EA040302B1 - ANTIBODIES TO CD40 - Google Patents

Description

Link to related applications

This application claims priority to U.S. Provisional Applications Nos. 62/252615, filed November 9, 2015, and 62/186076, filed June 29, 2015, the disclosures of which are incorporated herein by reference.

Background of the Invention

Recent studies have demonstrated that human cancers and chronic infections can be treated with agents that modulate the patient's immune response to malignant or infected cells. See, for example, Reck & Paz-Ares (2015) Semin. oncol. 42:402. Anti-CD40 agonist antibodies such as CP-870893 and dacetuzumab (SGN-40) have been tested for the treatment of cancer based on the belief that they can enhance such an immune response. See, for example, Kirkwood et al. (2012) C. A. Cancer J. Clin. 62:309; Vanderheide & Glennie (2013) Clin. Cancer Res. 19:1035. Recent experiments in mice have demonstrated that anti-CD40 antibodies with increased specificity for the inhibitory Fc receptor, FcyRIIb, have increased antitumor efficacy. See, for example, WO 2012/087928; Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012) Proc. Nat'l Acad. Sci USA 109:10966; Wilson et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754.

There is a need for improved anti-human CD40 agonist antibodies for the treatment of cancer and chronic infections in humans. Such antibodies preferably have increased specificity for the inhibitory Fc receptor, FcyRIIb, compared to activating Fc receptors and exhibit increased antitumor and/or anti-infective activity.

Brief summary of the present invention

Provided herein are isolated humanized mouse monoclonal antibodies that specifically bind to human CD40 (the mature sequence is shown in SEQ ID NO:1), optionally containing modified Fc regions that increase the specificity of binding to the FcyRIIb receptor. In certain embodiments, the invention relates to antibodies to huCD40 or antigen-binding fragments thereof that compete for binding, cross-link or bind to the same epitope as one or more of the antibodies 12D6 (SEQ ID NOS: 3 and 4), 5F11 (SEQ ID NOs: 23 and 24), 8E8 (SEQ ID NOs: 40 and 41), 5G7 (SEQ ID NOs: 52 and 53) and 19G3 (SEQ ID NOs: 58 and 59), including chimeric antibodies, human antibodies or humanized antibodies .

In certain embodiments, the anti-human CD40 antibodies of the present invention, or antigen-binding fragments thereof, bind to an epitope containing or consisting of one or more sequences selected from the group consisting of

WGCLLTAVHPEPPTACRE (residues 11-28 of SEQ ID NO: 1) (antibody 12D6), EPPTACREKQYLINS (residues 21-35 of SEQ ID NO: 1) (antibodies 12D6, 5G7 and 19G3) and ECLPCGESE (residues 58-66 of SEQ ID NO: 1) (antibody 5F11).

In certain embodiments, an antibody of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises the CDRH1, CDRH2, and CDRH3 sequences and the light chain comprises the CDRL1, CDRL2, and CDRL3 sequences derived at least in part from the same V gene segments. -region and gene segments J-region of the germline, as antibodies to huCD40 12D6, 5F11, 8E8, 5G7 or 19G3, as described in table. 3. Specifically, the antibody may contain CDR sequences derived from the same mouse germline as antibody 12D6 (heavy chain CDR sequences derived at least in part from the VH1-39_01 mouse V region germline and J region germline IGHJ4, and light chain CDR sequences derived at least in part from the mouse V region germline VK1-110_01 and the J region germline IGKJ1), antibody 5F11 (heavy chain CDR sequences derived at least in part from mouse V region germline VH1-4_02 and J region germline IGHJ3, and light chain CDR sequences derived at least in part from mouse V region germline VK3-5_01 and J region germline IGKJ5), antibody 8E8 (Heavy chain CDR sequences derived at least in part from the VH1-80_01 mouse V region germline and IGHJ2 J region germline and light chain CDR sequences, derived at least in part from the VK1-110_01 mouse V region germline and IGKJ2 J region germline), 5G7 antibody (heavy chain CDR sequences derived at least in part from the VH1-18_01 mouse V region germline and IGHJ4 J region germline, and light chain CDR sequences derived at least in part from mouse VK10-96_01 germline and IGKJ2 J region germline), or antibody 19G3 (heavy chain CDR sequences derived from at least partially from the VH5-9-4_01 mouse V region germline and IGHJ3 germline J region, and light chain CDR sequences derived at least partially from the VK1-117_01 mouse V region germline and J germline -region IGKJ2).

In various embodiments, an antibody of the present invention contains a heavy

- 1 040302 chain and light chain, where the heavy chain contains the sequences CDRH1, CDRH2 and CDRH3 and the light chain contains the sequences CDRL1, CDRL2 and CDRL3 selected from the group consisting of: CDR of antibody 12D6-03, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-108, respectively, of SEQ ID NO: 5, and CDRL1, CDRL2, and CDRL3 contain residues 24-39, 55-61, and 94-102, respectively, of SEQ ID NO: 6; CDR of antibody 12D6-22, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-108 respectively of SEQ ID NO: 7, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 9; CDR of antibody 12D6-23, where CDRH1, CDRH2 and CDRH3 contain residues 3135, 50-66 and 99-108 respectively of SEQ ID NO: 10, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94-102 respectively SEQ ID NO: 11; CDR of antibody 12D6-24, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-108 respectively of SEQ ID NO: 12, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 9; CDR antibody 5F11-17, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-106 respectively of SEQ ID NO: 25, and CDRL1, CDRL2 and CDRL3 contain residues 24-38, 54-60 and 93- 101 respectively SEQ ID NO: 26; CDR antibody 5F11-23, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-106 respectively of SEQ ID NO: 27, and CDRL1, CDRL2 and CDRL3 contain residues 24-38, 54-60 and 93- 101 respectively SEQ ID NO: 28; CDR antibody 5F11-45, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-106 respectively of SEQ ID NO: 29, and CDRL1, CDRL2 and CDRL3 contain residues 24-38, 54-60 and 93- 101 respectively SEQ ID NO: 30; CDRs of antibody 8E8-56, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-111 respectively of SEQ ID NO: 42, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 43; CDR of antibody 8E8-62, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-111 respectively of SEQ ID NO: 44, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 45; CDRs of antibody 8E8-67, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-111 respectively of SEQ ID NO: 46, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 47; CDRs of antibody 8E8-70, wherein CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-111 respectively of SEQ ID NO: 48, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 49; CDR of antibody 8E8-71, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-111 respectively of SEQ ID NO: 50, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 51; CDR of antibody 5G7-22, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-102 respectively of SEQ ID NO: 54, and CDRL1, CDRL2 and CDRL3 contain residues 24-34, 50-56 and 89- 97 respectively SEQ ID NO: 55; CDR of antibody 5G7-25, where CDRH1, CDRH2 and CDRH3 contain residues 3135, 50-66 and 99-102 respectively of SEQ ID NO: 56, and CDRL1, CDRL2 and CDRL3 contain residues 24-34, 50-56 and 89-97 respectively SEQ ID NO: 57; CDR of antibody 19G3-11, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-101 respectively of SEQ ID NO: 60, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94- 102 respectively SEQ ID NO: 62; and the CDR of the antibody 19G3-22, where CDRH1, CDRH2 and CDRH3 contain residues 31-35, 50-66 and 99-101 respectively of SEQ ID NO: 63, and CDRL1, CDRL2 and CDRL3 contain residues 24-39, 55-61 and 94 -102 respectively SEQ ID NO: 64.

In various embodiments, an antibody of the present invention comprises a heavy chain comprising a variable domain selected from the group consisting of 12D6 (residues 1-119 of SEQ ID NO: 3), 5F11 (residues 1-117 of SEQ ID NO: 23), 8E8 ( residues 1-122 of SEQ ID NO: 40), 5G7 (residues 1-113 of SEQ ID NO: 52) and 19G3 (residues 1-112 of SEQ ID NO: 58) with constant regions containing an FcyRIIb-specific Fc region selected from group consisting of IgG1f (SEQ ID NO: 65), SE (SEQ ID NO: 66), SELF (SEQ ID NO: 67), P238D (SEQ ID NO: 68), V4 (SEQ ID NO: 69), V4 D270E (SEQ ID NO: 70), V7 (SEQ ID NO: 71), V8 (SEQ ID NO: 72), V9 (SEQ ID NO: 73), V9 D270E (SEQ ID NO: 74), V11 (SEQ ID NO: 75) and V12 (SEQ ID NO: 76).

In certain embodiments, the antibody comprises specific heavy chain variable domains and light chain variable domains selected from the group consisting of 12D6-03 (residues 1-119 and 1-112 of SEQ ID NO: 5 and SEQ ID NO: 6, respectively), 12D6 -22 (residues 1-119 and 1-112 of SEQ ID NO: 7 and SEQ ID NO: 9, respectively), 12D6-23 (residues 1-119 and 1-112 of SEQ ID NO: 10 and SEQ ID NO: 11, respectively) , 12D6-24 (residues 1-119 and 1-112 of SEQ ID NO: 12 and SEQ ID NO: 9, respectively), 5F11-17 (residues 1-117 and 1-111 of SEQ ID NO: 25 and SEQ ID NO: 26 respectively), 5F11-23 (residues 1-117 and 1-111 of SEQ ID NO: 27 and SEQ ID NO: 28 respectively), 5F11-45 (residues 1-117 and 1-111 of SEQ ID NO: 29 and SEQ ID NO : 30), 8E8-56 (residues 1-122 and 1-112 of SEQ ID NO: 42 and SEQ ID NO: 43, respectively), 8E862 (residues 1-122 and 1-112 of SEQ ID NO: 44 and SEQ ID NO: 44 and SEQ ID NO: 45, respectively), 8E8-67 (residues 1-122 and 1-112 of SEQ ID NO: 46 and SEQ ID NO: 47, respectively), 8E8-70 (residues 1-122 and 1-112 of SEQ ID NO: 48 and SEQ ID NO: 49), 8E8-71 (os 1-122 and 1-112 SEQ ID NO: 50 and SEQ ID NO: 51, respectively), 5G7-22 (residues 1-113 and 1-107 of SEQ ID NO: 54 and SEQ ID NO: 55, respectively), 5G7- 25 (residues 1-113 and 1-107 of SEQ ID NO: 56 and SEQ ID NO: 57, respectively), 19G3-11 (residues 1-112 and 1-112 of SEQ ID NO: 60 and SEQ ID NO: 62, respectively), and 9G3-22 (residues 1-112 and 1-112 of SEQ ID NO: 63 and SEQ ID NO: 64, respectively). Any of these antibodies may further comprise a heavy chain constant region comprising an FcyRIIb-specific Fc region, wherein said heavy chain constant region is selected.

- 2 040302 from the group consisting of IgGlf (SEQ ID NO: 65), SE (SEQ ID NO: 66), SELF (SEQ ID NO: 67), P238D (SEQ

ID NO: 68), V4 (SEQ ID NO: 69), V4 D270E (SEQ ID NO: 70), V7 (SEQ ID NO: 71), V8 (SEQ ID NO: 72), V9 (SEQ ID NO: 73 ), V9 D270E (SEQ ID NO:74), V11 (SEQ ID NO:75) and V12 (SEQ ID NO:76). Any of these antibodies may further comprise the kappa light chain constant region of SEQ ID NO: 77.

In specific embodiments, an antibody of the present invention comprises a humanized antibody 12D6-24 comprising a light chain of SEQ ID NO: 9 and a heavy chain selected from the group consisting of any of SEQ ID NO: 13-22, or a humanized antibody 5F11-45, containing a light chain of SEQ ID NO: 30 and a heavy chain selected from the group consisting of any of SEQ ID NO: 31-39. Specific antibodies include 12D6-24 SE (SEQ ID NOs: 9 and 14), 12D6-24 SELF (SEQ ID NOs: 9 and 15), 12D6-24 P238D (SEQ ID NOs: 9 and 13), 12D6-24 V4 ( SEQ ID NOs: 9 and 16), 12D6-24 V4 D270E (SEQ ID NOs: 9 and 17), 12D6-24 V8 (SEQ ID NOs: 9 and 18), 12D6-24 V9 (SEQ ID NOs: 9 and 19 ), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20), 12D6-24 V11 (SEQ ID NOs: 9 and 21), 12D6-24 V12 (SEQ ID NOs: 9 and 22), 5F11-45 SE ( SEQ ID NOs: 30 and 31), 5F11-45 SELF (SEQ ID NOs: 30 and 32), 5F11-45 V4 (SEQ ID NOs: 30 and 33), 5F11-45 V4 D270E (SEQ ID NOs: 30 and 34 ), 5F11-45 V8 (SEQ ID NOs: 30 and 35), 5F11-45 V9 (SEQ ID NOs: 30 and 36), 5F11-45 V9 D270E (SEQ ID NOs: 30 and 37), 5F11-45 VI1 ( SEQ ID NOs: 30 and 38) and 5F11-45 V12 (SEQ ID NOs: 30 and 39), where the sequences are provided for the light and heavy chains, respectively.

In additional embodiments, the anti-huCD40 antibodies of the present invention comprise heavy and light chains having at least 80%, 85%, 90%, and 95% sequence identity with the heavy and light chain sequences of 12D6-24 SE (SEQ ID NOs: 9 and 14), 12D6 -24 SELF (SEQ ID NOs: 9 and 15), 12D6-24 P238D (SEQ ID NOs: 9 and 13), 12D6-24 V4 (SEQ ID NOs: 9 and 16), 12D6-24 V4 D270E (SEQ ID NOs: : 9 and 17), 12D6-24 V8 (SEQ ID NOs: 9 and 18), 12D6-24 V9 (SEQ ID NOs: 9 and 19), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20), 12D6 -24 V11 (SEQ ID NOs: 9 and 21), 12D6-24 V12 (SEQ ID NOs: 9 and 22), 5F11-45 SE (SEQ ID NOs: 30 and 31), 5F11-45 SELF (SEQ ID NOs: 30 and 32), 5F11-45 V4 (SEQ ID NOs: 30 and 33), 5F11-45 V4 D270E (SEQ ID NOs: 30 and 34), 5F11-45 V8 (SEQ ID NOs: 30 and 35), 5F11- 45 V9 (SEQ ID NOs: 30 and 36), 5F11-45 V9 D270E (SEQ ID NOs: 30 and 37), 5F11-45 V11 (SEQ ID NOs: 30 and 38) or 5F11-45 V12 (SEQ ID NOs: 30 and 39).

In additional embodiments, the anti-huCD40 antibodies of the present invention comprise heavy and light chains essentially consisting of the heavy and light chain sequences 12D6-24 SE (SEQ ID NOS: 9 and 14), 12D6-24 SELF (SEQ ID NOS: 9 and 15 ), 12D6-24 P238D (SEQ ID NOs: 9 and 13), 12D6-24 V4 (SEQ ID NOs: 9 and 16), 12D6-24 V4 D270E (SEQ ID NOs: 9 and 17), 12D6-24 V8 ( SEQ ID NOs: 9 and 18), 12D6-24 V9 (SEQ ID NOs: 9 and 19), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20), 12D6-24 V11 (SEQ ID NOs: 9 and 21 ), 12D6-24 V12 (SEQ ID NOs: 9 and 22), 5F11-45 SE (SEQ ID NOs: 30 and 31), 5F11-45 SELF (SEQ ID NOs: 30 and 32), 5F11-45 V4 (SEQ ID NOs: 30 and 33), 5F11-45 V4 D270E (SEQ ID NOs: 30 and 34), 5F11-45 V8 (SEQ ID NOs: 30 and 35), 5F11-45 V9 (SEQ ID NOs: 30 and 36) , 5F11-45 V9 D270E (SEQ ID NOs: 30 and 37), 5F11-45 V11 (SEQ ID NOs: 30 and 38), or 5F11-45 V12 (SEQ ID NOs: 30 and 39).

In certain embodiments, anti-huCD40 antibodies of the present invention that contain V4 or V9 Fc sequence variants further comprise a D270E sequence variant. Such antibodies include humanized 12D6-24 V4 D270E (SEQ ID NOs: 9 and 17), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20), 5F11-45 V4 D270E (SEQ ID NOs: 30 and 34) and 5F11 -45 V9 D270E (SEQ ID NOs: 30 and 37), where the sequences are provided for the light and heavy chains, respectively. In alternative embodiments, the anti-human CD40 antibodies of the present invention comprise antibodies comprising heavy and light chains substantially consisting of the sequences of these heavy and light chains, or comprising heavy and light chains having at least 80%, 85%, 90%, and 95% identity. sequences with these sequences. In certain embodiments, the anti-huCD40 antibodies of the present invention comprise modified Fc regions with greater specificity for binding to FcyRIIb compared to binding to activating receptors than antibodies with native Fc regions. In certain embodiments, the A/I ratio for an anti-huCD40 antibody of the present invention is less than 5, and in preferred embodiments, less than 1.

In certain embodiments, an anti-huCD40 antibody of the present invention comprises one or more heavy chains and one or more light chains, such as two heavy chains and two light chains.

The present invention further relates to nucleic acids encoding the variable regions of the heavy and/or light chains of the anti-CD40 antibodies of the present invention or antigen-binding fragments thereof, expression vectors containing the nucleic acid molecules, cells transformed with the expression vectors, and methods for producing antibodies by expressing the antibodies by the cells. , transformed with expression vectors, and recovery of antibodies.

The present invention also relates to pharmaceutical compositions containing the anti-huCD40 antibodies of the present invention, or antigen-binding fragments thereof, and a carrier.

The present invention relates to a method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of an anti-huCD40 antibody of the present invention, or an antigen-binding fragment thereof, such that the subject's immune response is enhanced. In certain embodiments, the individual is afflicted with a tumor and there is an increase in the immune response against the tumor. In another embodiment, the individual is afflicted with a viral infection, such as a chronic viral infection, and an antiviral immune response is enhanced.

The present invention also provides a method for suppressing tumor growth in a subject, comprising administering to the subject an anti-huCD40 antibody of the present invention, or an antigen-binding fragment thereof, such that tumor growth is suppressed.

The present invention further relates to a method of treating cancer, such as by immunotherapy, comprising administering to an individual in need thereof a therapeutically effective amount of an anti-huCD40 antibody of the present invention, or an antigen-binding fragment thereof, such as in the form of a pharmaceutical composition, thereby treating the cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer. bowel cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasia, lymphoma, leukemia, myeloma, sarcoma, and virus-associated malignant tumor. In certain embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

In certain embodiments, methods for modulating immune system function and methods of treatment described herein comprise administering an anti-huCD40 antibody of the present invention in combination or as a bispecific reagent with one or more additional therapeutic agents, e.g., an anti-PD1 antibody, an anti-PD antibody. -L1, an anti-LAG3 antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-CD137 antibody, an anti-CD27 antibody, an anti-CSF-1R antibody, an anti-CTLA-4 antibody, a TLR agonist, or a small molecular weight IDO antagonist, or TGFp. In specific embodiments, the huCD40 therapeutic is combined with a PD1 and/or PD-L1 therapeutic, e.g., treatment with an antibody or antigen-binding fragment thereof that binds to human PD1, or an antibody or antigen-binding fragment thereof that binds to human PD-L1.

Brief description of the drawings

In FIG. 1 shows the sequence of the human IgG1f constant domain (SEQ ID NO: 65), numbered 118-446 to better illustrate the Fc sequence variants described herein (Table 4). Residues subject to variation are shown in bold and the amino acid modified is shown in bold below the residue. The D270E replacement is underlined. The C-terminal lysine residue (K) in FIG. 1 and in SEQ ID NO: 65, as well as all other heavy chain sequences and heavy chain constant domains described in the sequence listing, is deleted. However, in other embodiments, especially the nucleic acid constructs encoding the heavy chains and heavy chain constant domains of the anti-huCD40 antibodies of the present invention, these sequences contain an additional lysine residue at the C-terminus of the protein or nucleotides encoding additional lysine at the 3' end of the nucleic acid. acids.

Fig. 2 is a Venn diagram illustrating epitope groups (samples) on human CD40 bound by the antibodies of the present invention, as well as blocking CD40L binding. Antibodies with overlapping ovals or circles compete for binding to human CD40, and antibodies falling within a rectangle block CD40L from binding to human CD40.

