provided herein, that an antibody 3.1.1 of the invention encompasses any combination of the light and / or heavy variable regions shown herein. That is, an antibody can comprise any combination of variable regions, including, but not limited to: 3.1.1 H (SEQ ID NO: 1) /3.1.1L (SEQ ID NO: 3), 3.1.1H (SEQ ID NO: 1) /3.1.1L-L4M-L83V (SEQ ID NO: 10), 3.1.1H-A78T-V88A-V97A (SEQ ID NO: 9) /3.1.1L (SEQ ID NO: 3) and, more preferably, 3.1.1H-A78T-V88A-V97A (SEQ ID N °: 9V3.1.1L-L4M-L83V (SEQ ID NO: 10).) However, the invention is by no means limited to these or any other particular combinations In certain embodiments, treatment of the tumor inhibits cancer cell proliferation, inhibits or prevents an increase in tumor weight or volume and / or causes a decrease in tumor weight or volume. Tumor treatment prolongs patient survival In certain embodiments, tumor growth is inhibited by at least 50%, 55%, 60%, 65%, 70% or 75% compared to untreated. embodiments, the tumor is CD40 positive In some embodiments, the tumor is CD40 negative The tumor may be a solid tumor or a non-solid tumor such as a lymphoma In some embodiments, an anti-CD40 antibody is administered to a patient having a tumor that is cancerous, patients that can be treated with antibodies Anti-CD40 or antibody portions include, but are limited to, patients who have been diagnosed with: brain cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, cancer uterine, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colorectal cancer, colon cancer, gynecological tumors (eg, uterine sarcomas, fallopian tube carcinoma, endometrial carcinoma, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (for example, cancer of the thyroid, parathyroid or adrenal glands), sarcomas of the soft tissues , leukemia, myeloma, multiple myeloma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid infant tumors, Hodgkin's disease, lymphomas lymphocytes, non-Hodgkin's lymphoma, bladder cancer, liver cancer, kidney cancer, kidney or ureter cancer (eg, renal cell carcinoma, renal pelvis carcinoma) or neoplasms of the central nervous system (eg, lymphoma) of the main CNS, tumors of the spinal axis, brainstem glioma or pituitary adenomas), glioma or fibrosarcoma. As used herein, the term "patient" refers to a human or non-human mammal expressing a cross-reactive CD40 (e.g., a primate, a cynomolgus monkey or rhesus). Preferably the patient to be treated is human. As used herein, the term "intermittent dosing regimen" means a dosage regimen comprising administering a CD40 agonist antibody, followed by a rest period.

As used herein, the term "rest period" means a period of time during which a CD40 agonist antibody is not administered to the patient. For exampleIf the antibody has been administered daily, there will be a rest period if the daily administration is discontinuous, for example, for a number of days or weeks. If a dose is administered with a different program, a rest period will be given if that dosage is discontinuous for some time. If not, a rest period may occur if the antibody concentration is maintained at a subtherapeutic level. In one embodiment, the antibody is not administered after the second rest period, ie, if the method of the invention involves two cycles, it is not necessary to administer the drug after the second rest cycle. Preferably, during the rest period, the plasma concentration of the antibody is maintained at a subtherapeutic level. The dosage period and / or the dose of the antibody may be the same or different between the cycles. The total treatment time (ie, the number of cycles for the treatment) will vary from patient to patient based on reasonable medical criteria and the particular factors of the patient to be treated. In general, the treatment is administered until a satisfactory response is obtained. In certain embodiments of the invention, the treatment period comprises 2-20, 2-15, 2-10, 2-7, 2-5 cycles or 2-3 cycles.

The antibody can be administered in any desired manner, including, for example, intravenous, subcutaneous, intramuscular, parenteral, intratumoral and transdermal administration. In one embodiment, the CD40 antibody is administered intravenously. In another, it is administered using a microneedle device; such devices are well known and include, for example, the device described in WO 03/084598. If administered in combination with a DNA replication inhibitor, e.g., cisplatin, the antibody can be administered before, during or after administration of the inhibitor. In one aspect, the invention relates to an aqueous solution for intravenous injection, with the pH from about 5.0 to 6.0, preferably a pH of about 5.5. Such a solution can be formulated with sodium acetate (trihydrate), acetic acid (glacial), Polysorbate 80, sodium chloride and water. It is preferred that the antibody solution be stored at refrigerated temperatures between 2 ° C and 8 ° C, and not freeze. In accordance with the present invention, methods are also provided for treating a tumor in a patient in need of such treatment, comprising administering to said patient a combination of a therapeutically effective amount of a CD40 agonist antibody and a therapeutically effective amount of an inhibitor of DNA replication, for example, a platinum derivative. In certain embodiments, a CD40 agonist antibody functions in synergistic combination with the platinum-derived compound, especially cisplatin, so that the antitumor effect of the combination is greater than would be expected from the administration of each compound separately. The platinum derivatives are well-known groups of compounds that show their antitumor activity by interfering with DNA replication. In certain embodiments, platinum derivatives are selected from the group consisting of cisplatin (cis-diaminodichloroplatinum, See Merck Index), carboplatin and oxaliplatin. The invention wbe more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the invention. All bibliographic citations are incorporated as reference. EXAMPLES Example 1: Effects of the Antibody on Lymphatic Nodule Cells from Cancer Patients The effects of a human anti-CD40 antibody were examined (21.4.1) on lymph node cells obtained from cancer patients stimulated with autologous tumor cells. Tumors and lymph node cells were collected from patients with renal cell carcinoma, non-small cell lung cancer, transitional cell carcinoma of the bladder, colon cancer, prostate cancer and cancer of the head and neck. The lymph node cells were introduced into the culture along with tumors treated with irradiated collagenase (recovered from the same patient) in the presence or absence of 21.4.1 (1 μg / ml, 6.7 nM). Proliferation was assessed using 3H-thymidine 96 hours later. The number of cells that produce INF was assessed through ELISPOT, after restimulation. The antibody increased the number of INF? + Positive T cells in cultures of lymph node cells stimulated with tumor antigen. In addition, the proliferation of these lymph node cells in response to the tumor antigen was increased 3-4 times. The antibody improved the proliferation and the ability to produce cytokine from lymph node cells obtained from cancer patients when stimulated with tumor antigen. Example 2: Binding of the Antibody to the Fe Receptor The binding of an anti CD40 antibody (21.4.1) to Fe receptors in cynomolgus and human leukocytes was examined. Flow cytometry studies indicated that types of FcR, Fc? RII (CD32) and Fc? RIII (CD16) were expressed in human leukocytes, as well as very low levels of Fc? RI (CD64). The binding of 21.4.1 to Fe (FcR) receptors was determined in cynomolgus or human peripheral blood leukocytes using 125l-21.4.1 and a control mAb of human Ig1. Human leukocytes from cynomolgus leukocytes or normal donors were isolated from whole blood using plasma gel and washed thoroughly to allow dissociation of serum immunoglobulins bound to the receptor. Centrifugation was used in a sucrose buffer to separate free and cell-bound antibodies. Studies were carried out at 4 ° C in. presence of sodium azide to prevent internalization of the receptor.