Fig. 3A and B show dendritic cell activation as determined by IL-6 secretion by anti-CD40 agonist antibodies as a function of the Fc sequence. See Example 7. A range of antibodies were constructed containing the 12D6-24 mAb variable domain and various human IgG1f constant regions, including IgG1f, SE, SELF, P238D, V4, V8, V9, and V12 variants. In FIG. 3A shows data obtained using cells from a single donor, and FIG. 3B shows data obtained using cells from another donor.

In FIG. 4 shows cell activation, as determined by cell surface CD54, by anti-CD40 agonist antibodies, as a function of variable domain sequence. See example 7. A number of antibodies were constructed containing the human IgG1f-V12 constant region and variable domains from the parent mAbs (mouse) to CD40 12D6, 5G7, 8E8, 19G3 and 5F11. The results are plotted as median fluorescence intensity (MFI) as a function of antibody concentration.

In FIG. 5 shows the percentage of FcyR bound by various antibodies of the present invention, including antibodies with D270 substitutions. See Example 8. Antibody names containing .alpha.-soup are supernatants of antibody-producing cells, while others are purified antibodies. Data are provided as percent maximum receptor binding for each combination of antibody and receptor as determined by the ForteBio Octet system. Cm.,

- 4 040302 eg Example 3. Each group of three columns represents, from left to right, binding to hCD32a/FcyRIIa-H131 (10 μM) (shaded bars), hCD32b/FcyRIIa-R131 (10 μM) (black bars) and hCD32b/ FcyRIIb (1 μM) (white bars).

Detailed disclosure of the present invention

The present invention relates to isolated antibodies, specifically monoclonal antibodies, eg humanized or human monoclonal antibodies, that specifically bind to human CD40 (huCD40) and have agonist activity. The sequences of various humanized anti-huCD40 mouse monoclonal antibodies are provided. In certain embodiments, the antibodies described herein are derived from specific mouse heavy and light chain germline sequences and/or contain specific structural features, such as CDR regions containing specific amino acid sequences. In other embodiments, antibodies compete for CD40 binding with anti-CD40 antibodies or bind to the same epitope as anti-CD40 antibodies for which sequences are provided herein. In certain embodiments, the sequence of the heavy chain Fc region is modified to specifically enhance binding to FcyRIIb.

In addition, provided herein are methods for preparing such antibodies, immunoconjugates and bispecific molecules containing such antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated containing these antibodies or fragments. Also provided herein are methods of using antibodies to enhance an immune response, alone or in combination with other immunostimulatory agents (eg, antibodies) and/or therapeutics against cancers or infections. Thus, the anti-huCD40 antibodies described herein can be used in the treatment of a wide range of therapeutic applications, including, for example, tumor growth inhibition and treatment of chronic viral infections.

Definitions

To facilitate understanding of the present description, some terms are first defined. In the course of the detailed description, additional definitions are provided.

CD40 is a member of the TNF receptor superfamily 5 (TNFRSF5). Unless otherwise indicated or clear from the context, reference CD40 herein refers to human CD40 (huCD40), and anti-CD40 antibodies refers to anti-human CD40 antibodies. Human CD40 is further described in GENE ID NO: 958 and MIM (Mendelian Inheritance in Man): 109535. The sequence of human CD40 (NP_001241.1), including the 20 amino acid signal sequence, is given in SEQ ID NO: 1.

CD40 interacts with the CD40 ligand (CD40L), also referred to as TNFSF5, gp39 and CD154. Unless otherwise indicated or clear from the context, CD40L references herein refer to human CD40L (huCD40L). Human CD40L is further described in GEN ID NO: 959 and MIM: 300386. The sequence of human CD40L (NP_000065.1) is given in SEQ ID NO:2.

Unless otherwise indicated or clear from the context, the term antibody, as used herein, can include whole antibodies and any antigen-binding fragments (eg, antigen-binding parts) or individual chains. In one embodiment, the antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain natural IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains, CH1, C _H 2 and C _H 3. In certain natural antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and light chain constant region. The light chain constant region consists of a single domain, CL. The V _H and V _L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). Each of VH and V _L consists of three CDRs and four framework regions (FR) arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

Typically, antibodies bind to the antigens they recognize specifically with high affinity, as reflected by a dissociation constant (K _D ) of 10-7-10 ^-11 M or less. Any K _D greater than about 10 ^-6 M, as a rule, is considered as indicating non-specific binding. As used herein, an antibody that specifically binds to an antigen refers to an antibody that binds to an antigen and is substantially identical to high affinity antigens, meaning binding to a K _D of 10 ^-7 M or less, preferably 10 ^-8 M or less, even more preferably 5 x 10' ⁹ M or less, and most preferably 10 ^-8 M to 10 ^-10 M or less, but does not bind with high affinity to unrelated antigens. An antigen is substantially identical to a given antigen if it exhibits a high degree of sequence identity with the given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity with that of the antigen. As an example, an antibody that specifically binds to human CD40 can also cross-react with CD40 of certain non-human primate species (e.g., cynomolgus monkey), but cannot cross-react with CD40 of other species or antigen other than CD40.

Unless otherwise indicated, immunoglobulins can form any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype in certain species is divided into subclasses: IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice.

Immunoglobulins, such as human IgG1, exist as several allotypes that differ from each other by no more than a few amino acids. Unless otherwise indicated, the antibodies of the present invention comprise an IgG1f constant domain (SEQ ID NO:65). Unless otherwise indicated, antibodies may include, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human antibodies and non-human antibodies; fully synthetic antibodies and single chain antibodies.

As used herein, the term antigen-binding portion or antigen-binding fragment of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, human CD40). Examples of binding fragments included in the term antigen-binding portion/fragment of an antibody include (i) a Fab fragment, a monovalent fragment consisting of V _L , VH, _CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the V _L and VH domains of one of the arms of the antibody, and (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain. An isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs linked by a synthetic linker may also contain an antigen-binding domain of an antibody if it is capable of binding the antigen.

The invention also includes single chain antibody constructs. Although the two domains of the Fv fragment, VL and VH, code for different genes, they can be linked by recombinant methods with a synthetic linker that allows them to form a single protein chain, in which the VL and VH regions pair to form a monovalent molecule known as single-stranded Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. sci. USA 85:5879-5883). I believe that such single-chain antibodies are also included in the term antigennegative part/fragment of an antibody. These and other possible designs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are prepared by conventional methods known to those skilled in the art, and the fragments are screened for suitability in the same manner as intact antibodies. Antigen-binding portions/fragments can be obtained by recombinant DNA technologies or by enzymatic or chemical cleavage of intact immunoglobulins.

Unless otherwise indicated, the word fragment, when used in relation to an antibody, as in the claims, refers to an antigen-binding fragment of an antibody such that the antibody or fragment has the same meaning as the antibody or antigen-binding fragment thereof.

A bispecific or bifunctional antibody is an artificial hybrid antibody with two different heavy/light chain pairs that provides two antigen-binding sites with specificity for different antigens. Bispecific antibodies can be made in a number of ways, including hybridoma fusion or Fab' fragment binding. See, for example, Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992) J. Immunol. 148, 15471553.

As used herein, the term monoclonal antibody refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or nucleic acid encoding the antibody, and they are reproduced without deliberately making any sequence changes. Thus, the term human monoclonal antibody refers to a monoclonal antibody containing variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are generated by hybridoma means, for example, obtained by fusion of a B cell derived from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse with a genome containing a heavy chain transgene and a light chain transgene). human) with an immortalized cell.

As used herein, the term recombinant human antibody includes all human antibodies that are produced, expressed, produced, or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., mouse) that are transgenic or transchromosomal for immunoglobulin genes. human, or from a hybridoma derived from it, (b) antibodies isolated from a host cell transformed to express antibodies, for example from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies, produced, expressed, obtained or isolated by any other means, which involve the splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that use specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during maturation of the antibody. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains an antigen-binding domain that encodes different genes that rearrange to form an antibody specific for a foreign antigen. In addition to the permutation, the variable region can further be modified by several single amino acid substitutions (referred to as a somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. With a further response to the antigen, the constant region changes (i.e., isotype switching occurs). Thus, somatically mutated and permuted nucleic acid sequences that encode immunoglobulin light chain and heavy chain polypeptides in response to an antigen may not be identical to the original germline sequences, but instead be substantially identical or similar (e.g., at least 80% identical).

A human antibody (HuMAb) refers to an antibody with variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term human antibody is not intended to include antibodies in which human framework sequences are grafted with CDR sequences derived from the germline of another mammalian species, such as a mouse. The terms human antibody and wholly human antibody are used interchangeably.

A humanized antibody refers to an antibody in which certain, most or all of the amino acids outside the CDR domains of a non-human antibody, such as a mouse antibody, have been replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of the antibody, certain, most or all of the amino acids outside the CDR domains are replaced with amino acids from human immunoglobulins, while certain, most or all of the amino acids in one or more CDR regions remain unchanged. Minor amino acid additions, deletions, insertions, substitutions or modifications are acceptable provided they do not eliminate the ability of the antibody to bind a particular antigen. The humanized antibody retains antigenic specificity similar to that of the original antibody.

A chimeric antibody refers to an antibody in which the variable regions are from one species and the constant regions are from another species, such as an antibody in which the variable regions are from a mouse antibody and the constant regions are from a human antibody. A hybrid antibody refers to an antibody with different types of heavy and light chains, such as a mouse heavy chain (original) and a humanized light chain, or vice versa.

As used herein, an isotype refers to the class of antibody (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that heavy chain constant region genes encode.

An allotype refers to naturally occurring variants within a particular isotype group where the variants differ in one or more amino acids. See, for example, Jefferis et al. (2009) mAbs 1:1.

The phrases antigen-recognizing antibody and antigen-specific antibody are used interchangeably herein with the term antibody that specifically binds to an antigen.

As used herein, isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., isolated

- 7 040302 an antibody that specifically binds to CD40 essentially does not contain antibodies that specifically bind to antigens other than CD40). However, an isolated antibody that specifically binds to a CD40 epitope may cross-react with other CD40 proteins from other species.

Effector functions resulting from the interaction of an antibody Fc region with certain Fc receptors include, but are not necessarily limited to, C1q binding, complement-mediated cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC, and antibody-dependent cell-mediated phagocytosis. (ADCP) and decreased expression of cell surface receptors (eg B cell receptor; BCR). Typically, such effector functions require the combination of an Fc region with an antigen-binding domain (eg, an antibody variable domain).

An Fc or FcR receptor is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody include the FcyR family of receptors, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and one inhibitory (FcyRIIb or equivalently FcyRIIB) receptors. The various properties of human FcyR are summarized in Table 1. 1. Most innate effector cell types co-express one or more activating FcyR and inhibitory FcyRIIb, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not inhibitory FcyRIIb in either mice or mice. in people. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activating Fc receptors it binds to.

Table 1. Properties of human FcyR

cy allelic options Affinity for human IgG Preference isotypes Distribution in cells cyRI one described High (Kd ~10 nM) IgGl=3>4»2 Monocytes, macrophages, activated neutrophils, dendritic cells? cyRIIA 131 Low to Medium IgGl>3>2>4 Neutrophils, monocytes, macrophages, eosinophils, dendritic cells, platelets R131 Low IgGl>3>4>2 cyRIIIA 158 Medium IgGl=3»4>2 NK cells, monocytes, macrophages, mast cells, eosinophils, dendritic cells? F158 Low IgGl=3»4>2 cyRIIb 1232 Low IgGl=3=4>2 B cells, monocytes, macrophages, dendritic cells, mast cells T232 Low IgGl=3=4>2

Fc region (crystallizable fragment region) or Fc domain or Fc refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or with the first component (C1q) of the classical complement system. Thus, the Fc region contains the constant region of the antibody, excluding the region of the first constant domain of the immunoglobulin (eg, CH1 or Cl). In the IgG, IgA, and IgD antibody isotypes, the Fc region contains CH2 and CH3 constant domains in each of the two antibody heavy chains; The Fc regions of IgM and IgE contain three heavy chain constant domains (CH2-4 domains) in each polypeptide chain. In IgG, the Fc region contains the Cy2 and Cy3 immunoglobulin domains and a hinge between Cy1 and Cy2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between those two amino acids) to the C-terminus of the heavy chain, where the numbering is carried out according to the EU index as in Kabat. Kabat et al. (1991) Sequences of Proteins of

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Immunological Interest, National Institutes of Health, Bethesda, MD; fig. 3c-3f of US Patent Publication No. 2008/0248028. The C _H 2 domain of the human IgG Fc region is located from about amino acid 231 to about amino acid 340, while the CH3 domain is in the Fc region on the C-terminal side of the CH2 domain, for example, it is located from about amino acid 341 to about amino acid 447 of IgG (including C-terminal lysine). As used herein, the Fc region can be a native sequence Fc, including any allotype or Fc variant (eg, a non-natural Fc). Also, Fc may refer to this region alone or within an Fc-containing protein, such as a binding protein containing an Fc region, also referred to as an Fc fusion protein (eg, antibody or immunoadhesin).

A native sequence Fc region or a native sequence Fc contains an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Native sequence human Fc regions include native sequence human IgG1 Fc region; Native-sequence human IgG2 Fc region; Native sequence human IgG3 Fc region and native sequence human IgG4 Fc region, as well as natural variants thereof. Native sequence Fcs include various Fc allotypes. See, for example, Jefferis et al. (2009) mAbs 1:1.

The term epitope or antigenic determinant refers to a site on an antigen (eg, huCD40) to which an immunoglobulin or antibody specifically binds. Epitopes in protein antigens can be formed from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids located due to the tertiary folding of the protein (usually a conformational epitope). Epitopes formed by contiguous amino acids are generally, but not always, retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Typically, an epitope contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

The term epitope mapping refers to a method for identifying molecular determinants on an antigen involved in antibody-antigen recognition. Methods for determining which epitopes bind to a given antibody are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, where overlapping or contiguous peptides from (e.g., from CD40) are tested for reactivity with a given antibody (e.g., an anti-CD40 antibody). ); x-ray diffraction crystallography; 2D nuclear magnetic resonance; yeast display (see example 6); and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)) (see example 5).

The term binds to the same epitope in relation to two or more antibodies means that the antibodies bind to the same region of amino acid residues, as determined by this method. Methods for determining binding of antibodies to the same epitope on CD40 as antibodies described herein include, for example, epitope mapping methods such as X-ray crystal analyzes of antigen:antibody complexes, which provide atomic resolution of the epitope, and hydrogen -deuterium exchange (HDX-MS). Other methods monitor antibody binding to fragments of an antigen (e.g., proteolytic fragments) or to mutated variants of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered indicative of a component of the epitope, such as alanine-scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081) or yeast display of mutant target sequence variants (see example 6). In addition, computer combinatorial epitope mapping methods can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies with the same or very similar VH and V _L or with the same CDR sequences are expected to bind to the same epitope.

Antibodies that compete with another antibody for binding to a target refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, for example, whether and to what extent one antibody inhibits the binding of another antibody to a target, can be determined using known competition experiments. In certain embodiments, an antibody competes with another antibody and inhibits its binding to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Depending on which antibody is the blocking antibody, the level of inhibition or competition may differ (eg, colds the antibody that is first incubated with the target). Competition analyzes can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb. Protoc; 2006; doi:10.1101/pdb.prot4277 or Chapter 11 Using Antibodies by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes ( for example, as is evident from the steric hindrance).

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Other competitive binding assays include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli et al. (1983) Methods in Enzymology 9:242); biotin-avidin solid phase direct EIA (see Kirkland et al. (1986) J. Immunol. 137:3614); labeled direct solid phase assay, labeled solid phase direct sandwich assay (see Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); labeled solid phase direct RIA using label I-125 (see Morel et al. (1988) Mol. Immunol. 25(1):7); solid phase direct EIA with biotinomavidin (Cheung et al. (1990) Virology 176:546); and labeled direct RIA (Moldenhauer et al. (1990) Scand. J. Immunol. 32:77).

As used herein, the terms specific binding, selective binding, selectively binds, and specifically binds refer to the binding of an antibody to an epitope on a predetermined antigen, but not to other antigens. Typically, antibody (i) binds to an equilibrium dissociation constant (K _D ) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less, when determined, for example, by Surface Plasmon Resonance (SPR) technology in the BIACORE® 2000 Surface Plasmon Resonance Device using a predetermined antigen, such as recombinant human CD40, as an analyte and an antibody as a ligand, or a Scatchard assay of antibody binding to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least twice its binding affinity for a non-specific antigen (eg BSA, casein) other than the predefined antigen or a very similar antigen. Thus, an antibody that specifically binds to human CD40 refers to an antibody that binds to soluble or cell-bound human CD40 with a KD of 10 ^-7 M or less, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less. An antibody that cross-reacts with cynomolgus CD40 refers to an antibody that binds to cynomolgus CD40 with a KD of 10 ^-7 M or less, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or even less.

As used herein, the term k _a cc _oc . or k _a refers to the association rate constant of a particular antibody-antigen interaction, while the term k _diss . or k _d as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. As used herein, the term K _D refers to the equilibrium dissociation constant, which is obtained from the ratio of kd and k _a (eg, kd/k _a ) and is expressed as a molar concentration (M). The K _D values for an antibody can be determined by methods well known in the art. Preferred methods for determining the K _D of an antibody are Biolayer Interferometry (BLI) analysis, preferably using the ForteBio Octet RED device (see Example 3), Surface Plasmon Resonance, preferably using a biosensor system such as the BIACORE® Surface Plasmon Resonance System (see Example 3). example 4) or flow cytometry and Scatchard analysis.

The term EC50 in the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof refers to the concentration of the antibody or antigen-binding fragment thereof that induces a response of 50% of the maximum response, i. half between maximum response and baseline.

The term binds to immobilized CD40 refers to the ability of an antibody described herein to bind to CD40, eg expressed on a cell surface or bound to a solid support.

As used herein, the term cross-react refers to the ability of an antibody described herein to bind to CD40 of other species. For example, an antibody described herein that binds to human CD40 can also bind to CD40 of other species (eg, cynomolgus CD40). As used herein, cross-reactivity can be determined by detecting specific reactivity with purified antigen in binding assays (eg, SPR, ELISA) or binding or other functional interaction with cells physiologically expressing CD40. Methods for determining cross-reactivity include standard binding assays as described herein, for example by BIACORE® Surface Plasmon Resonance (SPR) analysis using a BIACORE® 2000 SPR device (Biacore AB, Uppsala, Sweden) or flow cytometric methods.

As used herein, the term natural, as applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in the body (including viruses), that can be isolated from a source in nature, and that has not been purposefully modified in the laboratory by humans, is naturally occurring.

A polypeptide refers to a chain containing at least two consecutively linked

- 10 040302 amino acid residues with no upper chain length limit. One or more amino acid residues in a protein may be subject to modifications, such as glycosylation, phosphorylation, or disulfide bonding, as non-limiting examples. A protein may contain one or more polypeptides.

As used herein, the term nucleic acid molecule is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded and may be cDNA.

Conservative sequence modifications of the antibody sequence provided herein are also provided, i.e. modifications of the nucleotide and amino acid sequence that do not eliminate the binding of the antibody encoded by the nucleotide sequence, or containing the amino acid sequence, to the antigen. For example, modifications can be made by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan , histidine). Thus, a theoretically calculated non-essential amino acid residue in an anti-CD40 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art. See, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. sci. USA 94:412-417 (1997).

Alternatively, in another embodiment, all or part of the coding sequence of an anti-CD40 antibody can be randomly mutated, eg, by saturation mutagenesis, and the resulting modified anti-CD40 antibody can be screened for improved binding activity.

In relation to nucleic acids, the term significant homology means that two nucleic acids or their specified sequences, when optimally aligned and compared, are identical with the corresponding nucleotide insertions or deletions of at least about 80% of the nucleotides, typically at least about 90 to 95%, and more preferably at least about 98 to 99.5% of nucleotides. Alternatively, substantial homology exists when the regions under selective hybridization conditions hybridize to a complementary strand.

In relation to polypeptides, the term significant homology means that two polypeptides or their specified sequences, when optimally aligned and compared, are identical with corresponding amino acid insertions or deletions of at least about 80% amino acids, typically at least about 90 to 95 %, and more preferably at least about 98 to 99.5% amino acids.

The percent identity of two sequences is a function of the number of identical positions in the sequences when the sequences are optimally aligned (e.g., % homology = number of identical positions/total number of positions 100), where the optimal alignment is determined by considering the number of gaps and the length of each gap that are needed enter for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm as described in the non-limiting examples below.