The specific binding to FcR of 21.4.1 was tested using in excess an isotype of unlabeled human IgG2 confronting the antibody as a competitor. The specific binding of 126l-21.4.1 to FcR in human leukocytes (n = 5 donors) was -1.0 ± 8.5% and the specific binding to FcR in cynomolgus leukocytes (n = 4) was 15 ± 13%. The addition of 21.4.1 unmarked 500 times in excess, which would block any specific binding of 125l-21.4.1 to leukocyte CD40 receptors as well as to FcR, demonstrated a specific binding of 49% and 67% of 125l-21.4. 1 to CD40 receptors in human and cynomolgus leukocytes, respectively (% specific binding to CD40 was calculated by subtracting% FcR binding from total% specific binding). As a control, 125 I-IgG1 systematically demonstrated specific binding to human and cynomolgus leukocytes. The specific binding of the IgG1 control antibody to FcR in human and cynomolgus leukocytes explained 56% and 51% of the total bound radioactivity, respectively. These studies indicate that the antibody shows a minimal specific binding to Fe receptors in human and cynomolgus leukocytes. Example 3: Complete Blood Cytokine Release Assay The ability to induce the release of cytokines from unstimulated human whole blood in an anti CD40 antibody (21.4.1) was tested using a whole blood in vitro assay, which has correlation with the induction of antibody-mediated cytokine release in humans. 21.4.1 was tested at 1, 10 and 100 μg / ml, together with a murine antihuman CD3 IgG1 as a positive control that induces the release of cytokine via a Fe-mediated pathway, and LPS as the second positive control that induces cytokines . by stimulating macrophages. The donors used included individuals that responded to both murine antibody and LPS (4 donors), as well as individuals that only responded to LPS (3 donors). The heparinized whole blood was cultured with 21.4.1 for 5 hours and the plasma was collected and analyzed for tumor necrosis factor (TNF-a), interferon gamma alpha (INF-?) And interleukin 6 (IL-6) by ELISA (using commercially available equipment). The cultures were also incubated for 48 hours and the interleukin 1 beta (IL-1ß) was analyzed. No cytokines were detected in the plasma of human blood cultured with 1 or 10 μg / ml of 21.4.1. Only one donor treated with 100 μg / ml of the antibody showed low but appreciable levels of two cytokines (34 pg / ml TNF-a and 90 pg / ml IL-6). Subsequently, this donor was re-tested and showed no detectable induction of TNF-a or IL-6. There was no increase in INF? or IL-1ß in none of the examples. These studies indicate that 21.4.1 does not induce inflammatory cytokines in human whole blood. Example 4: Pharmacodynamics and Pharmacokinetics of the Antibody. A CD40 antibody (21.4.1) was administered intravenously at various doses (1 mg / kg n = 4.3 mg / kg n = 4.5 mg / kg n = 2 and 10 mg / kg n = 2) to monkeys cynomolgus. Heparinized blood was taken from the monkeys at various pre and postdose moments. The blood was aliquoted and stained. Data were acquired using the FACSCalibur Becton Dickinson and analyzed with the CellQuest program. The results were calculated as the times that the median fluorescence intensity increases compared to the predose values. The expression of MHC Class II, which reflects the activation status and the ability to present antigen of B cells, increased 2.5 to 3 times in 24 hours after dosing for all doses tested, without observing a clear relationship between dose and response. The expression of CD23, another marker of B cell activation, was evaluated in 2 animals at 3 mg / kg, and one animal at 10 mg / kg. The expression of CD23 increased > 20 times in 24 hours after dosing without observing dose effect. The upward regulation of both surface markers persisted (increased> 2 times) if the levels of 21.4.1 continued above 1 μg / ml.

The levels of the co-stimulatory molecule of CD86 and CD71 (transferrin receptor) also showed moderate upregulation, if the expression of CD80 did not change significantly. 21.4.1 upregulates the surface markers in B cells in vivo of cynomolgus. The expression of MHC Class II and CD23 in cells CD20 + increases with treatment, and 1 mg / kg (corresponding to a Cma of »20 μg / ml and an exposure of > 0.1 μg / ml for 4 days) seems to produce a pharmacodynamic response of saturation in B cells of cynomolgus. The duration of this response was longer at higher doses. The pharmacokinetic properties of an anti-CD40 antibody (21.4.1) in cynomolgus monkeys were examined after intravenous (IV) administration of a single dose of 1, 3, 5 or 10 mg / kg. The 21.4.1 was characterized by a low systematic removal (from 0.0133 to 0.0635 ml / min / kg) and a small volume of constant state distribution (0.0459 to 0.0757 l / kg), giving as result in an apparent mean elimination half-life of 0.75 to 2.0 days (Table 1). The pharmacokinetics of 21.4.1 appeared to be dose dependent during the dose interval examined. Elimination values generally decreased with increasing dose from 1 to 10 mg / kg and the apparent mean elimination half-life increased from 0.75 days, to 1 mg / kg, to 2.0 days, to 10 mg / kg. The volume of distribution in constant state was similar in different doses (average of 0.0575 l / kg). The dose-dependent elimination observed may be in part due to the binding of 21.4.1 to CD40 receptors that are widely expressed in normal tissues and to the subsequent internalization and elimination of the antibody and receptor complex. The development of the primate anti-human antibody (PAHA) response can also contribute to accelerated elimination in some monkeys. PAHA was evaluated only after the individual serum concentrations of 21.4.1 reached the lower limit of quantification (LLOQ, from English "Lower Limit of Quantitation", 0.03 μg / ml), since the presence of 21.4.1 in the test serum interferes with the PAHA assay. Anti-21.4.1 antibodies were detected in all monkeys in the dose groups of 3, 5 and 10 mg / kg in 14 to 28 days after administration of the antibody.

Table 1 Mean Pharmacokinetic Parameters (± SD) of 21.4.1 in Cynomolgus Monkeys After Single IV Administration at 1.3.5 and 10 mg / kg Example 5: Antitumor Activity of the Antibody The tumor growth inhibitory activity of a CD40 antibody (21.4.1) was determined in beige mice with SCID injected subcutaneously with tumor cells alone (1x107) or with T cells (5x105) and human DC ( 1x105) from the same donor. The ratio of tumor cells to T cells and DC was 100: 1: 5. Unless indicated otherwise, the results are presented in terms of the tumor size in mm2 at a predetermined fixed time (from the kinetic experiments) to be the time at which the tumor growth in control animals reached a size of 300-400. mm2 and it was no longer human to continue the experiment. In all cases, only one injection of 21.4.1 was administered, which had a T -? / 2 of > 30 days in beige mice with SCID.

Example 5 (a): Effects of the Antibody on Human Tumors CD40 (-) The effects of a CD40 antibody (21.4.1) on the growth of CD40 (-) tumors (e.g., erythroleukemia and colon carcinoma) were examined. Specifically, K562 tumors were chosen to assess the efficacy of 21.4.1 against a CD40 (-) lower immunogenic tumor (class I and class II negative). Beige mice with SCID were injected subcutaneously with the erythroleukemic tumor CD40 (-), K562 (ATCC CCL-243) alone or in the presence of T cells from human peripheral blood and DCs. The animals received a single IP injection of 21.4.1 either at the same time as the tumor injection or 5 days later using various dose levels. A single IP injection of 21.4.1 resulted in dose-dependent inhibition of K562 tumor growth if the immune cells were presented as illustrated on Day 21 after the tumor challenge (Figure 1). The amount of 21.4.1 to cause a 50% inhibition of tumor growth was 0, 005 mg / kg, which corresponded to a Cmax serum concentration of 0.05 μg / ml. Similar results were seen with the CD40 (-) colon carcinoma, Lovo (ATCC CCL-229). The results were identical when 21.4.1 was administered on Day 0 or Day +5 relative to the tumor challenge. The growth of these CD40 (-) tumors was not inhibited by 21.4.1 in the absence of immune cells. 