The percent identity of two nucleotide sequences can be determined using the GAP program of the GCG software package, using the NWSgapdna.CMP matrix, and a gap creation penalty of 40, 50, 60, 70, or 80, and a gap extension penalty of 1, 2, 3, 4 , 5, or 6. The percent identity of two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which is built into the ALIGN program (version 2.0), using weight tables for PAM120 residue substitutions, a gap extension penalty of 12, and a gap creation penalty of 4. In addition, the percent identity of two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970 )), which is built into the GAP program of the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and a gap creation penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap penalty Change skip 1, 2, 3, 4, 5 or 6.

The nucleic acid and protein sequences described herein can be

- 11 040302 additionally used as a search sequence for searching in public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST searches for nucleotides can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST searches for proteins can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default settings of the respective programs (for example, XBLAST and NBLAST).

The nucleic acids may be in whole cells, in a cell lysate, or in partially purified or substantially purified form. A nucleic acid is isolated or purified to substantially purity when it is purified from other cellular components or other contaminants, e.g. other cellular nucleic acids (e.g. other parts of the chromosome) or proteins, by standard methods including alkaline/SDS treatment, separation in CsCl, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a plasmid, which means a closed circular double-stranded DNA into which additional sections of DNA can be ligated. Another type of vector is the viral vector, where additional stretches of DNA can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) when introduced into a host cell can integrate into the host cell's genome and thus replicate along with the host's genome. In addition, certain vectors are capable of controlling the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). Typically, expression vectors useful in recombinant DNA technologies are often in the form of plasmids. In the present description, the plasmid and vector can be used interchangeably since the plasmid is the most widely used form of the vector. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

As used herein, the term recombinant host cell (or simply host cell) is intended to refer to a cell that contains a nucleic acid that is not naturally present in that cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to an individual specific cell, but also to the progeny of such a cell. Since certain modifications may occur due to mutation or environmental exposure in subsequent generations, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term host cell as used herein.

The immune response refers to the biological response of vertebrates against foreign agents, which protects the body from these agents and the diseases they cause. The immune response is mediated by the action of cells of the immune system (eg, T-lymphocytes, B-lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells or by the liver (including antibodies , cytokines and complement) resulting in the selective targeting, binding, damage, destruction and/or elimination from the mammalian body of invasive pathogens, pathogen-infected cells or tissues, malignant or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells, such as a CD4+ or CD8+ T cell, or inhibition or deletion of _Treg cells. Effector T cells (Eff) refers to T cells (eg, CD4+ and CD8+ T cells) with cytolytic activity, as well as helper T cells (Th) cells that secrete cytokines and activate and control other immunocytes, but do not includes regulatory T cells (T _reg cells).

As used herein, the term T cell mediated response refers to a response mediated by T cells, including effector T cells (eg, CD8 ⁺ cells) and helper T cells (eg, CD4 ⁺ cells). The T cell mediated response includes, for example, cytotoxicity and T cell proliferation.

As used herein, the term cytotoxic T lymphocyte (CTL) response refers to an immune response induced by cytotoxic T cells. The CTL response is predominantly mediated by CD8+ T cells.

An immunomodulator or immunoregulator refers to an agent, such as a component of a signal transduction pathway, that may be involved in the modulation, regulation, or modification of an immune response. Modulation, regulation, or modification of an immune response refers to any change in a cell of the immune system or the activity of such a cell (eg, an effector T cell). Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of different types of cells, an increase or decrease in the activity of these cells, or other changes that may occur in the immune system. Inhibiting and stimulating immunomodulators have been identified, some of which can function intensively in the tumor microenvironment. In preferred embodiments, the immunomodulator is located on the surface of the T cell. An immunomodulatory target or immunoregulatory target is an immunomodulator that is targeted for binding and whose activity is altered by the binding of a substance, agent, fragment, compound or molecule. Immunomodulatory targets include, for example, receptors on cell surfaces (immunomodulatory receptors) and receptor ligands (immunomodulatory ligands).

Immunotherapy refers to the treatment of an individual who is afflicted with, or at risk of developing, or suffering from a relapse of a disease, in a manner including inducing, enhancing, suppressing, or otherwise modifying an immune response.

Immunostimulatory therapy refers to treatment that results in an enhancement (induction or enhancement) of an immune response in an individual, for example, for the treatment of cancer.

Potentiating an endogenous immune response means increasing the effectiveness or activity of an existing immune response in an individual. This increase in efficiency and activity can be achieved, for example, by overcoming mechanisms that suppress the host's endogenous immune response or by stimulatory mechanisms that enhance the host's endogenous immune response.

As used herein, the term bound refers to the association of two or more molecules. The bond may be covalent or non-covalent. The relationship may also be genetic (eg, recombinantly fused molecules). Such linkages can be made using a wide variety of methods accepted in the art, such as chemical conjugation and production of recombinant proteins.

As used herein, administration refers to the physical administration of a composition containing a therapeutic agent to an individual using any of the various delivery methods and systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and topical administration, typically by injection, and includes, as non-limiting examples, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraocular, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein may be administered via a non-parenteral route, eg, a topical, epidermal, or mucosal route, eg, intranasally, orally, vaginally, rectally, sublingually, or topically. The introduction can also be carried out, for example, once, many times and/or over one or more extended periods.

As used herein, the terms inhibit or block are used interchangeably and include partial and complete inhibition/blocking of at least about 50%, such as at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% .

As used herein, cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that grow into nearby tissues and can metastasize to the distal parts of the body through the lymphatic system or bloodstream.

As used herein, the terms treat and treatment refer to any intervention or methods administered to, or active agent in, an individual for the purpose of reversing, alleviating, ameliorating, suppressing, or slowing or preventing the progression, development, increase in severity, or recurrence of a symptom. , complications, conditions or biochemical indications associated with the disease. Prophylaxis refers to administration to an individual who has not been diagnosed with a disease to prevent the onset of the disease or to minimize its effects if it does.

The term effective dose or effective dosage is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A therapeutically effective amount or therapeutically effective dosage of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes the regression of a disease, as indicated by a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of periods without symptoms. disease or prevents deterioration or disability due to the disease. A prophylactically effective amount or prophylactically effective dosage of a drug is an amount of a drug that, when administered alone or in combination with another therapeutic agent to an individual at risk of developing a disease or relapse of a disease, inhibits the development or relapse of a disease. The ability of a therapeutic or prophylactic agent to achieve regression of a disease or to suppress the development or recurrence of a disease can be assessed using a number of methods known to the practitioner, such as during clinical trials in humans, in animal model systems predictive of efficacy in humans, or by evaluating the activity of the agent. in in vitro assays.

As an example, an anti-cancer agent is a drug that slows down the progression of a cancer or causes the cancer to regress in an individual. In preferred embodiments, the therapeutically effective amount of the drug causes the cancer to regress until the cancer is eliminated. Regression of a malignant tumor means that administration of an effective amount of a drug, alone or in combination with an antineoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one symptom of the disease, an increase in the frequency and duration of periods without symptoms of the disease. , preventing deterioration or disability due to the disease, or otherwise reducing the intensity of the symptoms of the disease in the patient. Pharmacological efficacy refers to the ability of a drug to bring about regression of a malignant tumor in a patient. Physiological safety refers to a tolerably low level of toxicity or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from the administration of a drug.

By way of example, for the treatment of tumors, a therapeutically effective amount or dose of a drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to untreated individuals. In the most preferred embodiments, the therapeutically effective amount or dose of the drug completely inhibits cell growth or tumor growth, eg, preferably inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Inhibition of tumor growth may not occur immediately after treatment, but may occur only after a certain period of time or after repeated administration. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the practitioner. In other preferred embodiments described herein, tumor regression can be observed and can last for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

As used herein, the term combination therapy, unless otherwise clear from the context, is intended to include concurrent administration of two or more therapeutic agents, and includes, as non-limiting examples, simultaneous dosing. Specifically, combination treatment includes co-administration (eg, administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that the administration of one therapeutic agent is somehow conditioned by the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after another therapeutic agent has been administered and has been maintained for the prescribed period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

The terms patient and subject refer to any person receiving prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat an individual with cancer.

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The following subsections describe the various aspects described in this document in more detail.

I. Antibodies to CD40

The present invention describes anti-huCD40 agonist antibodies with desirable properties for use as therapeutic agents in the treatment of diseases such as cancer. These properties include one or more of the ability to bind with high affinity to human CD40, tolerably low immunogenicity in humans, the ability to preferentially bind to FcyRIIb, and the absence of sequence mobility that can reduce the chemical stability of the antibody.

Anti-CD40 antibodies described herein by sequence bind to specific epitopes on human CD40, as can be determined as described in Examples 5 and 6. Other antibodies that bind to the same or closely related epitopes would likely also have these desired properties. , and they can be detected by performing competitive binding experiments.

Anti-huCD40 antibodies that compete with anti-huCD40 antibodies described herein

Anti-huCD40 antibodies that compete with the antibodies of the present invention for binding to huCD40 can be induced using immunization protocols similar to the immunization protocols described herein (Examples 1 and 2). Antibodies that compete for binding with huCD40 antibodies described herein by sequence can also be generated by immunizing mice or other non-human animals with human CD40 or a construct containing its extracellular domain (residues 21-193 of SEQ ID NO: 1 ), or by immunization with a human CD40 fragment containing an epitope bound by the anti-huCD40 antibodies described herein. The resulting antibodies can be screened for the ability to block the binding of 12D6, 5F11, 8E8, 5G7 and/or 19G3 to human CD40 by methods well known in the art, such as blocking binding to a fusion protein from the extracellular domain of CD40 and an immunoglobulin Fc domain in an ELISA, or block the ability bind to cells expressing huCD40 on their surface, for example, via FACS. In various embodiments, the test antibody is contacted with a CD40-Fc fusion protein (or with cells expressing huCD40 on their surface) before, simultaneously with, or after the addition of 12D6, 5F11, 8E8, 5G7, or 19G3. For example, sorting experiments (Example 4) can be performed to determine if a test antibody is in the same sample as antibodies described herein by sequence, antibodies described herein by sequence as control antibodies, and antibodies tested as tested antibodies. Antibodies that reduce the binding of antibodies described herein by sequence to human CD40 (as Fc fusion or on cell), especially at approximately stoichiometric concentrations, are likely to bind the same, overlapping or adjacent epitopes and thus may also have the required functional properties of 12D6, 5F11, 8E8, 5G7 or 19G3.

Thus, provided herein are anti-huCD40 antibodies that inhibit the binding of anti-huCD40 antibodies described herein to huCD40 on cells of at least 10, 20, 30, 40, 50, 55, 60, 65, 70, 75 at least by 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, for example, as determined by ELISA or FACS, for example using the assay described in the next paragraph.

An exemplary competition experiment to determine test antibody blocking binding to (eg, competition with) a control antibody can be performed as follows: cells expressing CD40 are plated in a 96-well plate with 105 cells per sample well. The plate is placed on ice, followed by the addition of unconjugated test antibody at concentrations ranging from 0 to 50 μg/ml (triple titration starting at the highest concentration of 50 μg/ml). Extraneous IgG can be used as an isotype control of the first antibody and added at the same concentrations (triple titration, starting with the highest concentration of 50 µg/ml). As a positive control for complete blocking (100% inhibition), a sample previously incubated with 50 μg/ml of unlabeled control antibody can be added, and as a negative control (no competition; 0% inhibition), a sample without antibody can be used in the primary incubation. After 30 minutes of incubation, a labeled, eg biotinylated, control antibody is added at a concentration of 2 μg/ml per well without washing. Samples are incubated for an additional 30 min on ice. Unbound antibodies are removed by washing the cells with FACS buffer. Cell-bound labeled control antibody is detected using an agent that detects the label, such as conjugated with

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Streptavidin PE (Invitrogen cat. no. S21388) for biotin detection. Samples are placed on a FACS Calibur flow cytometer (BD, San Jose) and analyzed using Flowjo software (Tree Star, Inc, Ashland, OR). The results can be presented as % inhibition.

Typically, the same experiment is then carried out in reverse, eg the test antibody is the control antibody and the control antibody is the test antibody. In certain embodiments, an antibody at least partially (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%) or completely (100%) blocks another antibody from binding to a target, for example, human CD40 or a fragment thereof, and regardless of whether the inhibition occurs when the control antibody is one or the other antibody. The control antibody and the test antibody cross-block each other from binding to the target when the antibodies compete with each other in both directions, for example in competition experiments in which the control antibody is added first and in competition experiments in which the test antibody is added first.

Anti-huCD40 antibodies are considered to compete with anti-huCD40 antibodies described herein if they inhibit the binding of 12D6, 5F11, 8E8, 5G7 and/or 19G3 to human CD40 by at least 10, 20, 30, 40, 50, 60, 70 , 80, 90, or 100% when present in approximately equal concentrations, for example in competition experiments such as those described in Example 4. Unless otherwise indicated, an antibody is considered to compete with an antibody selected from the group consisting of anti-CD40 antibodies. of the present invention if it reduces the binding of the selected antibody to human CD40 (SEQ ID NO:1) by at least 20% when used at approximately the same molar concentration with the selected antibody as determined in competitive ELISA experiments as described in the two previous paragraphs.

Anti-huCD40 antibodies that bind to the same epitope Anti-huCD40 antibodies that bind to the same or similar epitopes as the antibodies described herein can be induced using immunization protocols similar to those described herein (Examples 1 and 2). The resulting antibodies can be screened for high affinity binding to human CD40 (Example 3). The selected antibodies can then be examined in a yeast display assay, in which huCD40 sequence variants are presented on the surface of yeast cells (Example 6), or in hydrogen-deuterium exchange experiments (Example 5), to determine the exact epitope bound by the antibody.

Epitope determination can be carried out by any method known in the art. In various embodiments, anti-huCD40 antibodies are considered to bind to the same epitope as an anti-huCD40 mAb described herein if they come into contact with one or more of the same residues in at least one region of huCD40; if they come into contact with most of the residues at least one in one region of huCD40; if they come into contact with most of the residues in each region of huCD40; if they come into contact with most of the contacts all over huCD40; if they come into contact in all the same areas of human CD40; if they come into contact with all residues in any of the human CD40 regions; or if they come into contact with all the same remnants in all the same areas. Epitope regions are clusters of residues in the primary sequence.

Methods for detecting antibodies that bind the same epitope on huCD40 to the antibodies described herein include X-ray analyzes of crystals of antigen:antibody complexes that provide atomic resolution of the epitope. In other methods, binding of an antibody to fragments of an antigen or mutant variants of an antigen is determined, where loss of binding due to modification of an amino acid residue in the sequence of the antigen is often considered indicative of a component of the epitope. Also, methods can be based on the ability of the antibody of interest to affinity isolate specific short peptides (in native three-dimensional form or in denatured form) from combinatorial phage display peptide libraries or from a protease cleavage product of a target protein. The peptides are then considered as guides to determine the epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping, which have been shown to map conformational discontinuous epitopes.

An epitope, or a region containing an epitope, can also be identified by screening for binding to a number of overlapping CD40-spanning peptides. Alternatively, the method of Jespers et al. can be used to direct the selection of antibodies with the same epitope and thus similar properties to anti-CD40 antibodies described herein. (1994) Biotechnology 12:899. First, using phage display, the heavy chain of the anti-CD40 antibody is paired with a repertoire of light chains (preferably human) with a selection of a CD40 binding antibody, and then a new light chain is paired with a repertoire of heavy chains (preferably a human) with a selection of a CD40 binding antibody (preferably a human) in order to the same epitope or epitope region as the anti-huCD40 antibodies described herein. Alternatively, variants of an antibody described herein can be generated by mutagenesis of cDNA encoding heavy and

- 16 040302 antibody light chains.

Also, alanine-scanning mutagenesis as described by Cunningham & Wells (1989) Science 244:1081, or some other form of point mutagenesis of CD40 amino acid residues (e.g., the yeast display method provided in Example 6) can be used to determine the functional epitope of an anti-CD40 antibody. .

An epitope or epitope region (an epitope region is a region containing an epitope or overlapping with an epitope) bound by a specific antibody can also be determined by assaying the binding of the antibody to peptides containing CD40 fragments. A number of overlapping peptides spanning the sequence of CD40 (e.g., human CD40) can be synthesized and screened for binding, e.g., in a direct ELISA, a competitive ELISA (where a peptide is assayed for its ability to prevent antibody from binding to CD40 bound to a well of a microtiter plate), or chip. Such peptide screening methods cannot detect certain discontinuous functional epitopes, such as functional epitopes containing amino acid residues that are not consecutive in the primary sequence of the CD40 polypeptide chain.

An epitope can also be identified by MS based protein footprinting such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). HDX-MS can be performed, for example, as further described in Wei et al. (2014) Drug Discovery Today 19:95, the methods of which are specifically incorporated herein by reference. Example 5 FPOP can be performed as described, for example, in Hambley & Gross (2005) J. American Soc. Mass Spectrometry 16:2057, the methods of which are specifically incorporated herein by reference.

The epitope bound by anti-CD40 antibodies can also be determined by structural methods such as X-ray crystal structure determination (e.g., WO 2005/044853), molecular modeling, and nuclear magnetic resonance (NMR) spectrometry, including NMR determination of the H-D exchange rates of labile hydrogen atoms of CD40 amides. when it is in free form and when it is complexed with the antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31:11335; Zinn-Justin et al. (1993) Biochemistry 32:6884).

With respect to X-ray crystallography, crystallization can be carried out by any of the methods known in the art (e.g., Giege et al. (1994) Acta Crystallogr. D50:339; McPherson (1990) Eur. J. Biochem. 189:1), including microvolume ( e.g. Chayen (1997) Structure 5:1269), hanging drop vapor diffusion (e.g. McPherson (1976) J. Biol. Chem. 251:6300), seeding and dialysis. It is desirable to use a protein preparation at a concentration of at least about 1 mg/ml, and preferably from about 10 mg/ml to about 20 mg/ml. Crystallization is best carried out in a precipitating solution containing polyethylene glycol 1000-20000 (PEG; average molecular weight in the range from about 1000 to about 20000 Da), preferably from about 5000 to about 7000 Da, more preferably about 6000 Da, with concentrations in the range of about from 10% to about 30% (w/v). It may also be desirable to include a protein stabilizer, such as glycerol, at a concentration in the range of about 0.5% to about 20%. Also in the precipitating solution, a suitable salt such as sodium chloride, lithium chloride, or sodium citrate may be desirable, preferably in a concentration in the range of about 1 mM to about 1000 mM. The precipitant is preferably buffered at a pH of about 3.0 to about 5.0, preferably about 4.0. The specific buffers available in the precipitant solution may vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of suitable buffers include, but are not limited to, HEPES, Tris, MES, and acetate. Crystals can be grown over a wide range of temperatures including 2, 4, 8 and 26°C.

Antibody:antigen crystals can be studied by well-known X-ray diffraction techniques and can be detailed using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Blundell & Johnson (1985) Meth. Enzymol 114 & 115, H. W. Wyckoff et al., eds., Academic Press; US Patent Application Publication No. 2004/0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst D56:1313-1323), the descriptions of which are hereby incorporated by reference in their entirety.

Unless otherwise indicated and within the context of the claims, an antibody-bound epitope is an epitope as determined by HDX-MS methods, essentially as described in Example 5.

Anti-CD40 antibodies with high affinity binding

In certain embodiments, the anti-huCD40 antibodies of the present invention bind to huCD40 with high affinity, similar to the anti-huCD40 antibodies described herein, increasing the likelihood that they are effective therapeutic agents. In various embodiments, the anti-huCD40 antibodies of the present invention bind to huCD40 with a K _D of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM, or 100 pM. In other embodiments

- 17 040302 huCD40 antibodies of the present invention bind to huCD40 with a KD of 2 nM to 100 pM. Standard assays for evaluating antibody binding ability to huCD40 include ELISA, RIA, Western blotting, biolayer interferometry (BLI) (see Example 3) and SPR BIACORE® assay (see Example 4).