21.4.1 prevents the growth of CD40 (-) tumors when immune cells are present, suggesting an improvement in immune mediated antitumor activity. This was demonstrated against a carcinoma of the colon and an erythroleukemic tumor. This antitumor activity was also demonstrated using antibody 3.1.1 for colon carcinoma and for antibody 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V (IC50 <0.01 mg / kg) in the erythroleukemic tumor. Thus, the data described herein demonstrate that the antibody 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V has the in vivo activity of antibody 3.1.1. In addition, these in vivo tumor results argue that given similar data obtained in vitro if the two antibodies were compared, antibody 3.1.1 and 3.1.1H-A78T-V88A-V97A 3.1.1 L-L4M-L83V would function from a similar way in vivo. Thus, the results obtained using 3.1.1 apply to 3.1.1 H-A78T-V88A-V97A / 3.1.1L-L4M-L83V in this I and other tests. Example 5 (b): Effects of the Antibody on the Growth of Prostate and Human Breast Tumors. The effects of an anti-CD40 antibody (21.4.1) on the prevention of the growth of breast and prostate tumors were examined. Beige mice with SCID were challenged with the human breast tumor, BT 474 (ATCC HTB-20), subcutaneously, together with T cells from human peripheral blood and DC. The animals received a single dose of 21.4.1 (IP) at the same time as the tumor injection. As shown in Figure 2, a single injection of 21.4.1 prevented the growth of BT 474 cells in the presence of immune cells. The amount of 21.4.1 needed to cause a 50% reduction in tumor growth was 0.005 mg / kg, which corresponded to a Cmax serum concentration of 0.05 μg / ml. Similar results were observed against the human prostate cancer cell line, PC-3 (ATCC CRL-1435). This was also demonstrated using antibody 3.1.1 and can be expected for 3.1.1 H-A78T-V88A-V97A / 3.1.1 L-L4M-L83V. 21.4.1 prevents the growth of human prostate and breast tumors. Example 5 (c): Antitumor Effects of the Antibody on Tumors CD40 (+). The effects of an anti CD40 antibody (21.4.1) on the antitumor activity against CD40 (+) tumors and the changes, on the efficacy in the presence and absence of immune cells were studied. Beige mice with SCID were injected subcutaneously (SC) with Raji CD40 (+) Raji B cell lymphoma (ATCC CCL-86) followed by a single dose of 21.4.1 (IP) at the same time as tumor injection. Some animals were also injected with human T cells and DC. Tumor growth was assessed on Day 21. As shown in Figure 3, the amount of 21.4.1 to cause a 50% inhibition of tumor growth in the absence of immune cells was 0.02 mg / kg, which corresponded at a Cmax serum concentration of 0.2 μg / ml. If the tumor cells are injected together with immune cells, they will be decreased 20 times to 0.001 mg / kg. { serum concentration Cmax = 0.01 μg / ml) the amount of 21.4.1 needed to cause a 50% inhibition of tumor growth.

These results illustrate that 21.4.1 has direct antitumor activity against CD40 (+) tumors. This observation was also made for 3.1.1 H-A78T-V88A-V97A / 3.1.1L-L4M-L83V (IC50 <0.01 mg / kg). This anti-tumor activity for antibody 21.4.1 was improved when immune cells were present and this was also demonstrated with antibody 3.1.1 and expected for antibody 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M- L83V. Example 5 (d): Antitumor Effects of the Antibody on B Cell Lymphoma. The ability of an anti CD40 antibody according to the invention (21.4.1) to delay mortality in a systematic tumor model CD40 (+) was evaluated. used a B-cell lymphoma. Beige mice with SCID were injected IV with the Daudi B-cell lymphoma (ATCC CCL-213). 21.4.1 was administered as a single injection (PI) at the same time as the injection of the tumor. The mortality was monitored for 58 days. As shown in Figure 4, a single injection of 21.4.1 prevented the mortality induced by a systematically administered tumor cell line. 21.4.1 delays mortality in a CD40 (+) systematic tumor model using a B-cell lymphoma. This was also demonstrated using 3.1.1 and similar results are expected for 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V. Example 6: Therapeutic effects of the Antibody in Combination with Cisplatin.

The therapeutic effects of an anti CD40 antibody (21.4.1) on preventing the growth of human breast tumors alone and in the presence of cisplatin were examined. Beige mice with SCID were injected subcutaneously with the breast tumor, BT 474. The antibody (1 mg / kg, IP) and / or cisplatin (2.5 mg / kg, IP) were administered as a single injection once the tumors reached a size of 200 mm2. Tumor growth was measured on Day 84 after the challenge. As shown in Figure 4, a single injection of 21.4.1 or cisplatin prevented tumor growth. However, the combination of both treatments leads to complete regression of the tumor in 7/8 animals. 21.4.1 prevents tumor growth if administered only once the tumors are established and causes tumor regression when administered in combination with cisplatin. This is also shown using antibody 3.1.1 and is also similar for 3.1.1 H-A78T-V88A-V97A / 3.1.1L-L4M-L83V. Example 7: Multidose Pharmacokinetics of the Antibody In a multiple dose study, 21.4.1 was administered intravenously to cynomolgus monkeys (2 / sex / dose) at doses of 0.3, 1.0 and 10 mg / kg on Days 1, 3, 5, 7 and 9 during 5 total doses. Blood was collected on Days 1 and 9 before dosing and 0.5, 6 and 24 hours after dosing and before dosing and 0.5 hours after dosing on Day 5 to measure drug concentrations in serum. The systematic exposure to 21.4.1, measured by mean Cmax and mean AUC (o-24), increased with the dose increase from 0.3 to 10 mg / kg on both Day 1 and Day 9 ( Table 2). Similar exposures (average Cmax and mean AUC) were observed on Days 1 and 9 in the dose groups of 0.3 and 1 mg / kg. In the dose group of 10 mg / kg, the values of Cma? mean and AUC (o-24) mean increased 2.6 and 2.8 times, respectively, from Day 1 to Day 9. No differences in exposure related to gender were observed.

Table 2 Mean Pharmacokinetic Parameters (± SD) of 21.4.1 in Monkeys Cynomolgus on Days 1 and 9 After Administration IV Every Two Days 3 N = 2 / sex / dose Example 8: Antibody Formulation The CD40 antibody was concentrated to approximately 11.0 mg / ml ± 0.8 mg / ml using an ultrafiltration unit containing isolated cassettes of 30 kDa molecular weight . The concentrate was then diafiltered in 20 mM sodium acetate / 140 mM sodium chloride, buffer pH 5.5. A 2% polysorbate 80 solution was added to the concentrated dialfiltrate to achieve a final concentration of 0.02% Polysorbate 80.

Claims (20)

1. CLAIMS 1. A method for treating cancer in a patient in need of said treatment, comprising administering to said patient a CD40 agonist antibody or a fragment thereof, wherein said antibody is administered according to a regimen of intermittent dosing of at least two cycles, each cycle comprising (a) a period of dosing during which a therapeutically effective amount of said CD40 agonist antibody is administered to said patient and then, (b) a rest period.
2. 2. The method of claim 1, wherein the therapeutically effective amount produces a plasma concentration of said antibody from 0.01 μg / ml to 10 μg / ml for at least three hours and the rest period is for less 1 week.
3. 3. The method according to claim 1, wherein the therapeutically effective amount produces a plasma concentration of 0.03 μg / ml to 1.0 μg / ml.
4. 4. The method according to claim 1, wherein the therapeutically effective amount maintains a plasma concentration of 0.1 μg / ml to 0.3 μg / ml.
5. 5. The method according to claim 1, wherein the antibody is selected from the group consisting of an antibody having the amino acid sequence of the antibody: 3.1.1, 3.1.1H-A78T, 3.1.1H- A78T-V88A-V97A, 3.1.1 H-A78T-V88A-V97A / 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15. 1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1 H-D16E, 23.29.1, 24.2.1, 3.1.1L -L4M-L83V and 23.28.1L-C92A.
6. 6. The method according to claim 1, wherein the antibody comprises a variable region or a CDR of an antibody selected from the group consisting of an antibody called: 3.1.1, 3. 1.1 H-A78T, 3.1.1 H-A78T-V88A-V97A, 3.1.1 H-A78T-V88A-V97A / 3.1.1 L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4. 1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23. 25.1, 23.28.1, 23.28.1H-D16E, 23.29.1, 24.2.1, 3.1.1 L-L4M-L83V and 23. 28.1 L-C92A.