Anti-CD40 antibody sequence variants

You can allow a certain variability in the sequences of the antibodies described herein, and still retain the desired properties of the antibodies. CDR regions were established using the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Thus, the present invention further provides anti-huCD40 antibodies comprising CDR sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CDR sequences of the antibodies described herein. (eg 12D6, 5F11, 8E8, 5G7 and 19G3 and their humanized derivatives). The present invention also relates to huCD40 antibodies comprising heavy and/or light chain variable domain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to heavy chain variable domain sequences. and/or light chains of the antibodies described herein (eg, 12D6, 5F11, 8E8, 5G7 and 19G3 and their humanized derivatives).

Anti-CD40 antibodies with common CDR sequences or derived from the same mouse germline

Given that antigen binding specificity is predominantly determined by CDRs, antibodies with common CDR sequences to the antibodies described herein (eg, 12D6, 5F11, 8E8, 5G7, and 19G3) are highly likely to have their desired properties. In certain embodiments, an anti-huCD40 antibody of the present invention comprises heavy and light chain variable regions derived from the same mouse germline V and J region sequences as antibodies 12D6, 5F11, 8E8, 5G7, or 19G3. The 12D6 antibody contains a heavy chain derived from the mouse germ lines VH1-39_01 and IGHJ4 and a light chain from the germ lines VK1-110_01 and IGKJ1. The 5F11 antibody contains a heavy chain derived from the mouse germ lines VH1-4_02 and IGHJ3 and a light chain from the germ lines VK3-5_01 and IGKJ5. The 8E8 antibody contains a heavy chain derived from the mouse germ lines VH180_01 and IGHJ2 and a light chain from the germ lines VK1-110_01 and IGKJ2. The 5G7 antibody contains a heavy chain derived from the mouse germ lines VH1-18_01 and IGHJ4 and a light chain from the germ lines VK10-96_01 and IGKJ2. The 19G3 antibody contains a heavy chain derived from the mouse germ lines VH5-9-4_01 and IGHJ3 and a light chain from the germ lines VK1-117_01 and IGKJ2. The sequences of the D regions of the germline heavy chains (constituting part of CDRH3) are not indicated as their identity is often difficult to ascertain due to their high variability, and thus the antibodies of the present invention may contain heavy chains derived from the listed V- and J germlines. -regions and any germline D-region. Other antibodies that bind to human CD40 and are derived from some or all of these germline sequences are highly likely to be sequence-contiguous, especially those derived from the same V-region genes, and thus would be expected to also share the same same required properties.

As used herein, a mouse antibody contains heavy or light chain variable regions that are derived from a particular germline sequence if the antibody variable regions are derived from a system that uses mouse germline immunoglobulin genes and the antibody sequence is sufficiently close to that germline so that it is more likely to come from that germline than from any other. Such systems include immunizing a mouse with an antigen of interest. The mouse germline immunoglobulin sequence(s) from which the antibody sequence is derived can be identified by comparing the amino acid sequence of the antibody with the amino acid sequence of mouse germline immunoglobulins and selecting the germline immunoglobulin sequence that is closest in sequence (e.g., with the highest % identity) to antibody sequence. A mouse antibody that is derived from a particular germline immunoglobulin sequence may contain different amino acids compared to the germline sequence, for example due to natural somatic mutations or the deliberate introduction of a site-specific mutation. However, in general, the mouse antibody of choice is at least 90% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin (eg, V region) gene. In certain instances, a mouse antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene (e.g., V region). Typically, an antibody derived from a particular mouse germline sequence contains no more than 10 amino acids that differ from the amino acid sequence encoded by the germline immunoglobulin gene (eg, the V region). In certain cases, the mouse antibody may contain no more than 5 or even no more than 4, 3, 2 or 1 amino acids that differ from the amino acid sequence encoded by the germline immunoglobulin gene (eg, V region).

II. Engineered and modified antibodies

Regions VH and V _L

Also provided are engineered and modified antibodies that can be obtained using as a starting material for constructing a modified antibody, where this modified antibody may have properties changed compared to the original antibody, antibodies with one or more of the VH and/or V _L sequences described in this document. An antibody can be engineered by modifying one or more residues in one or both of the variable regions (eg, VH and/or VL), for example, in one or more CDRs and/or one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues in the constant region(s), such as changing the effector function(s) of the antibody.

One type of variable region construction that can be performed is CDR grafting. Such grafting is specifically used in humanized non-human anti-CD40 antibodies that compete for binding with anti-huCD40 antibodies described herein and/or bind to the same epitope as anti-huCD40 antibodies described herein. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDR differ more between antibodies than those outside the CDR. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific control antibodies by constructing expression vectors that contain CDR sequences from a particular control antibody grafted onto another antibody's framework sequences with different properties (see e.g., Riechmann, L. et al (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; US Pat. Nos. 5,225,539 to Winter;

Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) The Repertoire of Human Germline Vh Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

Preferred framework sequences for use in the antibodies described herein are framework sequences that are structurally similar to the framework sequences used in the antibodies described herein. CDR1, -2 and -3 VH sequences and CDR1, -2 and -3 VL sequences can be grafted onto framework regions with sequences identical to those found in the germline immunoglobulin gene from which the framework sequences are derived, or CDR sequences can be grafted onto framework regions. regions that contain up to 20, preferably conservative, amino acid substitutions compared to germline sequences. For example, it has been found that in certain cases, in order to maintain or enhance the ability of an antibody to bind an antigen, it is advisable to carry out mutations of residues in the framework regions (see, for example, US patents Nos.

The engineered antibodies described herein include antibodies in which modifications have been made to the V _H and/or V _L framework residues, for example, to improve the properties of the antibodies. Often, such backbone modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to back-mutate one or more framework residues to the appropriate germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody with the germline sequences from which the antibody is derived. To return the framework sequences to their germline configuration, somatic mutations can be back-mutated to the germline sequence, for example by site-directed mutagenesis or PCR-mediated mutagenesis. Such backmutated antibodies are also intended to be included.

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Another kind of framework modification involves mutating one or more residues in the framework region, or even in one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication No. 2003/0153043 to Carr et al.

Another form of variable region modification is the mutation of amino acid residues in the CDR regions to improve one or more binding properties (eg, affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s) and affect antibody binding or other functional property of interest. Preferably conservative modifications are made. Mutations may be additions, deletions or, preferably, amino acid substitutions. In addition, as a rule, no more than one, two, three, four or five residues are changed in the CDR region.

Methionine residues in the CDRs of antibodies can be oxidized, leading to potential chemical degradation and a consequent decrease in antibody activity. Thus, anti-CD40 antibodies are also provided in which one or more methionine residues in the CDRs of the heavy and/or light chains are replaced with amino acid residues that do not undergo oxidative degradation. Similarly, deamidation sites can be removed from anti-CD40 antibodies, in particular in the CDR. Potential glycosylation sites are preferably removed from the antigen-binding domain to prevent glycosylation that may interfere with antigen binding. See, for example, US Pat. No. 5,714,350.

Binding to target antigens

In various embodiments, an antibody of the present invention is modified to selectively block antigen binding in tissues and environments where antigen binding is detrimental, but allow antigen binding where it may be beneficial. In one embodiment, a blocking peptide mask is prepared that specifically binds to an antigen-binding surface of an antibody and prevents antigen binding, where the mask is linked to each of the binding arms of the antibody via a peptidase-cleavable linker. See, for example, US Pat. No. 8,518,404 issued to CytomX. Such constructs are suitable for the treatment of malignant tumors in which protease levels are significantly elevated in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows dissociation of the masking/blocking peptide allowing selective antigen binding in the tumor rather than in peripheral tissues where antigen binding can cause undesirable side effects.

Alternatively, in a related embodiment, a bivalent binding compound (mask ligand) containing two antigen-binding domains is designed that binds to two antigen-binding surfaces of a (bivalent) antibody and interferes with antigen binding, where the two mask binding domains are linked to each other (but not to the antibody) a cleavable linker, for example cleavable with a peptidase. See, for example, International Patent Application Publication No. WO 2010/077643 filed with Tegopharm Corp. Masking ligands may contain the antigen to which the antibody is intended to bind, or may be derived from or independently derived from it. Such masking ligands are useful in the treatment of cancers in which protease levels are significantly elevated in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing avidity for antibody antigen-binding surfaces. The resulting dissociation of the masking ligand and antibody allows selective antigen binding in the tumor rather than in peripheral tissues where antigen binding can cause unwanted side effects.

Fc and modified Fc

The antibodies of the present invention may contain the variable domains of the invention combined with constant domains containing various Fc regions selected based on the biological activities (if appropriate) of the antibody for the intended use. Salfeld (2007) Nat. Biotechnol. 25:1369. For example, human IgG can be classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, and each contains an Fc region with a unique binding profile to one or more of the Fcy receptors (activating receptors FcyRI (CD64), FcyRIIA, FcyRIIC (CD32a ,c); FcyRIIIA and FcyRIIIB (CD16a,b) and the inhibitory FcyRIIB receptor (CD32b) and the first complement component (C1q). Human IgG1 and IgG3 bind to all Fcy receptors; binds to IgG2 FcyRIIA _H131 and with less affinity to FcyRIIAR ₁₃₁ , FcyRIIIA _V158 , IgG4 binds to FcyRI, FcyRIIA, FcyRIIB, FcyRIIC and FcyRIIIA _V158 , and the inhibitory receptor FcyRIIB has lower affinity for IgG1, IgG2 and IgG3 than all other Fcy receptors Bruhns et al (2009) Blood 113:3716. Studies have shown that FcyRI does not bind to IgG2 and FcyRIIIB does not bind to IgG2 or IgG4.

- 20 040302 same. Generally, in terms of ADCC activity, human IgGl = IgG3 >> IgG4 = IgG2. As a result, for example, for use in a drug where ADCC is needed, the constant domain of IgG1, rather than IgG2 or IgG4, can be selected; IgG3 can be selected if activation of FcyRIIIA-expressing NK cells, monocytes, or macrophages is required; and IgG4 can be selected if the antibody is to be used to desensitize allergic patients. IgG4 can also be selected if it is desired that the antibody lack all effector functions.

The variable regions of the anti-huCD40 antibodies described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc, which can be of any allotype or isoallotype, e.g., for IgG1: G1m , G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v);. See, for example, Jefferis et al. (2009) mAbs 1:1). The choice of allotype may be influenced by issues related to potential immunogenicity, such as minimizing the formation of antibodies to the drug.

In preferred embodiments, the anti-CD40 antibodies of the present invention comprise an Fc that binds or potently binds to an FcyRIIb that can provide an enhanced agonistic effect. See, for example, WO 2012/087928; Li & Ravetch (2011) Science 333:1030; Wilson et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754. The variable regions described herein can be linked to Fc variants that increase affinity for the inhibitory FcyRIIb receptor, for example to enhance apoptosis inducing activity or an adjuvant. Li & Ravetch (2012) Proc. Natl. Acad. sci. USA 109:10966; U.S. Patent Application Publication 2014/0010812. Such variants may provide antibodies with immunomodulatory activity associated with FcyRIIb ⁺ cells, for example, including B cells and monocytes. In one embodiment, the Fc variants provide selectively increased affinity for FcyRIIb for one or more activating receptors. Such variants may also exhibit increased FcR-mediated cross-linking, resulting in increased therapeutic efficacy. Modifications to alter binding to FcyRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328 and 332 according to the EU index . Exemplary substitutions for enhancing affinity for FcyRIIb include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 26E, 26D, 26D, 26D, 287D 327D, 327E, 328F, 328W, 328Y and 332E. Exemplary replacements include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing FcyRIIb binding include 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E, and 267E-328F. In particular, variants S267E, G236D, S239D, L328F and I332e, including the dual human IgG1 variant S267E-L328F, are of particular importance for specifically enhanced affinity for the inhibitory FcyRIIb receptor. Chu et al. (2008) Mol. Immunol. 45:3926; US Patent Application Publication 2006/024298; WO 2012/087928. Increased specificity for FcyRIIb (as opposed to FcyRIIa _R131 ) can be achieved by P238D substitution and other mutations (Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; WO 2012/1152410), as well as V262E and V264E (Yu et al. (2013) J. Am. Chem. Soc. 135:9723 and WO 2014/184545. See Table 4.

Half-life extension

In certain embodiments, an antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn. In one embodiment, the antibody is altered in the CH1 or CL region to contain a recycling receptor binding epitope taken from two loops of the CH2 domain of the IgG Fc region as described in US Pat. Other exemplary Fc variants that increase FcRn binding and/or improve pharmacokinetic properties include substitutions at positions 259, 308 and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al. (2004) J. Biol. Chem. 279(8): 6213-6216, Hinton et al. (2006) Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al. (2001) Journal of Biological Chemistry 276(9):6591 -6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H (Dall'Acqua et al. (2002) Journal of Immunology 169: 5171-5180, Dall'Acqua et al (2006) Journal of Biological Chemistry 281:23514-23524). See US Pat. No. 8,367,805.

As a way to increase affinity for FcRn, thus increasing the circulating half-life of the antibody, modification of certain conserved IgG Fc residues (1253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J Immunol 182:7663). See WO 98/023289. A combination Fc variant containing M428L and N434S has been shown to increase FcRn binding and increase serum half-life five-fold. Zalevsky et al. (2010) Nat. Biotechnol. 28:157. Combination variant Fc containing modifications T307A, E380A and N434A,

- 21 040302 also increases the half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol. 18:1759. In addition, combination Fc variants have also been shown to prolong half-life, including variants M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and

M428L-N434S. See WO 2009/086320.

In addition, the Fc combination variant containing M252Y, S254T, and T256E increased the half-life approximately 4-fold. Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514. A related IgG1 modification providing increased affinity for FcRn but reduced pH dependence (M252Y-S254T-T256E-H433K-N434F) was used to generate an IgG1 construct (MST-HN Abdeg) for use as a competitive agent to prevent other antibodies from binding to FcRn , resulting in reduced clearance of these other antibodies, endogenous IgG (eg, in autoimmune conditions) or other exogenous (therapeutic) mAbs. Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.

Other modifications to increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6277375; 6821505; WO 97/34631 and WO 2002/060919.

In certain embodiments, hybrid IgG isotypes can be used to increase FcRn binding and potentially increase half-life. For example, an IgG1/IgG3 fusion variant can be constructed by replacing the IgG1 positions of the _CH 2 and/or _CH 3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, a fusion variant of an IgG antibody can be constructed that contains one or more substitutions, for example, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments described herein, an IgG1/IgG2 fusion variant can be constructed by substituting CH2 and/or CH3 regions at IgG2 positions with amino acids from IgG1 at positions where the two isotypes differ. Thus, an IgG antibody fusion variant can be constructed that contains one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (relative to the glycine insertion at position 236) and 327A. See US Pat. No. 8,629,113. IgG1/IgG2/IgG4 sequence fusions have been generated that are expected to increase serum half-life and improve expression. US Patent No. 7,867,491 (sequence number 18 therein).

The serum half-life of the antibodies of the present invention can also be increased by pegylation. The antibody can be pegylated, for example to increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody or antibody fragment is typically reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG moieties are bound to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol is intended to include any of the forms of PEG that are used to derivatize other proteins, such as mono-(C ₁ -C ₁₀ )-alkoxy or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the pegylation antibody is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be used for the antibodies described herein. See for example EP 0154316 issued by Nishimura et al. and EP 0401384 issued by Ishikawa et al.

Alternatively, in certain circumstances it may be desirable to reduce the half-life of an antibody of the present invention rather than increase it. Human IgG1 Fc modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A/R, I253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29: 2819) may decrease binding to FcRn, thus reducing half-life (increasing clearance) for use in situations where rapid clearance is preferred, such as medical imaging. Kenanova et al. (2005) Cancer Res. 65:622. Other means of increasing clearance include formatting the antigen-binding domains of the present invention as antibody fragments with no ability to bind to FcRn, such as Fab fragments. This modification can reduce the circulating half-life of an antibody from two weeks to several hours. If necessary, selective pegylation of antibody fragments can then be used to fine-tune (increase) the half-life of antibody fragments. Chapman et al. (1999) Nat. Biotechnol. 17:780. Antibody fragments can also be fused to human serum albumin, for example in a fusion protein construct, to increase half-life. Yeh et al. (1992) Proc. Nat'l Acad. sci. USA 89:1904. Alternatively, a bispecific antibody can be designed with a first antigen-binding domain of the present invention and a second antigen-binding domain that binds to human serum albumin (HSA). See International Patent Application Publication WO 2009/127691 and the patent references cited therein. Alternatively, specialized polypeptide sequences, such as XTEN polypeptide sequences, can be added to antibody fragments to increase half-life. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; publication of an international patent application WO

- 22 040302

2010/091122.

Additional Fc options

In general, when using an IgG4 constant domain, it is preferred to include an S228P substitution that reproduces the IgG1 hinge sequence and thus stabilizes IgG4 molecules, for example, reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925.

Potential protease cleavage sites at the hinges of IgG1 constructs can be eliminated by modifications to D221G and K222S, increasing antibody stability. WO 2014/043344.

The affinities and binding properties of Fc variants for their ligands (Fc receptors) can be determined by a number of in vitro assay methods (biochemical or immunological assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE® SPR assay) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel chromatography). filtration). These and other methods can use a label on one or more of the analyzed components and/or use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

In other embodiments, the glycosylation of the antibody is modified to increase or decrease effector function. For example, one can generate an aglycosylated antibody lacking all effector functions by mutating a conserved asparagine residue at position 297 (eg, N297A), thus abolishing complement binding and FcyRI. Bolt et al. (1993) Euro. J. Immunol. 23:403. Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 with glycosylation abolished at position 297).

Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, e.g., aglycosylated antibodies resulting from N297A/C/D/ or H mutations or produced in systems (e.g., E. coli) that do not glycosylate proteins, can be further mutated to restore FcyR binding, e.g., S298G and /or T299A/G/ or H (WO 2009/079242) or E382V and M428I (Jung et al. (2010) Proc. Natl. Acad. Sci. USA 107:604).

It is also possible to use glycoengineering to modify the anti-inflammatory properties of the IgG construct by replacing the α2,6-sialic acid content of the carbohydrate chains associated with the Asn297 Fco region, where an increased proportion of α2,6-sialylated forms leads to increased anti-inflammatory activity. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513. Conversely, reducing the proportion of antibodies with a2,6-sialylated carbohydrates may be useful in cases where anti-inflammatory properties are undesirable. Methods for modifying the α2,6-sialic acid content of antibodies, for example by selective removal of α2,6-sialylated forms or by enzymatic modification, are provided in US Patent Application Publication No. 2008/0206246. In other embodiments, the amino acid sequence of the Fc region can be modified to mimic the effect of a2,6 sialylation, for example including the modification of F241A. WO 2013/095966.

III. Physical properties of antibodies

The antibodies described herein may contain one or more glycosylation sites in any of the variable regions of the light or heavy chains. Such glycosylation sites can lead to increased immunogenicity of the antibody or to a change in the pK of the antibody due to altered antigen binding (Marshall et al. (1972) Ann. Rev. Biochem. 41:673-702; Gala and Morrison (2004) J. Immunol. 172: 5489-94 Wallick et al (1988) J Exp Med 168:1099-109 Spiro (2002) Glycobiology 12:43R-56R Parekh et al (1985) Nature 316:452-7 Mimura et al (2000) Mol Immunol 37:697706). Glycosylation is known to occur in motifs containing the N-X-S/T sequence. In some cases, it is preferable to produce an anti-huCD40 antibody that does not contain a glycosylated variable region. This can be achieved by selecting antibodies that do not contain a glycosylation motif in the variable region, or by mutating residues in the glycosylation region.

In certain embodiments, the antibodies described herein do not contain asparagine isomerism regions. Deamidation of asparagine can occur along the N-G or D-G sequences and lead to the production of an isoaspartic acid residue, which introduces a bend in the polypeptide chain and reduces its stability (the action of isoaspartic acid).

Each antibody has a unique isoelectric point (pI), which typically ranges from pH 6 to 9.5. Typically, the pI of an IgG1 antibody is in the pH range of 7-9.5, and the pI of an IgG4 antibody is typically in the pH range of 6-8. It has been suggested that antibodies with a pI outside the normal range may be misfolded and unstable in vivo.

Thus, it is preferable to obtain an anti-CD40 antibody with a pI value that falls within the normal range. This can be achieved by selecting an antibody with a pI in the normal range or by mutating charged residues on the surface.

Each antibody has a characteristic melting point, where a higher melting point means greater overall in vivo stability (Krishnamurthy & Manning (2002) Curr. Pharm. Biotechnol. 3:361-71). As a rule, it is preferable that T _M1 (initial unfolding temperature) is more than 60°C, preferably more than 65°C, even more preferably more than 70°C. The melting point of an antibody can be determined using differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett. 68:47-52) or circular dichroism (Murray et al. (2002) J Chromatogr Sci 40:343-9). In a preferred embodiment, antibodies are selected that are not susceptible to rapid degradation. Antibody degradation can be determined using capillary electrophoresis (CE) and MALDI-MS (Alexander & Hughes (1995) Anal. Chem. 67:3626-32).

In another preferred embodiment, antibodies are selected with minimal aggregation effects that may lead to the initiation of an undesirable immune response and/or altered or adverse pharmacokinetic properties. In general, antibodies are acceptable with an aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less. Aggregation can be determined in several ways, including size exclusion chromatography (SEC), high performance liquid chromatography (HPLC), and light scattering.

IV. Nucleic acid molecules

Another aspect described herein relates to nucleic acid molecules that encode the antibodies described herein. The nucleic acids may be in whole cells, in a cell lysate, or in partially isolated or substantially pure form. A nucleic acid is isolated or substantially pure when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (eg, other chromosomal DNA, eg, chromosomal DNA that is associated with isolated DNA in nature) or proteins. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

Nucleic acids described herein can be obtained by standard methods of molecular biology. For antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying human immunoglobulin genes as described below), cDNAs encoding the light and heavy chains of hybridoma-derived antibodies can be obtained by standard PCR amplification or cDNA cloning methods. For antibodies derived from an immunoglobulin gene library (eg, using phage display techniques), the nucleic acid encoding the antibody can be isolated from the library.

Once DNA fragments encoding the V _H and VL regions have been obtained, these DNA fragments can be further modified by standard recombinant DNA techniques, for example, converting variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv gene. With these modifications, the V _L or VH encoding DNA fragments are operably linked to other DNA fragments encoding other proteins, such as an antibody constant region or a flexible linker. As used in this context, the term operably linked is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments are in the same reading frame.

Isolated DNA encoding the V _H region can be converted into a full length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, C _H 1 , CH2 and/or CH3). Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 ) and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, such as an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the C _H 1 heavy chain constant region.

The isolated DNA encoding the V _L region can be converted into a full length lung gene.

- 24 040302 chain (also in the Fab light chain gene) by operably linking the V _L encoding DNA to another DNA molecule encoding the light chain constant region, C _L . Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91- 3242) and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

To obtain the scFv gene, the V _H and V _L encoding DNA fragments are operably linked to another flexible linker encoding fragment, for example, encoding the amino acid sequence (Gly ₄ -Ser) ₃ , such that the VH and V _L sequences can be expressed as a contiguous single stranded protein with V _L and V H regions linked by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883 McCafferty et al (1990) Nature 348:552-554).

V. Obtaining antibodies

Various antibodies of the present invention, such as antibodies that compete with the anti-human CD40 antibodies described herein or bind to the same epitope as they do, can be made by a number of known methods, such as the standard somatic cell hybridization method described by Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization methods are preferred, other methods for producing monoclonal antibodies can in principle also be used, such as viral or oncogenic transformation of B lymphocytes, phage display using human antibody gene libraries.

The preferred animal system for producing antibodies is the murine system. Obtaining hybridomas in mice is a very well established method. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion methods are also known.

The chimeric or humanized antibodies described herein can be derived from the sequence of a mouse monoclonal antibody prepared as described above. DNA encoding immunoglobulin heavy and light chains can be obtained from a mouse hybridoma of interest and engineered to contain non-mouse (eg, human) immunoglobulin sequences by standard molecular biology techniques. For example, to generate a chimeric antibody, mouse variable regions can be linked to human constant regions by methods known in the art (see, for example, US Pat. No. 4,816,567 to Cabilly et al.). To make a humanized antibody, the mouse CDR region can be incorporated into a human scaffold by methods known in the art (see, for example, U.S. Patent No. 5,225,539 issued to Winter and U.S. Patent Nos. 5,530,101;

In one embodiment, the antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed to human CD40 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and collectively referred to herein as human Ig mice.

The HuMAb® mouse (Medarex, Inc.), in conjunction with targeting mutations that inactivate the endogenous μ and k loci, carries human immunoglobulin miniloci genes that encode unrearranged immunoglobulin heavy (μ and γ) and light k chain sequences (see e.g., Lonberg, et al (1994) Nature 368(6474):856-859). Thus, reduced expression of IgM or to mouse is observed in mice, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity monoclonal human IgGKs (Lonberg, N. et al. (1994), supra ; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci. 764:536-546). The production and use of HuMAb mice and the genomic modifications carried by such mice are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are thus specifically incorporated by reference in their entirety. See also US Pat. Nos. 5,545,806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429, all issued by Lonberg and Kay; US Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all Lonberg and Kay; and PCT Publication No. WO 01/14424 Korman et al.

- 25 040302

In certain embodiments, the antibodies described herein are induced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as KM mice, are described in detail in PCT Publication WO 02/43478 Ishida et al.

In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to induce the anti-huCD40 antibodies described herein. For example, an alternative transgenic system referred to as Xenomouse® (Abgenix, Inc.) may be used; such mice are described, for example, in US patent No. 5939598; 6075181; 6114598; 6150584 and 6162963 issued by Kucherlapati et al.

In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to induce the anti-huCD40 antibodies described herein. For example, mice carrying a human heavy chain transchromosome and a human light chain transchromosome, referred to as TC mice, can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. sci. USA 97:722-727. In addition, cows carrying human heavy and light chain transchromosomes are described and can be used to induce anti-huCD40 antibodies described herein (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894).

Additional mouse systems described in the art for inducing human antibodies, e.g., anti-human huCD40 antibodies, include (i) VelocImmune® mice (Regeneron Pharmaceuticals, Inc.) in which endogenous mouse heavy and light chain variable regions are replaced by homologous recombination with variable regions human heavy and light chains operably linked to endogenous mouse constant regions such that chimeric antibodies (human V/mouse C) are induced in mice and then converted to fully human antibodies by standard recombinant DNA techniques; and (ii) a MEMO® mouse (Merus Biopharmaceuticals, Inc.), wherein the mouse carries unrearranged human heavy chain variable regions but a single rearranged common human light chain variable region. Such mice and their use for antibody induction are described, for example, in WO 2009/15777, US 2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO 2012/148873, US 2012 /0070861 and US 2012/0073004.

The human monoclonal antibodies described herein can also be generated by phage display methods screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are well known in the art. See for example: US Pat. Nos. 5,223,409; 5403484 and 5571698 issued by Ladner et al.; US Pat. Nos. 5,427,908 and 5,580,717 issued to Dower et al.; US Pat. Nos. 5,969,108 and 6,172,197 issued to McCafferty et al.; and US Patent Nos. 5,885,793; 6521404; 6544731; 6555313; 6582915 and 6593081 issued by Griffiths et al.

The human monoclonal antibodies described herein can also be generated using SCID mice in which human immunocytes have been reconstituted so that a human antibody response can be obtained upon immunization. Such mice are described in, for example, US Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

Methods of immunization

To generate fully human anti-CD40 antibodies, mice or transgenic or transchromosomal mice carrying human immunoglobulin genes (e.g., HCo12, HCo7, or KM mice) can be immunized with a purified or enriched preparation of CD40 antigen and/or CD40-expressing cells as described for others. antigens, for example in Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can be immunized with DNA encoding human CD40. Preferably, mice are 6-16 weeks old at first infusion. For example, for intraperitoneal immunization of mice, a purified or enriched preparation (5-50 μg) of recombinant human CD40 antigen can be used. In the event that immunization with a purified or enriched CD40 antigen preparation does not induce antibodies, mice can also be immunized with CD40 expressing cells, such as a cell line, to stimulate an immune response.

Experience with various antigens has shown that HuMAb transgenic mice respond best when they are first immunized with antigen intraperitoneally (i/p) or s/c in Ribi's adjuvant followed by i/p immunizations with antigen in Ribi's adjuvant. once every two weeks (up to 10 in total). The immune response during an immunization protocol can be monitored using plasma samples obtained through retro-orbital blood sampling. Plasma can be screened by ELISA and FACS (as described below) and mice with sufficient anti-human CD40 immunoglobulin titers can be used for fusions. Mice can be stimulated intravenously with antigen followed by sacrifice after 3 days and removal of the spleen and lymph nodes. It is believed that 2-3 fusions may be required for each immunization. How

- 26 040302 As a rule, 6 to 24 mice are immunized for each antigen. As a rule, HCo7 lines are used,

HCo12 and KM. In addition, the HCo7 and HCo12 transgenes can be crossed with each other in the same mouse with two different human heavy chain transgenes (HCo7/HCo12).

Obtaining hybridomas producing anti-CD40 monoclonal antibodies

To obtain hybridomas producing the monoclonal antibodies described herein, splenocytes and/or lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for antigen-specific antibodies. For example, single cell suspensions of spleen lymphocytes from immunized mice can be monofused with non-secreting mouse Sp2/0 myeloma cells (ATCC, CRL 1581) using 50% PEG. Cells are seeded at approximately 2x105 in a flat-bottom microtiter plate followed by incubation for two weeks in selective media containing 10% FetalClone serum, 18% 653 conditioned media, 5% Origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate , 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin, and 1x HAT (Sigma). After approximately two weeks, cells can be cultured in medium in which HAT has been replaced with HT. Individual wells can then be screened by ELISA for human IgM and IgG monoclonal antibodies. After the beginning of a significant growth of the hybridoma, it is possible to observe the environment, as a rule, after 10-14 days. Antibody-secreting hybridomas can be replated on the plate, rescreened, and when positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

VII. Antibody production

Preparation of transfectomes producing anti-CD40 monoclonal antibodies

The antibodies of the present invention, including specific antibodies for which sequences are provided, and other related anti-CD40 antibodies, can be generated in a transfectome host cell using, for example, a combination of recombinant DNA technologies and gene transfection techniques, as is well known in the art ( Morrison, S. (1985) Science 229:1202).

For example, to express antibodies or fragments thereof, DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing an antibody of interest) and these DNAs can be inserted into expression vectors. so that the genes are operably linked to transcription and translation control sequences. In this context, the term operably linked is intended to mean that the antibody gene is ligated into the vector such that the transcription and translation control sequences in the vector serve their intended function of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into a single expression vector. The antibody genes are inserted into the expression vector(s) by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present). The variable regions of the light and heavy chains of the antibodies described herein can be used to generate full length antibody genes of any antibody isotype by inserting them into expression vectors already encoding the heavy chain and light chain constant regions of the desired isotype such that the V _H segment is operably linked into the vector to the CH segment(s), and the V _L segment is operably linked in the vector to the CL segment. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (eg, a signal peptide of a non-immunoglobulin protein).

In addition to the antibody chain genes, recombinant expression vectors can carry regulatory sequences that control the expression of antibody chain genes in the host cell. The term regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of host cell for transformation, the level of expression of the desired protein, and so on. Preferred crucible regulatory sequences for expression in mammalian host cells include viral elements that cause high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (eg, adenovirus major late promoter (AdMLP)) and polyomaviruses. Alternatively, non-viral regulatory sequences such as a ubiquitin promoter or a β-globin promoter can be used. In addition, regulatory elements composed of sequences from various sources can be used, such as the SRa promoter system, which contains sequences from the early SV40 promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol Cell Biol 8:466-472).

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates isolation of host cells into which it is introduced (see, for example, US Pat. For example, typically, the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate in the host cell into which the vector is introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard methods. The various forms of the term transfection are intended to include a wide variety of methods commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, transfection with DEAE-dextran, and the like. Although it is theoretically possible to express the antibodies described herein in prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, and most preferably in mammalian host cells, is most preferred, since such eukaryotic cells, and especially mammalian cells, with more likely than prokaryotic cells to translate and secrete a correctly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be inefficient in obtaining high yields of active antibodies (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13). The antibodies of the present invention can also be produced in glycoengineered strains of the yeast Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24:210.

Preferred mammalian host cells for expressing the recombinant antibodies described herein include Chinese hamster cells (CHO cells) (including CHO dhfr- cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216 -4220 used with a DHFR selectable marker, for example as described in R. J. Kaufman and P. A. Sharp (1982) Mol Biol 159:601-621), NSO myeloma cells, COS cells and SP2 cells. Particularly for use with NSO myeloma cells, another preferred expression system is the GS gene expression system described in WO 87/04462, WO 89/01036 and EP 338841. When recombinant expression vectors encoding antibody genes are introduced into host cells mammals, antibodies are obtained by culturing the host cells for a period of time sufficient to allow expression of the antibodies in the host cells or, more preferably, the secretion of antibodies into the culture medium in which the host cells are grown. Antibodies can be isolated from the culture medium by standard protein isolation techniques.

The N- and C-terminus of the polypeptide chains of the antibodies of the present invention may differ from the expected sequence due to post-translational modifications observed in most cases. For example, heavy chains of antibodies often lack C-terminal lysine residues. Dick et al. (2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine and, to a lesser extent, glutamic acid residues on the light and heavy chains of therapeutic antibodies are often converted to pyroglutamic acid residues. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al. (2011) J. Biol. Chem. 286:11211.

The amino acid sequences of the various anti-huCD40 agonist antibodies of the present invention are provided in the sequence listing shown in Table 1. 8. For the above reasons, C-terminal lysine is not included in any of the sequences in the heavy chain sequence listing or heavy chain constant domains. However, in an alternative embodiment, each heavy chain of the anti-huCD40 antibodies of the present invention and/or the genetic construct encoding such antibodies or their heavy or light chains contains this additional lysine residue at the C-terminus of one or both of the heavy chains.

VII. Analyzes

The antibodies described herein can be tested for binding to CD40, for example by standard ELISA. Briefly, microtiter plates cover

- 28 040302 Feed with purified CD40 at 1-2 μg/ml in PBS and then block with 5% bovine serum albumin in PBS. Antibody dilutions (eg, plasma dilutions from immunized CD40 mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS/Tween and then incubated with a secondary reagent (e.g., for human antibodies or antibodies otherwise containing a human heavy chain constant region, Goat anti-human IgG Fc-specific polyclonal reagent) conjugated with horseradish peroxidase (HRP) in for 1 hour at 37°C. After washing, the plates are stained with ABTS substrate (Moss Inc, product: ABTS-1000) and analyzed on a spectrophotometer at OD 415-495. The sera from the immunized mice are then further screened by flow cytometry for binding to a cell line expressing human CD40, but not to a control cell line that does not express CD40. Briefly, antibody binding to CD40 is assessed by incubating CD40-expressing CHO cells with anti-CD40 antibody at a 1:20 dilution. Cells are washed and binding is detected using PE-labeled anti-human IgG Ab. Flow cytometry analyzes were performed using FACScan flow cytometry (Becton Dickinson, San Jose, CA). Preferably, the mice that develop the highest titers are used for fusions. Similar experiments can be performed using detection antibodies to mouse molecules if mouse antibodies to huCD40 are detected.

An ELISA as described above can be used to screen for antibodies and thus hybridomas that produce antibodies that are positive with the CD40 immunogen. Then, hybridomas producing antibodies that bind, preferably with high affinity, to CD40 can be subcloned and further characterized. Then, one of the clones of each hybridoma that retains the reactivity of the original cells (via ELISA) can be selected for cell bank generation and antibody purification.

To purify anti-CD40 antibodies, selected hybridomas can be grown in 2 liter flasks with agitation to purify monoclonal antibodies. Supernatants can be filtered and concentrated followed by affinity chromatography on Protein A Sepharose (Pharmacia, Piscataway, NJ). To prove purity, eluted IgGs can be checked by gel electrophoresis and high performance liquid chromatography. The buffer solution can be replaced with PBS and the concentration can be determined at OD ₂₈₀ using an extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and stored at -80°C.

To determine if anti-CD40 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of biotinylated MAbs can be detected with a streptavidin labeled probe. Competition studies can be performed using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies using CD40-coated ELISA plates as described above.

To determine the isotype of isolated antibodies, isotyping ELISAs can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of microtiter plates can be coated overnight at 4° C. with 1 μg/ml of antibodies to human immunoglobulins. After blocking with 1% BSA, the plates are reacted with 1 μg/ml or less of the tested monoclonal antibodies or purified isotype controls at ambient temperature for 1-2 hours. The wells can then be reacted with human IgG1 specific or human IgM conjugated alkaline phosphatase probes. The plates are then stained and analyzed as described above.

Flow cytometry can be used to test the binding of monoclonal antibodies to live cells expressing CD40. Briefly, cell lines expressing membrane-bound CD40 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with labeled phycoerythrin (PE) with an anti-IgG antibody under the same conditions as staining with the primary antibody. Samples can be analyzed with a FACScan using light and side scatter properties with single cell transmission and detection of labeled antibody binding. In addition to or instead of flow cytometry analysis, an alternative analysis using fluorescence microscopy can be used. Cells can be stained exactly as described above and analyzed by fluorescence microscopy. This method provides visualization of individual cells, but may be less sensitive depending on the density of the antigen.

Anti-huCD40 antibodies can be further tested for reactivity with the CD40 antigen by Western blotting. Briefly, cell extracts can be obtained from cells expressing CD40 and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG antibodies with alkaline phosphatase and color development performed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-CD40 antibodies include standard assays known in the art, such as Biolayer Interferometry (BLI) analysis and BIACORE® Surface Plasmon Resonance (SPR) analysis using the BIACORE® 2000 SPR device (Biacore AB, Uppsala, Sweden).

In one embodiment, the antibody specifically binds to the extracellular region of human CD40. An antibody can specifically bind to a particular domain (eg, functional domain) of the extracellular domain of CD40. In certain embodiments, the antibody specifically binds to the extracellular region of human CD40 and the extracellular region of cynomolgus CD40. Preferably, the antibody binds to human CD40 with high affinity.

VIII. Bispecific molecules

The antibodies described herein can be used to generate bispecific molecules. An anti-CD40 antibody, or antigen-binding fragment thereof, can be derivatized or coupled to another functional molecule, such as another peptide or protein (such as another antibody or receptor ligand), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. . An antibody described herein can in fact be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be included in the term bispecific molecule as used herein. To obtain a bispecific molecule described herein, an antibody described herein can be operably linked (e.g., by chemical linkage, genetic linkage, non-covalent association, or otherwise) with one or more other binding molecules, such as another antibody, fragment antibodies, peptide, or binding mimetic so that a bispecific molecule is obtained.

Thus, provided herein are bispecific molecules having at least one first binding specificity for CD40 and a second binding specificity for a second target epitope. In one of the embodiments described herein, in which the bispecific molecule is multispecific, the molecule may additionally have a third binding specificity.

In one embodiment, the bispecific molecules described herein, as molecules providing binding specificities, comprise at least one antibody or fragment thereof, including, for example, Fab, Fab', F(ab') ₂ , Fv, or single chain Fv. The antibody can also be a light chain or heavy chain dimer or any minimal fragment thereof such as Fv or a single chain construct as described in Ladner et al., US Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules described herein are murine, chimeric, and humanized monoclonal antibodies.

The bispecific molecules described herein can be obtained by conjugation of constituent molecules that confer binding specificities by methods known in the art. For example, each binding specificity moiety of a bispecific molecule can be prepared separately and then conjugated to each other. When binding specificities are mediated by proteins or peptides, a variety of binding or crosslinking agents for covalent conjugation can be used. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-( 2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(M-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see e.g. Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al (1985) Proc Natl Acad Sci USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. no. 78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugators are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When binding specificity is conferred by antibodies, they can be conjugated by sulfhydryl linkage of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in a single vector and expressed and translated in a single host cell. This method is particularly useful when the bispecific molecule is mAbxmAb, mAbxFab, FabxF(ab') ₂ or a χFab ligand fusion protein. The bispecific molecule described herein can be a single chain molecule containing one single chain antibody and a binding determinant, or a single chain bispecific molecule containing two binding determinants. A bispecific molecule may contain at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in US Pat. Nos. 5,260,203; 5455030; 4881175; 5132405; 5091513; 5476786; 5013653; 5258498 and 5482858.