7. 7. The method according to claim 1, wherein the antibody binds to the same epitope as a CD40 antibody selected from the group consisting of an antibody designated: 3.1.1, 3.1.1H-A78T, 3. 1.1 H-A78T-V88A-V97A, 3.1.1 L-L4M-L83V, 3.1.1 H-A78T-V88A-V97A / 3.1.1 L- L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.29.1, 24.2.1, and 23.28.1 L- C92A.
8. 8. The method according to claim 1, wherein the antibody competes with a CD40 antibody selected from the group consisting of an antibody named: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T- V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H- D16E, 23.29.1, 24.2.1, 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V and 23.28.1L-C92A.
9. 9. The method according to claim 1, wherein the antibody has the amino acid sequence of an antibody selected from the group consisting of 21.4.1, 3.1.1 and 3.1.1H-A78T-V88A-V97A / 3.1.1L- L4M-L83V.
10. 10. A method for treating a tumor in a patient in need of said treatment, comprising administering to said patient a CD40 agonist antibody or a fragment thereof, wherein said antibody is administered according to a dosage regimen intermittent of at least two cycles, each cycle comprising (a) a dosing period of 1-5 days during which 0.03 to 3.0 mg / kg / day of the antibody is administered and then (b) a Rest period of 1 to 8 weeks.
11. 11. The method according to claim 10, wherein 0.1 to 1.0 mg / kg / day or 0.1 to 0.3 mg / kg / day of the antibody is administered.
12. 12. A method for treating the tumor in a patient in need of such treatment, comprising administering to said patient a combination of a therapeutically effective amount of a CD40 agonist antibody and a therapeutically effective amount of an inhibitor of the replication of DNA
13. 13. A liquid pharmaceutical formulation, stable and suitable for parenteral administration comprising a CD40 agonist antibody at a pH of 5.0 to 6.0 and a pharmaceutically acceptable carrier, said formulation being stable for a period of at least three months
14. 14. The formulation of claim 13, having a concentration of said CD40 antibody of at least about 5 mg / ml.
15. 15. The formulation of claim 13, comprising an anti-CD40 antibody, sodium acetate, sodium chloride and polysorbate 80.
16. 16. The formulation of claim 13, wherein said anti-CD40 antibody has the amino acid sequence of an antibody selected from the group consisting of 21.4.1, 3.1.1 and 3.1.1H-A78T-V88A-V97A / 3.1.1L-L4M-L83V.
17. 17. A method for treating a tumor in a patient in need of said treatment, comprising administering to a patient a CD40 agonist antibody or a fragment thereof in a dosage of less than 1 mg / kg in which the concentration in serum Cma? of said antibody in said patient is less than 50 μg / ml.
18. 18. The method of claim 17, wherein the dosage is between 0.1 and 0.3 mg / kg and wherein the concentration in serum Cma? of said antibody in said patient is between 0.5 and 10 μg / ml.
19. 19. A method for improving the immune response in a patient in need thereof, comprising administering to said patient a CD40 agonist antibody or a fragment thereof, wherein said antibody is administered according to a regimen of intermittent dosing of at least two cycles, each cycle comprising (a) a period of dosing during which a therapeutically effective amount of said CD40 agonist antibody is administered to said patient and then, (b) a rest period.
20. 20. The use of a CD40 agonist antibody, or a fragment thereof, for the manufacture of a medicament for treating cancer in a patient in need of such treatment by administering to said patient said CD40 agonist antibody or a fragment thereof. same, wherein said antibody is administered according to an intermittent dosing regimen of at least two cycles, each cycle comprising (a) a dosage period during which a therapeutically effective amount of said CD40 agonist antibody is administered to said patient, and then (b) a rest period.