Binding of bispecific molecules to their specific targets can be confirmed by art-accepted methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg, growth inhibition), or Western blot assay. Typically, each of these assays detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

IX. Compositions

Further provided are compositions, eg pharmaceutical compositions, comprising one or more anti-CD40 antibodies or antigen-binding fragments thereof, as described herein, formulated together with a pharmaceutically acceptable carrier. Such compositions may contain one or a combination (eg, two or more different) antibodies or immunoconjugates or bispecific molecules described herein. For example, the pharmaceutical composition described herein may contain a combination of antibodies (or immunoconjugates or bispecific molecules) that bind to different epitopes on the target antigen or that have complementary activity.

In certain embodiments, the composition comprises an anti-CD40 antibody at a concentration of at least 1, 5, 10, 50, 100, 150, 200, or 1-300 or 100-300 mg/mL.

The pharmaceutical compositions described herein can also be administered in a combination drug, for example combined with other agents. For example, the combination drug may comprise an anti-CD40 antibody as described herein combined with at least one other anti-cancer agent and/or T-cell stimulating (eg, activating) agent. Examples of therapeutic agents that can be used in a combination drug are described in more detail below in the section on the use of antibodies described in this document.

In certain embodiments, the therapeutic compositions described herein may contain other compounds, drugs, and/or agents used to treat cancer. Such compounds, drugs and/or agents may include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some instances, the therapeutic compositions may contain, for example, one or more anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L), anti-LAG-3 antibody, anti-CD73 antibody, anti-CD137 antibody, anti-CD27 antibody, anti-CSF-1R antibody, TLR agonist or small molecule antagonist of IDO or TGFe.

As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically acceptable. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, such as an antibody, immunoconjugate, or bispecific molecule, may be coated with some material to protect the compound from acids and other environmental conditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the parent compound and does not provide any undesirable toxicological effect (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical composition described herein may also contain a pharmaceutically acceptable antioxidant. Examples of Pharmaceutically Acceptable Antioxidants

- 31 040302 include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, through the use of coating agents such as lecithin, maintaining the required particle size in the case of dispersions, and through the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization methods above and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonicity agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions described herein is contemplated. Additional active compounds can also be included in the compositions.

In general, therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, through the use of coating agents such as lecithin, maintaining the required particle size in the case of dispersions, and through the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent with one or a combination of the ingredients listed above, if necessary, followed by microfiltration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization), whereby a powder of the active ingredient and any desired additional ingredient is obtained from their previously sterile filtered solution.

The amount of active ingredient that can be combined with a carrier material to form a single dose dosage form varies depending on the individual being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to form a single dose dosage form is generally the amount of the composition that provides a therapeutic effect. Typically, from 100%, this amount is in the range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dosing regimens are adjusted to provide the optimum desired response (eg, therapeutic response). For example, a single bolus administration may be given, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased as dictated by the requirements of the therapeutic situation. It is particularly preferred for ease of administration and dosage uniformity to formulate parenteral compositions into unit dosage forms. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for individuals to be treated; where each unit contains a predetermined amount of the active compound, calculated to provide the desired therapeutic effect, in association with the desired pharmaceutical carrier. The characteristics of the unit dosage form described herein are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect that is desired to achieve and (b) the limitations inherent in the field of formulation of such an active compound for the treatment of sensitivity in individuals.

When the antibody is administered, the dosage range is from 0.0001 to 100 mg/kg or more, typically from 0.01 to 5 mg/kg of host body weight. For example, doses may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight, or be in the range of 1-10 mg/day. kg. An exemplary treatment regimen includes administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.

In certain methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated ranges. Typically, the therapeutic antibody is administered multiple times. The intervals between individual doses can be, for example, a week, a month, every three months or a year. The intervals may also be irregular as indicated by determinations of levels of antibody to the target antigen in the patient's blood. In certain methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 µg/ml, and in certain methods, about 25-300 µg/ml.

The antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, a relatively low dose is administered at relatively long intervals over a long period of time. Certain patients continue to receive treatment for the rest of their lives. In therapeutic use, a relatively high dose is sometimes needed at relatively short intervals until the progression of the disease is reduced or stopped, and preferably until the patient has shown a partial or complete reduction in the symptoms of the disease. The patient can then optionally be switched to a prophylactic regimen, although many immuno-oncological indications do not require continued treatment.

Actual dosage levels of active ingredients in the pharmaceutical compositions described herein may vary so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without toxicity to the patient. The selected dosage level depends on a number of pharmacokinetic factors, including the activity of the specific compositions used herein, or their esters, salts or amides, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, other drugs, compounds and /or materials used in combination with the specific composition used, age, sex, weight, condition, general health and previous medical history (anamnesis) of the patient being treated and similar factors well known in the medical field.

A therapeutically effective dose of the anti-CD40 antibody described herein preferably results in a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of symptom-free periods, or the prevention of aggravation or disability due to the disease. In the context of cancer, a therapeutically effective dose preferably prevents further deterioration of physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, unusual nevus characteristics, change in nevus appearance including asymmetry, border, color and/or diameter, newly pigmented skin area, abnormal nevus, dark area under the nail, breast lumps, changes nipples, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, difficulty breathing, blood spitting up, blood in the urine, blood in the stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, stomach fullness, flatulence, abdominal fluid, vaginal bleeding, constipation, abdominal distention, intestinal perforation, acute peritonitis (infection, fever, pain), pain, bloody vomiting, severe sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle spasm, intestinal metastases, metastases in the lung, bladder metastases, liver metastases, bone metastases, kidney metastases and pancreas metastases, difficulty swallowing, and the like. Therapeutic efficacy can be determined immediately after the first administration of the huCD40 agonist mAb of the present invention, or it can be observed after a certain period of time and/or after a series of doses. Delayed efficacy can only be observed after several months of treatment, up to 6, 9 or 12 months. In light of the delayed efficacy demonstrated by certain immuno-oncological agents, it is extremely important not to prematurely conclude that the huCD40 agonist mAb of the present invention is not therapeutically effective.

A therapeutically effective dose may prevent or delay the onset of cancer, as may be desirable when early or indicative signs of the disease are present. Laboratory tests used to diagnose cancer include chemical tests (including determinations of soluble CD40 or CD40L levels) (Hock et al. (2006) Cancer 106:2148; Chung & Lim (2014) J. Trans. Med. 12:102), hematological, serological and radiological agents. Thus, any clinical or biochemical assay that allows monitoring of any of the above can be used to determine the administration of a particular therapeutic agent at a therapeutically effective dose for the treatment of cancer. One of skill in the art can determine such amounts based on factors such as the size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration chosen.

The composition described herein can be administered by one or more routes of administration by one or more of a variety of methods known in the art. As one of skill in the art will appreciate, the route and/or route of administration will vary depending on the desired results. Preferred routes of administration of the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and topical administration, typically by injection, and includes, as non-limiting examples, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, an antibody described herein may be administered via a non-parenteral route, such as topical, epidermal, or mucosal routes of administration, eg, intranasally, orally, vaginally, rectally, sublingually, or topically.

Active compounds can be formulated with carriers that protect the compound from rapid release, for example in controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Patented or well-known specialists in this field a variety of ways to obtain such compositions. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered by medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition described herein can be administered using a needleless hypodermic injection device such as those described in US Patent Nos. 5,399,163; 5383851; 5312335; 5064413; 4941880; 4,790,824 or 4,596,556. Examples of well-known implants and modules for use with huCD40 antibodies described herein include US Pat. No. 4,487,603, which describes an implantable microinfusion pump for rate controlled drug release; US patent No. 4486194, which describes a therapeutic device for the introduction of drugs through the skin; US Pat. No. 4,447,233, which describes a drug infusion pump for drug delivery at a precise infusion rate; US Pat. No. 4,447,224, which describes an implantable variable-flow infusion device for continuous drug delivery; US patent No. 4439196, which describes an osmotic drug delivery system containing compartments with multiple chambers; and US Pat. No. 4,475,196, which describes an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In certain embodiments, the anti-huCD40 antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) precludes the use of many highly hydrophilic compounds. To allow the therapeutic compounds described herein to cross BBB (if desired), they can be formulated, for example, into liposomes. For a review of methods for preparing liposomes, see, for example, US Pat. Nos. 4,522,811; 5,374,548 and 5,399,331. Liposomes may contain one or more molecules that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685) . Illustrative targeting molecules include folate or

- 34 040302 biotin (see, for example, US patent 5416016 issued by Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Chemother. 39:180); surfactant protein A receptor (Briscoe et al.

(1995) Am. J Physiol. 1233:134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;

M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. killion; I.J. Fidler (1994) Immunomethods 4:273.

X. Applications and Methods

The antibodies, antibody compositions, and methods described herein have many in vitro and in vivo benefits, including, for example, enhancing the immune response as CD40 signaling agonists. In a preferred embodiment, the antibodies described herein are human antibodies or humanized antibodies. For example, the anti-huCD40 antibodies described herein can be administered to cells in culture, in vitro or ex vivo, or to human individuals, eg in vivo, to enhance immunity in a number of diseases. Thus, provided herein are methods for modifying an immune response in an individual, comprising administering to the individual an antibody, or an antigen-binding fragment thereof, as described herein such that there is an enhancement, stimulation, or enhancement of the immune response in the individual.

Preferred individuals include human patients who may require enhanced immune response. The methods are particularly suitable for treating human patients with a disorder that can be treated by enhancing an immune response (eg, a T-cell mediated immune response). In a specific embodiment, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-huCD40 antibodies described herein may be administered together with the antigen of interest, or the antigen may already be present in the individual to be treated (eg, tumor-bearing or virus-bearing individual). When anti-CD40 antibodies are administered together with another agent, they may be administered separately or simultaneously.

Also included are methods for detecting the presence of human CD40 antigen in a sample or for quantifying human CD40 antigen, comprising bringing the sample and a control sample into contact with a human monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human CD40, under conditions that allow formation of a complex between the antibody. or an antigen-binding fragment thereof and human CD40. Complex formation is then detected, where a difference in complex formation with the sample being compared to the control sample is indicative of the presence of human CD40 antigen in the sample. In addition, the anti-CD40 antibodies described herein can be used to purify human CD40 by immunoaffinity purification.

Given the ability of the anti-huCD40 antibodies described herein to enhance costimulation of T cell responses, such as those of antigen-specific T cells, methods are provided herein for using the antibodies described herein, in vitro and in vivo, to stimulate, enhance, or increase responses of antigen-specific T cells, for example, responses of antitumor T cells. CD4+ and CD8+ T cell responses can be enhanced using anti-CD40 antibodies. T cells can be T _e ff cells, for example, CD4+ cells, CD8+ T _e ff cells, helper (T _h ) T cells, and cytotoxic (T _c ) T cells.

Also included are methods of enhancing an immune response (e.g., an antigen-specific T cell response) in an individual, comprising administering to the individual an anti-huCD40 antibody described herein such that the immune response (e.g., an antigen-specific T cell response) in the individual is enhanced. In a preferred embodiment, the individual is a tumor-bearing individual and the immune response against the tumor is enhanced. The tumor may be a solid tumor or a fluid tissue tumor, such as hemoblastosis. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-LI positive. In certain embodiments, the tumor is PD-L1 negative. The individual may also be a virus-bearing individual, and the immune response against the virus is enhanced.

Also provided are methods for inhibiting tumor cell growth in an individual, comprising administering to the individual an anti-huCD40 antibody described herein such that tumor growth in the individual is inhibited. Also provided are methods for treating a chronic viral infection in an individual, comprising administering to the individual an anti-huCD40 antibody described herein such that the chronic viral infection in the individual is treated.

In certain embodiments, the anti-huCD40 antibody is administered to the individual as adjunctive therapy. Treatment of individuals with cancer with anti-huCD40 antibodies may result in a long-term response relative to current standard of care; long life expectancy of at least 1, 2, 3, 4, 5, 10 or more years,

- 35 040302 life without recurrence for a period of at least 1, 2, 3, 4, 5 or 10 years or more. In certain embodiments, treatment of an individual with cancer with an anti-huCD40 antibody prevents cancer recurrence or delays cancer recurrence, e.g., by

1, 2, 3, 4, 5 or 10 years or more. Treatment with anti-CD40 antibodies can be used as first or second line treatment.

These and other methods described herein are described in more detail below. malignant tumor

Provided herein are methods of treating a subject with a cancer comprising administering to the subject an anti-huCD40 antibody described herein such that the subject is treated, for example such that growth of the cancer is inhibited or retarded and/or tumor regression occurs. An anti-huCD40 antibody for inhibiting the growth of malignant tumors can be used alone. Alternatively, the anti-huCD40 antibody can be used in combination with another agent, such as other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Also provided is a combination with a PD-1 inhibitor such as an anti-PD-1 antibody or an anti-PD-L1 antibody. See, for example, Ellmark et al. (2015) Oncolmmunology 4:7 e101 1484.

Thus, provided herein are methods of treating cancer, for example, by inhibiting tumor cell growth, in a subject, comprising administering to the subject a therapeutically effective amount of an anti-huCD40 antibody as described herein, such as humanized based on 12D6, 5F11, 8E8, 5G7, or 19G3, or an antigen-binding fragment thereof. The antibody can be a humanized anti-huCD40 antibody (such as any of the humanized anti-huCD40 antibodies described herein), a chimeric human anti-huCD40 antibody, or a humanized non-human anti-huCD40 antibody, such as a human, chimeric, or humanized anti-huCD40 antibody that competes for binding or binds to the same epitope as at least one of the anti-huCD40 antibodies specifically described herein.

Cancers whose growth can be inhibited using the antibodies of the invention include those that are generally responsive to immunotherapy. Non-limiting examples of malignancies for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, cancer liver, colorectal cancer, endometrial cancer, kidney cancer (eg, renal cell carcinoma (RCC)), prostate cancer (eg, hormone-resistant prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme) , cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon and head and neck cancer (or carcinoma), stomach cancer, germ cell tumor, childhood sarcoma, natural killer sinonasal tumor, melanoma (eg. , metastatic malignant melanoma such as cutaneous or intraocular malignant melanoma nomu), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, female vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer , parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced malignancies, including asbestos-induced malignancies, virus-associated malignancies (eg, human papillomavirus (HPV) associated malignancies) tumor) and hematological malignancies originating from any of the two major blood cell lineages, such as the myeloid cell line (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid cell line (which produces B-, T-, NK, and plasma cells), such as all types of leukemias, lymphomas and myelomas, such as acute, chronic, lymphocytic and/or myelogenous leukemias such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloid leukemia (M1), myeloid leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosal lymphoid tissue lymphoma (MALT), anaplastic (eg, Ki 1 +) large cell lymphoma, T-cell lymphoma/adult leukemia, mantle cell lymphoma, angioimmunoblastic T-cell

- 36 040302 lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, progenitor T-lymphoblast lymphoma, T-lymphoma and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma , lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), tumors of hematopoietic origin of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma , diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, progenitor B-lymphoblast lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Cesari's syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenström's macroglobulinemia; myeloma such as IgG myeloma, light chain myeloma, nonsecretory myeloma, indolent myeloma (also called slow growing myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; tumors of hematopoietic origin of the myeloid line, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nerves, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosa, keratoacanthoma, seminoma, follicular cancer, and thyroid teratocarcinoma; as non-limiting examples of T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and medulla cells; large granular lymphocyte (LGL) leukemia, preferably of the T cell type; a/d T-NHL hepatolienal lymphoma; peripheral/postthymic T-cell lymphoma (pleiomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these malignant tumors. The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers that have not been treated with prior immunotherapy, e.g., blocking antibodies to CTLA-4 or PD-1) and recurrent cancers. tumors.

Notwithstanding the above, the anti-huCD40 agonist antibodies of the present invention have no use in the treatment of CD40-expressing hematological malignancies that may be aggravated by treatment with a CD40 agonist. It is known that certain cancers can express CD40 and thus be subject to such exacerbation, and thus can be categorically ruled out. In other embodiments, specific tumor samples are tested for CD40 expression and excluded from therapy with the anti-huCD40 agonist antibodies of the present invention based on the test results.

The anti-huCD40 antibody can be administered as a monotherapy or as an immunostimulatory therapy alone, or it can be combined with an immunogenic agent in a cancer vaccine strategy such as cancer cells, isolated tumor antigens (including recombinant protein, peptide and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF. Many experimental tumor vaccination strategies have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428, Foon, K. 2000, ASCO Educational Book Spring: 730-738, also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, the vaccine is produced using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. It has been shown that GM-CSF is a potent activator of antigen presentation during antitumor vaccination. Dranoff et al. (1993) Proc. Natl. Acad. sci. USA 90: 3539-43.

The study of gene expression and large-scale gene expression profiles in various tumors has led to the identification of so-called tumor-specific antigens. Rosenberg, S A (1999) Immunity 10: 281-7. In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cells from which the tumor originates, such as gp100 melanocyte antigens, MAGE and Trp-2 antigens. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. CD40 agonists can be used in combination with a group of recombinant proteins and/or peptides expressed by a tumor to generate an immune response to these proteins. The immune system is normal

- 37 040302 sees these proteins as its own antigens and is thus tolerant to them. The tumor antigen may include the protein telomerase, which is required for the synthesis of chromosome telomeres and which is expressed in more than 85% of human malignant tumors and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens can also be neoantigens expressed in malignant cells due to somatic mutations that alter the protein sequence or result in fusion proteins between two unrelated sequences (e.g., bcr-abl on the Philadelphia chromosome) or are the idiotype of B cell tumors.

Other tumor vaccines may contain proteins from viruses that cause human cancer, such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpes virus (KHSV). Another form of tumor specific antigen that can be used in conjunction with CD40 inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of tumor cell proteins and these HSPs are highly effective for delivery to antigen presenting cells to stimulate antitumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120 ).

Dendritic cells (DC) are efficient antigen-presenting cells that can be used to prime an antigen-specific response. DC can be prepared ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DC for immunization was also directly fused with tumor cells (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with CD40 agonist action to activate (initiate) stronger responses against tumors.

Also, the CD40 agonist action can be combined with standard cancer treatments (eg, surgery, radiation, and chemotherapy). Agonistic action against CD40 can be effectively combined with chemotherapeutic regimens. In these cases, the dose of the chemotherapeutic agent administered can be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is an anti-huCD40 antibody in combination with dacarbazine for the treatment of melanoma. Another example of such a combination is an anti-huCD40 antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of CD40 agonists and chemotherapy is that cell death, which is a consequence of the cytotoxic effect of most chemotherapeutic compounds, results in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with CD40 agonist action due to cell death are radiation, surgery, and hormonal deprivation. Each of these protocols provides a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with CD40 agonists. Inhibition of angiogenesis leads to the death of tumor cells, which can provide the entry of tumor antigen in the route of antigen presentation in the host.

Anti-huCD40 antibodies described herein can also be used in combination with bispecific antibodies directed to Fca or Fcy receptor-expressing tumor cell effector cells (see, for example, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to interact with two separate antigens. For example, bispecific antibodies to the Fc receptor/tumor antigen (eg, Her-2/neu) have been used to direct macrophages to tumor sites. This targeted action can more effectively activate a tumor-specific response. The T-cell link of this response can be supplemented with an agonistic effect against CD40. Alternatively, using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker, the antigen can be delivered directly to the DC.

Tumors escape the host's immunological surveillance by a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by tumors. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 10371050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these molecules can be used in combination with antibodies to huCD40 to counteract immunosuppressive agents and support the host's antitumor immune response.

Anti-CD40 antibodies are capable of effectively replacing the activity of helper T cells. Ridge et al. (1998) Nature 393: 474-478. Activating antibodies to costimulatory T cell molecules such as CTLA-4 (e.g. US Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol. 164: 21602169), CD137/4-1BB (Melero et al (1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff et al. (1999)

- 38 040302

Nature 397: 262-266) may also provide increased levels of T cell activation. Also, PD1 or PD-L1 inhibitors can be used in combination with huCD40 antibodies.

There are also several experimental treatment protocols that involve activation and expansion of antigen-specific T cells ex vivo and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against the tumor (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate a T cell response to infectious agents such as CMV. Ex vivo activation in the presence of anti-CD40 antibodies can increase the frequency and activity of adoptively transferred T cells.

Chronic viral infections

In another aspect, the invention described herein relates to a method of treating an infectious disease in an individual, comprising administering to the individual an anti-huCD40 antibody, or antigen-binding fragment thereof, such that the infectious disease in the individual is treated.

Similar to its use on tumors as noted above, antibody-mediated CD40 agonist activity can be used alone or as an adjuvant treatment in combination with vaccines to enhance the immune response to pathogens, toxins, and self antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include pathogens for which no effective vaccine currently exists, or pathogens for which conventional vaccines are less than fully effective. As non-limiting examples, they include HIV, hepatitis viruses (A, B, & C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. The CD40 agonist action is particularly useful against established infections with these pathogens, such as HIV, which presents altered antigens during infection. These new epitopes are recognized as foreign at the time of administration of anti-human CD40 antibodies, thus eliciting a strong T cell response.

Certain examples of pathogenic viruses that cause infections amenable to treatment by the methods described herein include HIV, hepatitis viruses (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

Certain examples of pathogenic bacteria that cause infections amenable to treatment by the methods described herein include Chlamydia, Rickettsial bacteria, Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci and Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria.

Certain examples of pathogenic fungi that cause infections amenable to treatment by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Certain examples of pathogenic parasites that cause infections amenable to treatment by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba spp., Giardia lambia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the methods described above, CD40 agonist action can be combined with other forms of immunotherapy, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that provides enhanced presentation of tumor antigens. See, for example, Holliger (1993) Proc. Natl. Acad. sci. USA 90:64446448; Poljak (1994) Structure 2:1121-1123.

Vaccine adjuvants

The anti-huCD40 antibodies described herein can be used to enhance an antigen-specific immune response by co-administering the anti-huCD40 antibody with an antigen of interest, such as a vaccine. Thus, provided herein are methods of enhancing an immune response to an antigen in an individual, comprising administering to the individual: (i) an antigen, and (ii) an anti-huCD40 antibody, or antigen-binding fragment thereof, such that the individual's immune response to the antigen is enhanced. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or a pathogen antigen. Non-limiting examples of such antigens include those described in the sections above, such as tumor antigens (or tumor vaccines) described above, or antigens of viruses, bacteria, or other pathogens described above.

Suitable routes for administering the antibody compositions (eg, human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) described herein in vivo and in vitro are well known in the art and can be selected by those skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable doses of molecules used depend on the age and weight of the individual and the concentration and/or composition of the antibody compositions.

As previously described, the anti-huCD40 antibodies described herein may be co-administered with one or more other therapeutic agents, for example, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody may be linked to the agent (as an immunocomplex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be co-administered with other known treatments, such as cancer therapy, such as radiation. Such therapeutic agents, among others, include antineoplastic agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which alone are effective only at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg/ml every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg/ml once every 21 days. Co-administration of anti-CD40 antibodies or antigen-binding fragments thereof described herein with chemotherapeutic agents provides two anti-cancer agents that act by different mechanisms leading to cytotoxic effects on human tumor cells. Such co-administration may solve problems due to the development of drug resistance or a change in the antigenicity of the tumor cells which may render them unreactive with the antibody.

Also included herein are kits containing compositions of the antibodies described herein (eg, human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. Additionally, the kit may contain at least one additional reagent, or it, or more than one additional human antibody described herein (eg, a human antibody with complementary activity that binds to a different epitope of the CD40 antigen from the first human antibody). As a rule, sets contain a label indicating the intended use of the contents of the set. The term label includes any document or written material supplied on or with the kit or otherwise accompanying the kit.

Methods of combined treatment

In addition to the combination therapies provided above, the anti-CD40 antibodies described herein can also be used in combination therapy, eg, in the treatment of cancer, as described below.

The present invention relates to combination treatments in which an anti-huCD40 antibody is co-administered with one or more additional agents, e.g., antibodies, that are effective in stimulating an immune response, thereby further enhancing, stimulating, or enhancing the immune response in an individual.

In general, an anti-huCD40 antibody described herein may be combined with (i) another costimulatory receptor agonist and/or (ii) an inhibitory signal antagonist on T cells, each of which results in amplification of an antigen-specific T cell response (immune checkpoint regulators). response). Most co-stimulatory and co-inhibitory molecules are members of the immunoglobulin superfamily (IgSF), and the anti-CD40 antibodies described herein can be administered with agents that target members of the IgSF family to enhance the immune response. One important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6. Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to related members of the TNF receptor family, which include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4 -1BBL, CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA , SHI, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNFe, TNFR2, TNFa, LTeR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY , NGFR (see, for example, Tansey (2009) Drug Discovery Today 00:1).

In another aspect, anti-huCD40 antibodies can be used in combination with cytokine antagonists that inhibit T cell activation (eg, IL-6, IL-10, TGF-P, VEGF) or other immunosuppressive cytokines; or cytokines that stimulate T cell activation to stimulate an immune response, for example in the treatment of proliferative diseases such as cancer.

In one aspect, a T cell response can be stimulated by a combination of an anti-huCD40 mAb of the present invention and one or more of (i) protein antagonists that inhibit T cell activation (e.g., immune response checkpoint inhibitors), such as CTLA-4, PD1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, SEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1 , TIM-1 and TIM-4, and (ii) protein agonists that stimulate T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS- L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Illustrative agents that modulate one of the above proteins and that can be combined with anti-huCD40 agonist antibodies, such as the antibodies described herein, for the treatment of cancer include: YERVOY®/ipilimumab or tremelimumab (anti-CTLA-4) , galiximab (to B7.1), BMS-936558 (to PD-1), pidilizumab/CT-011 (to PD-1), keytrudu (KEYTRUDA®)/pembrolizumab/MK-3475 (to PD-1), AMP224 (to B7-DC/PD-L2), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3 - WO 11 /109400), IMP321 (to LAG-3), urelumab/BMS-663513 and PF-05082566 (to CD137/4-1BB), varlilumab/CDX1127 (to CD27), MEDI-6383 and MEDI-6469 (to OX40), RG-7888 (to OX40L - WO 06/029879), ataccicept (to TACI), muromonab-CD3 (to CD3), ipilimumab (to CTLA-4).

Other molecules that can be combined with anti-huCD40 agonist antibodies for cancer treatment include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, anti-huCD40 agonist antibodies can be combined with KIR antagonists (eg, lirilumab).

Additional agents for combination treatments include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists such as CSF-1R antagonist antibodies, including RG7155 (WO 11/70024, WO 11/107553, WO 11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO 13/169264; WO 14/036357).

In general, the huCD40 agonist antibodies described herein can be used in conjunction with one or more of an agonist agent that binds positive co-stimulatory receptors, a blocking agent that attenuates signal transduction through inhibitory receptors, and one or more of an agent that systemically enhances frequency of antitumor T cells by means that bypass specific immunosuppressive pathways in the tumor microenvironment (eg, block inhibitory receptor involvement (eg, PD-L1/PD-1 interactions), deplete or inhibit _Treg (eg, using an anti-CD25 monoclonal antibody ( e.g., daclizumab) or via ex vivo depletion with anti-CD25 antibody beads), inhibit metabolic enzymes such as IDO or reverse/prevent anergy or T cell depletion) and agents that initiate innate immune activation and/or inflammation at sites tumors.

Provided herein are methods of stimulating an immune response in a subject, comprising administering to the subject a CD40 agonist, e.g., an antibody, and one or more additional immunostimulatory antibodies, such as a PD-1 antagonist, e.g. CTLA-4, eg, an antagonist antibody, and/or a LAG3 antagonist, eg, an antagonist antibody, such that stimulation of an immune response occurs in the individual, eg, inhibition of tumor growth or stimulation of an antiviral response. In one embodiment, an anti-huCD40 agonist antibody and an anti-PD-1 antagonist antibody are administered to the subject. In one embodiment, an anti-huCD40 agonist antibody and an anti-PD-L1 antagonist antibody are administered to the subject. In one embodiment, an anti-huCD40 agonist antibody and an anti-CTLA-4 antagonist antibody are administered to the subject. In one embodiment, at least one additional immunostimulatory antibody (e.g., PD-1 antagonist antibody, PD-L1 antagonist antibody, CTLA-4 antagonist antibody, and/or LAG3 antagonist antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody may be, for example, a chimeric or humanized antibody (eg, derived from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, and/or an anti-LAG3 mouse antibody).

Provided herein are methods for treating a hyperproliferative disease (eg, cancer) comprising administering an anti-huCD40 agonist antibody and an anti-PD-1 antagonist antibody to an individual. In certain embodiments, the anti-huCD40 agonist antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose, where subtherapeutic refers to monotherapy with the indicated agent. Also provided herein are methods of altering the adverse side effects associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering to an individual an anti-huCD40 agonist antibody and a subtherapeutic dose of an anti-PD-1 antibody. In certain embodiments, the individual is a human. In certain embodiments, the anti-PD-1 antibody is a human monoclonal antibody and the anti-huCD40 agonist antibody is a humanized monoclonal antibody, such as an antibody containing the CDRs or variable regions of the antibodies described herein.

Suitable for use in the methods described herein, PD-1 antagonists include, as non-limiting examples, ligands, antibodies (eg, monoclonal antibodies and bispecific antibodies), and polyvalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, such as an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is opdivo (OPDIVO®)/nivolumab (BMS936558) or an antibody that contains the CDRs or variable regions of one of the 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (KEYTRUDA®/pembrolizumab/formerly lambrolizumab) described in WO 2012/145493; AMP-514/MEDI-0680 described in WO 2012/145493; and CT-011 (pidilizumab; formerly CT-AcTibody or BAT; see, for example, Rosenblatt et al. (2011) J. Immunotherapy 34:409). Additional known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, U.S. Patent Nos. 7,635,757 and 8,217,149 and U.S. Patent Publication No. 2009/0317368. You can also use any of the antibodies to PD-1 described in WO 2013/173223. An anti-PD-1 antibody that competes for binding and/or binds to the same epitope on PD-1 as one of these antibodies can also be used in combination therapy.

In certain embodiments, an anti-PD-1 antibody binds to human PD-1 with a KD of 5x10'8 M or less, binds to human PD-1 with a KD of 1x10'8 M or less, binds to human PD-1 with a KD of 5x10'9 M or less, or binds to human PD-1 with a KD of 1x10'8 M to ^1x10'10 M or less.

Provided herein are methods for treating hyperproliferative diseases (eg, cancers) comprising administering an anti-huCD40 agonist antibody and an anti-PD-L1 antagonist antibody to an individual. In certain embodiments, the anti-huCD40 agonist antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods of modifying the adverse side effects associated with treatment of hyperproliferative diseases with an immunostimulatory agent, comprising administering an anti-huCD40 agonist antibody and a subtherapeutic dose of an anti-PD-L1 antibody to an individual. In certain embodiments, the individual is a human. In certain embodiments, the anti-PD-L1 antibody is a human monoclonal antibody and the anti-huCD40 agonist antibody is a humanized monoclonal antibody, such as an antibody containing the CDRs or variable regions of the antibodies described herein.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (listed in WO 2007/005874 and US Pat. No. 7,943,743 as 12A4), MSB0010718C (WO 2013/79174), or an antibody that contains the CDRs or variable regions 3G10, 12A4 , 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, as described in PCT Publication WO 07/005874 and US Pat. B7-H1) or MPDL3280A (also known as RG7446). Any of the anti-PD-L1 antibodies described in WO 2013/173223, WO 2011/066389, WO 2012/145493, US Pat. Nos. 7,635,757 and 8,217,149 and US Publication No. 2009/145493 can also be used. Anti-PD-L1 antibodies that compete with and/or bind to the same epitope as either of these antibodies can also be used in combination therapy.

In a further embodiment, the huCD40 agonist antibody of the present invention is combined with a PD-1/PD-L1 signaling antagonist, such as a PD-1 antagonist or a PD-L1 antagonist, in combination with a third immunotherapeutic agent. In one embodiment, the third immunotherapeutic agent is a GITR antagonist or OX-40 antagonist, such as anti-GITR antibodies or anti-OX40 antibodies described herein.

In another aspect, the immuno-oncological agent is a GITR agonist, such as an anti-GITR agonist antibody. Suitable anti-GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO 06/105021, WO 09/009116) and MK-4166 (WO 11/028683).

In another aspect, the immuno-oncological agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO 2006/122150, WO 07/75598, WO 08/36653, WO 08/36642), indoxymod or NLG-919 (WO 09/73620, WO 09/1156652, WO 11 /56652, WO 12/142237).

Methods for the treatment of hyperproliferative diseases are provided herein.

- 42 040302 (for example, malignant tumors) comprising administering to an individual an anti-huCD40 agonist antibody described herein and an anti-CTLA-4 antagonist antibody. In certain embodiments, the anti-huCD40 agonist antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose, where subtherapeutic refers to monotherapy with the indicated agent. Provided herein are methods of modifying adverse side effects associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering to an individual an anti-huCD40 agonist antibody and a subtherapeutic dose of an anti-CTLA-4 antibody. In certain embodiments, the individual is a human. In certain embodiments, the anti-CTLA-4 antibody is an antibody selected from the group consisting of: Yervoy (YERVOY®) (ipilimumab or the 10D1 antibody described in PCT Publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206) and antibodies to CTLA-4 described in the following publications: WO 98/42752; WO00/37504; US patent No. 6207156; Hurwitz et al. (1998) Proc. Natl. Acad. sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. You can also use any of the antibodies to CTLA-4 described in WO 2013/173223.

Provided herein are methods for treating hyperproliferative diseases (eg, cancers) comprising administering an anti-huCD40 agonist antibody and an anti-LAG-3 antibody to an individual. In further embodiments, the anti-huCD40 agonist antibody is administered at a subtherapeutic dose, the anti-LAG-3 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for modifying adverse side effects associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering to an individual an anti-huCD40 agonist antibody and a subtherapeutic dose of an anti-LAG-3 antibody. In certain embodiments, the individual is a human. In certain embodiments, the anti-LAG-3 antibody is a human monoclonal antibody and the anti-huCD40 agonist antibody is a humanized monoclonal antibody, such as an antibody containing the CDRs or variable regions of the antibodies described herein. Examples of anti-LAG3 antibodies include antibodies containing the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, as described in US Patent Publication No. US 2011/0150892 and WO 2014/008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-accepted anti-LAG-3 antibodies that can be used include IMP731, described in US 2011/007023. You can also use the IMP-321. Anti-LAG-3 antibodies that compete with and/or bind to the same epitope as either of these antibodies can also be used in combination therapy.

In certain embodiments, an anti-LAG-3 antibody binds to human LAG-3 with a KD of 5x10'8 M or less, binds to human LAG-3 with a KD of 1x10'8 M or less, binds to human LAG-3 with a KD of 5x10'9 M or less, or binds to human LAG-3 with a KD of 1x10'8 M to ^1x10'10 M or less.

Administration of huCD40 agonist antibodies described herein and antagonists, e.g. antagonist antibodies, of one or more secondary target antigens such as LAG-3 and/or CTLA-4 and/or PD-1 and/or PD -L1 can enhance the patient's immune response to malignant cells. Cancers whose growth can be inhibited using the antibodies of the present disclosure include those that generally respond to immunotherapy. Representative examples of cancers to be treated by combination therapy as described herein include those cancers specifically listed above in the description of anti-huCD40 agonist antibody monotherapy.

In certain embodiments, the combination of therapeutic antibodies described herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions with each of the antibodies in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-huCD40 agonist antibody can be administered sequentially, for example, an anti-CTLA-4 antibody can be administered first and an anti-huCD40 agonist antibody second, or an anti-huCD40 agonist antibody can be administered first and an anti-CTLA-4 antibody second. Additionally or alternatively, the anti-PD-1 antibody and the huCD40 agonist antibody can be administered sequentially, e.g. the anti-PD-1 antibody can be administered first and the huCD40 agonist antibody second, or the huCD40 agonist antibody can be administered first and the anti-PD-1 antibody second. Additionally or alternatively, the anti-PD-L1 antibody and the huCD40 agonist antibody can be administered sequentially, for example, the anti-PD-L1 antibody can be administered first and the huCD40 agonist antibody second, or the anti-huCD40 agonist antibody can be administered first and the anti-PD-L1 antibody second. . Additionally or alternatively, the anti-LAG-3 antibody and the huCD40 agonist antibody can be administered sequentially, for example, the anti-LAG-3 antibody can be administered first, and

- 43 040302 the huCD40 agonist antibody second, or the huCD40 agonist antibody can be administered first and the anti-LAG-3 antibody second.

In addition, if more than one dose of the combined preparation is administered sequentially, at each time of administration, the order of the sequential administration may be reversed or maintained in the same order, the sequential administrations may be combined with the simultaneous administrations, or any combination thereof. For example, the first administration of the combination of anti-CTLA-4 antibody and anti-huCD40 agonist antibody may be simultaneous, the second administration may be sequential, where the anti-CTLA-4 antibody is first and the huCD40 agonist antibody is second, and the third administration may be sequential, where the agonist the anti-huCD40 antibody is first and the anti-CTLA-4 antibody is second, and so on. Additionally or alternatively, the first administration of the combination of anti-PD-1 antibody and anti-huCD40 agonist antibody may be simultaneous, the second administration may be sequential, where the anti-PD-1 antibody is first and the anti-huCD40 agonist antibody is second, and the third administration may be sequential, where the anti-huCD40 agonist antibody is first and the anti-PD-1 antibody is second, and so on. Additionally or alternatively, the first administration of the combination of the anti-PD-L1 antibody and the anti-huCD40 agonist antibody may be simultaneous, the second administration may be sequential, where the anti-PD-L1 antibody is first and the anti-huCD40 agonist antibody is second, and the third administration may be sequential, where the anti-huCD40 agonist antibody is first and the anti-PD-L1 antibody is second, and so on. Additionally or alternatively, the first administration of the combination of the anti-LAG-3 antibody and the anti-huCD40 agonist antibody may be simultaneous, the second administration may be sequential, where the anti-LAG-3 antibody is first and the anti-huCD40 agonist antibody is second, and the third administration may be sequential, where the anti-huCD40 agonist antibody is first and the anti-LAG-3 antibody is second, and so on. Another exemplary dosing regimen may include a first administration that is sequential, where the anti-huCD40 agonist antibody is first and the anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-PD-L1 antibody to LAG-3) the second, and subsequent injections can be simultaneous.

Optionally, an anti-huCD40 agonist antibody as the sole immunotherapeutic agent, or a combination of an anti-huCD40 agonist antibody and one or more additional immunotherapeutic antibodies (e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody, and/or anti-LAG-3 antibodies) can be further combined with an immunogenic agent such as cancer cells, isolated tumor antigens (including recombinant protein, peptide and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides such as gp100 peptides, MAGE, Trp-2, MARTI antigens and/or tyrosinase or tumor cells to express the cytokine GM-CSF (described further below). A CD40 agonist and one or more additional antibodies (eg, a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker) can also be further combined with standard cancer treatment. For example, a CD40 agonist and one or more additional antibodies (eg, CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers) can be effectively combined with chemotherapy regimens. In these cases, the dose of the other chemotherapeutic agent administered with the combination of the present disclosure may be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of an anti-CD40 agonist antibody with or without and an additional antibody such as an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody. ) additionally in combination with decarbazine for the treatment of melanoma. Another example is the combination of an anti-huCD40 agonist antibody with or without and an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an LAG-3 antibody additionally in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of CD40 agonist and blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 with chemotherapy is that the cell death that results from the cytotoxic effect of most chemotherapeutic compounds should lead to increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with combined CD40 agonist action with or without and blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 due to cell death, include radiation, surgery, or hormonal deprivation. Each of these protocols results in the generation of a tumor antigen source in the host. Angiogenesis inhibitors can also be combined with combined CD40 agonist action and blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3. Inhibition of angiogenesis leads to the death of tumor cells, which can be a source of tumor antigen entry in the host antigen presentation pathway.

An anti-huCD40 agonist antibody as a single immunotherapeutic agent or a combination of a CD40 agonist and blocking antibodies against CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 can also be used in combination with bispecific antibodies directed to effector cells expressing Fca or Fcy receptors to tumor cells. See, for example,

- 44 040302 US patent No. 5922845 and 5837243. Bispecific antibodies can be used to interact with two different antigens. The T-cell link to this response can be supplemented by the use of combined CD40 agonist action and blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3.

In another example, an anti-CD40 agonist antibody as a single immunotherapeutic agent or a combination of an anti-CD40 antibody and an additional immunostimulatory agent, e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody, and/or an anti-PD-L1 antibody, and/or anti-LAG3 agents such as antibodies can be used in combination with an anti-neoplasia antibody such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexar® (tositumomab), Zevalin® (ibritumomab), Campas® (alemtuzumab), Lymphocide® (pertuzumab), Avastin® (bevacizumab) and Tarceva® (erlotinib), etc. By way of example, and without wishing to be bound by theory, treatment with an anti-cancer antibody or toxin-conjugated anti-cancer antibody can result in the death of cancer cells (e.g., tumor cells), which can enhance the immune response mediated by an immunostimulatory agent, e.g. an anti-CD40, TIGIT, CTLA-4, PD-1, PD-L1 or LAG-3 agent, eg an antibody. In an exemplary embodiment, treatment of a hyperproliferative disease (e.g., cancer) may include an anti-cancer agent, e.g., an antibody, in combination with an anti-huCD40 agonist antibody, and optionally, an additional immunostimulatory agent, e.g., an anti-CTLA-4 antibody and/or an anti- PD-1 and/or an anti-PD-L1 antibody and/or an anti-LAG-3 agent, eg an antibody, simultaneously or sequentially or in any combination thereof, which can enhance the host's antitumor immune response.

Provided herein are methods of altering adverse side effects associated with treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering to an individual an anti-huCD40 agonist antibody with or without and a subtherapeutic dose of an anti-CTLA-4 and/or anti-PD-1 antibody. , and/or to PD-L1, and/or anti-LAG-3 agents, eg antibodies. For example, the methods described herein provide a method for reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. As used herein, a non-absorbable steroid is a glucocorticoid that exhibits extensive first pass metabolism such that after hepatic metabolism, the bioavailability of the steroid is low, eg, less than about 20%. In one of the embodiments described herein, the nonabsorbable steroid is budesonide. Budesonide is a topical glucocorticosteroid that, after oral administration, is extensively metabolized, predominantly by the liver. Entocort EC® (AstraZeneca) is a pH and time dependent oral formulation of budesonide designed to optimize drug delivery to the ileum and throughout the colon. Entocort EC® is approved in the US for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. The usual oral dose of Entocort EC® for the treatment of Crohn's disease is 6 to 9 mg/day. Entocort EC® is released in the intestine, followed by absorption and retention in the intestinal mucosa. After its passage through the target tissue of the intestinal mucosa, Entocort EC® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with insignificant glucocorticoid activity. Thus, its bioavailability is low (about 10%). The low bioavailability of budesonide results in an improved therapeutic response compared to other glucocorticoids with less first pass metabolism. Budesonide results in fewer adverse effects, including less suppression of the hypothalamic-pituitary axis, than systemically acting corticosteroids. However, prolonged administration of Entocort EC® may result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR ^58th ed. 2004; 608-610.

In additional embodiments, a CD40 agonist with blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 (e.g., anti-CD40 immunostimulatory therapeutic antibodies and optionally anti-CTLA-4 antibodies and/or or to PD-1 and/or to PD-L1 and/or to LAG-3) or without it in combination with a non-absorbable steroid can be further combined with salicylate. Salicylates include 5-ASA agents such as sulfasalazine (azulfidine®, Pharmacia &UpJohn); olsalazine (Dipentum®, Pharmacia &UpJohn); balsalazide (colazal®, Salix Pharmaceuticals, Inc.); and mesalamine (asacol®, Procter & Gamble Pharmaceuticals; pentase®, Shire US; canase®, Axcan Scandipharm, Inc.; rovase®, Solvay).

According to the methods described herein, a salicylate administered in combination with an anti-huCD40 agonist antibody with an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody or without and non-absorbable steroid, may include any overlapping or sequential administration of salicylate and non-absorbable steroid in order to reduce the incidence of colitis induced by immunostimulatory antibodies. Thus, for example, methods for reducing the incidence of immunostimulatory antibody-induced colitis described herein include administering a salicylate and a nonabsorbable steroid simultaneously or sequentially (eg, salicylate is administered 6 hours after the nonabsorbable steroid) or any combination thereof. . In addition, the salicylate and the non-absorbable steroid may be administered by the same route (eg, both are administered orally) or by different routes (eg, the salicylate is administered orally and the non-absorbable steroid is administered rectally), which may differ from the route(s) used for administration. antibodies to huCD40; and antibodies to CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3.

The anti-huCD40 agonist antibodies and antibody combination therapies described herein can also be used in combination with other well known therapies that are selected for their particular utility against the indication being treated (eg, cancer). The combinations of huCD40 agonist antibodies described herein can be used with known pharmaceutically acceptable agent(s) in sequence.

For example, the anti-huCD40 agonist antibodies and antibody combination drugs described herein can be used in combination (e.g., simultaneously or separately) with additional treatments such as radiation, chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (taxol), doxorubicin, 5-fu or camptothecin + apo2L/TRAIL (6X combo)), one or more proteasome inhibitors (eg, bortezomib or MG132), one or more Bcl-2 inhibitors (eg, BH3I-2' (bcl-xl inhibitor), indolamindioxygenase-1 (IDO1) inhibitor (eg, INCB24360), AT-101 (derivative of R -()-gossypol), ABT-263 (small molecular weight compound), GX-15-070 (obatoclax) or MCL-1 antagonists (myeloid leukemia cell differentiation protein-1)), iAP antagonists (inhibitor of apoptosis protein) (for example, smac7, smac4, low smac molecular mimetic, synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC inhibitors (histone deacetylase), anti-CD20 antibodies (eg, rituximab), angiogenesis inhibitors (eg, bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., Avastin®), synthetic triterpenoids (see Hyer et al., Cancer Research 2005;65:4799-808), modulators of c-FLIP (cellular FLICE inhibitory protein) (e.g., natural and synthetic PPARy ligands ( peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (eg, sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3P inhibitors, IAP inhibitors and/or genotoxic drugs.

The anti-huCD40 agonist antibodies and antibody combination drugs described herein may additionally be used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds that can be used as antiproliferative cytotoxic agents include, by way of non-limiting examples, the following.

Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes): uramustine, chlormethine, cyclophosphamide (Cytoxane™) phosphamide, melphalan, chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphamine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

Antimetabolites (including but not limited to folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

Suitable antiproliferative agents for combination with anti-huCD40 agonist antibodies include, but are not limited to, taxanes, paclitaxel (paclitaxel is commercially available as Taxol™), docetaxel, discodermolide (DDM), dictiostatin (DCT), peloruside A, epothilones, epothilone A, epothilone B, epothilone C , epothilone D, epothilone E, epothilone F, furanoepothilone D, deoxykisepothilone B1, [17]-dehydrodeoxysepothilone B, [18]dehydrodeoxysepothilones B, C12.13-cyclopropylepothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16desmethylepothilone B, epothilone B10, discodermolide, patupilone (EPO-906), KOS-862, KOS-1584, ZKEPO, ABJ-789, XAA296A (discodermolide), TZT-1027 (soblidotin ), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), hemiasterlin (HTI-286), E-7974, cryptophycins, LY-355703, maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17-beta-acetoxy-2 -ethoxy-6-oxo-B-homoestra1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl(+)-discodermolides and cryptotilone 1 as well as other microtubule stabilizers known in the art.

In cases where, in combination with or prior to treatment with the huCD40 agonist antibodies described herein, aberrantly proliferating cells need to be brought into a state of

- 46 040302 rest, the patient can also be administered hormones and steroids (including their synthetic analogues), such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlortrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, zoladex™. When using the methods or compositions described herein, other agents used in the modulation of tumor growth or metastasis in the clinical setting, such as antimimetics, may also be administered as needed.

Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), for example, 1996 editions (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the description of which is incorporated herein by reference.

Administration of the chemotherapeutic agent(s) and/or radiation therapy can be carried out according to therapeutic protocols well known in the art. Those skilled in the art will appreciate that the administration of the chemotherapeutic agent(s) and/or radiation therapy may vary depending on the disease being treated and the known effect of the chemotherapeutic agent(s) and/or radiation therapy on that disease. It is also known to those skilled in the art that therapeutic protocols (eg, dose rates and times of administration) may vary based on the observed effect of the administered therapeutics on the patient and based on the observed disease response to the administered therapeutics.

XI. Characterization of specific anti-CD40 agonist antibodies of the present invention

The anti-CD40 agonist antibodies of the present invention were prepared as described in Example 1. 2. The numbering of the variable domains and CDR regions is the same for all antibodies derived from the same parent clone, eg, the humanized variants provided in this section do not contain any insertions or deletions.

Table 2. Variable domains and CDRs of antibodies

Clone Chain Variable domain CDR1 CDR2 CDR3 12D6 heavy chain 1-119 31-35 50-66 99-108 12D6 light chain 1-112 24-39 55-61 94-102 5F11 heavy chain 1-117 31-35 50-66 99-106 5F11 light chain 1-111 24-38 54-60 93-101 8E8 heavy chain 1-122 31-35 50-66 99-111 8E8 light chain 1-112 24-39 55-61 94-102 5G7 heavy chain 1-113 31-35 50-66 99-102 5G7 light chain 1-107 24-34 50-56 89-97 19G3 heavy chain 1-112 31-35 50-66 99-101 19G3 light chain 1-112 24-39 55-61 94-102

The invention also relates to huCD40 antibodies related to the antibodies described herein by sequence, by virtue of being derived from the same mouse germline sequences, specifically the V and J region gene segments. Mouse germline sequences for each of the antibodies described herein , provided in Table. 3.

- 47 040302

Table 3. Mouse germline sequences for anti-huCD40 mAbs

clone Chain V-region J-region 12D6 heavy chain VH1-3901 IGHJ4 12D6 light chain VK1-110-01 IGKJ1 5F11 heavy chain VH1-4_O2 IGHJ3 5F11 light chain VK3-5_01 IGKJ5 8E8 heavy chain VH1-8001 IGHJ2 8E8 light chain VK1-110-01 IGKJ2 5G7 heavy chain VH1-1801 IGHJ4 5G7 light chain VK10-96-01 IGKJ2 19G3 heavy chain VH5-9-4_01 IGHJ3 19G3 light chain VK1-117-01 IGKJ2

The invention also relates to humanized anti-huCD40 antibodies derived from the original mouse antibodies described herein by replacing mouse framework sequences with human framework sequences with additional mutations (backmutations) to restore affinity for the antigen (human CD40) that would otherwise be be lost upon humanization or with or without sequence motility removed. See example 3.

The invention also relates to antibody constructs containing the novel variable domain sequences described herein and constant domains with modified Fc regions with increased affinity for FcyRIIb relative to their affinity for other Fc receptors, e.g., activating receptors. Such anti-huCD40 agonist antibodies with increased specificity for FcyRIIb are expected to exhibit superior efficacy in the treatment of cancer and chronic infections. Li & Ravetch (2011) Science 333:1030; White et al. (2011) J. Immunol. 187:1754. In practice, such FcyRIIb-specific anti-CD40 agonist mAbs can exhibit an enhanced adjuvant effect, increasing maturation of dendritic cells, stimulating the proliferation and activation of cytotoxic CD8+ T cells, leading to an increased antitumor response. There. In practice, FcR-mediated signal enhancement of anti-CD40 agonist antibodies due to increased clustering or cross-linking of the receptors of the present invention may be a major component of therapeutic efficacy. Cross-linking of anti-CD40 agonist antibodies through interaction of the FcR with the Fc region of the antibody can increase signal strength and thus increase cell activation.

The relative binding affinity of antibodies for activating (A) versus inhibitory (I) Fc receptors can be expressed as an A/I ratio and is generally a function of the structure of the Fc region of an IgG antibody. See WO 2012/087928. Antibodies with increased binding specificity for the inhibitory FcyRIIb receptor have lower A/I ratios. Preferred huCD40 agonist antibodies of the present invention have A/I ratios of, for example, less than 5, 4, 3, 2, 1, 0.5, 0.3, 0.1, 0.05, 0.03, or 0.01.

Human IgG1 constant domains containing mutations to increase specificity for FcyRIIb are also provided in the sequence listing and are shown in Table 1. 4 and illustrated in FIG. 1. Sequence variants are defined with respect to the human IgG1f constant domain sequence shown in SEQ ID NO: 65. The nomenclature for the positions (numbering) of mutations in the Fc region is given according to the EU index, as in Kabat et al. (1981) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), which facilitates comparison of Fc sequences at equivalent positions in antibodies with different variable domain lengths. Edelman et al. (1969) Proc. Natl. Acad. sci. USA 63:78; WO 2012/130831 (using the same numbering system). It does not match the sequence numbering in the sequence list. In FIG. 1 is a graphical representation of Fc sequence variants from Table 1. 4, from which one of skill in the art can readily ascertain the corresponding positions in the sequences of the antibodies described herein. SE and SELF variants are described in Chu et al. (2008) Mol. Immunol. 45:3926. Variants P238D, V4, V7, V8, V9, V11 and V12 are described in Mimoto et al. (2013) Protein Engineering Design & Selection 26:589 (eg, Table 1 therein).

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Table 4. Fc sequence variants

Name option SEQ ID: Sequence Options IgGlf 65 SE 66 S267E SELF 67 S267E L328F P238D 68 P238D V4 69 P238D P271G V4- D270E 70 P238D D270E P271G V7 71 E233D P238D P271G A330R V8 72 G237D P238D H268D P271G V9 73 G237D P238D P271G A330R V9- D270E 74 G237D P238D D270E P271G A330R vil 75 G237D P238D H268D P271G A330R V12 76 E233D G237D P238D H268D P271G A330R

Yu et al. (2013) J. Am. Chem. soc. 135:9723 (and WO 2014/184545) describe additional Fc sequence variants with increased affinity for FcyRIIb, including V262E and V264E, for example for use in combination with S267E and L328F.

Generated additional variants with D270E mutations to reduce D270 isomerization that occurred in V4 and V9 Fc sequence variants that otherwise showed increased isomerization rates in accelerated degradation studies predicting "12-16% isomerization after two years at 4°C in PBS compared to "5-7% for other options. See table. 5 (which shows data from one experiment, but repeated experiments showed comparable values). Replacing the aspartic acid at position 270 with glutamic acid eliminates the possibility of this DG isomerization, resulting in antibodies that are chemically more stable and antibody preparations that are more homogeneous. Other D270 substitutions in V9, such as D270A, D270Q, D270S and D270T, effectively abolish binding to FcyRIIa and FcyRIIb receptors (Kd was greater than 5 μM). Although the D270E mutation reduces binding to the FcyRIIb receptor by an order of magnitude, the V9-D270E variant retained a favorable affinity bias for the FcyRIIb receptor compared to FcyRIIa and an acceptable absolute affinity for FcyRIIb. See example 8.

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Table 5. Affinity of Fc sequence variants for Fcy receptors (KD in nM)

Designation FcyRIIa-H131 FcyRIIa-R131 FcyRIIb IgGlf 530 850 3900 SE 520 22 98 SELF 1100 2 eleven P238D >5000 >5000 950 V4 >5000 1900 150 V4-D270E >5000 >5000 1800 V7 >5000 1600 84 V8 >5000 1400 93 V9 >5000 420 15 V9-D270A,Q,S,T >5000 >5000 >5000 V9-D270E >5000 >5000 150 VI1 - 450 15 V12 >5000 490 21

The agonist activity of various anti-CD40 antibodies of the present invention was determined. See example 7 and FIG. 3A, 3B and 4. Agonist activity was found to depend on both variable domain sequences (mAb clone number) that determine antigen binding (human CD40) and an Fc region sequence that determines Fc receptor binding (FcyRIIb).

The present invention is further illustrated by the following examples, which should not be considered as a further limitation. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

Example 1

Preparation of mouse monoclonal antibodies to human CD40.

Mouse monoclonal antibodies to human CD40 were generated using wild-type Balb/c mice (Charles Rivers Labs) expressing mouse antibody genes as follows.

Antigen

Soluble recombinant protein huCD40muFc was used as an antigen for immunization. The Mw of the soluble fusion protein was 91.6 kDa and consists of the extracellular portion of huCD40 fused at its C-terminus to mouse IgG2b Fc. This fusion protein is referred to herein as the huCD40-muFc fusion protein. The fusion protein was prepared by standard recombinant DNA techniques and expressed in transfected CHO cells which secreted soluble fusion protein into the culture supernatant. The CHO host cells used for transfection were obtained from Invitrogen (Cat. No. 11619-012). The secreted soluble fusion protein was isolated for use as an immunogen.

Immunization of mice

To generate mouse monoclonal antibodies to human CD40, Balb/c mice were immunized with isolated huCD40-muFc fusion protein. Mice were approximately 2-4 months of age at first antigen infusion. An isolated recombinant huCD40-muFc antigen preparation (10 μg isolated from transfected mammalian cells expressing the fusion protein) was used to immunize mice using two immunization protocols (A and B). Protocol A consisted of four weekly footpad (FP) immunizations, and protocol B consisted of seven weekly subcutaneous (sc)/intraperitoneal (i/p)/popliteal immunizations. In both cases, the immunogen was mixed 1:1 with RIBI adjuvant (Sigma, cat. no. M6536).

All mice received blood for analysis of antigen-specific antibody titers a week after the fourth immunization, and each mouse also underwent final blood sampling at the time of sacrifice. The immune response was monitored by retroorbital bleeding. The plasma was screened by ELISA analysis using the recombinant protein used for immunization. Mice following Protocol A received two final booster injections intravenously (IV) and in the ball of the foot (FP) using soluble antigen on days -2 and -3 before fusion. Protocol B mice were given two final booster injections intravenously as well as IP and sub-knee using soluble antigen on days -2 and -3 before confluence.

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All four mice after Protocol A (ID 291763, 291764, 291765, 291766) were sacrificed. Lymph node and spleen cells were isolated and mixed for fusion (fusions 3582 and 3583). After protocol B, only two mice were sacrificed (ID 294286 and 294288). The spleen and lymph nodes of each mouse were mixed and fused (fusions 3716 and 3717).

Obtaining hybridomas producing monoclonal antibodies to human CD40

Mouse splenocytes and lymph nodes isolated from high titer Balb/c mice were fused with a mouse myeloma fusion partner using an electric field using a Hybrimune electrofusion device and a large 0.9 ml fusion chamber (BTX Harvard Apparatus, Inc., Holliston , MA). Suspensions of single lymphocytes from immunized mice were fused with an equal number of non-secreting mouse P3X63 Ag8.6.53 myeloma cells (ATCC CRL 1580). The resulting cells were seeded at 2.0 x ^{10 4} cells/well in flat-bottomed microtiter plates in ClonaCell-HY Medium E selective medium (Cat. No. 03805; STEMCELL Technologies Inc., Vancouver BC, Canada) supplemented with aminopterin for hybridoma selection. Approximately 7 days later, the culture medium was changed to medium E (without aminopterin).

After a period of 10 to 12 days, individual wells were screened for the presence of mouse IgG antibodies/mouse kappa light chains using homogeneous HTRF analysis. In this assay, supernatants from 96-well fusion plates were mixed with self-labeled terbium cryptate goat anti-mouse IgG (Fcy-specific) (Cisbio US Inc. Bedford, MA) and goat anti-mouse IgG labeled with AlexaFluor 647 (Jackson ImmunoResearch; 109-605-098) Fab'2. Incubation was carried out for 1 hour. Then the plates were read on a Rubystar scanner. Then, hybridoma cells from wells positive for mouse IgG antibodies/mouse kappa light chains were screened by FACS using human CD40-transfected CHO cells and non-transfected CHO cells as a control (fusion 3582/3583), or by ELISA using recombinant protein followed by FACS on Daudi B cells and Jurkat T cells as a negative control (fusion 3716/3717). FACS positive baselines were transferred to 24-well plates. A few days later, cell supernatants from individual wells were re-screened by FACS to confirm IgG specificity for human CD40.

A panel of antigen-specific hybridomas were cloned by serial dilutions and re-screened by FACS using CHO transfectants or Daudi cells. Nineteen antibodies from the 3582/3583 fusions and twelve antibodies from the 3716/3717 fusions were isolated and tested for functional activity. From this panel of antibodies, five potent agonists were selected for re-subcloning. These five antibodies, namely 1802.3582.19G3.F10.E1, 1802.3583.5G7.F12.G3, 1802.3583.8E8.C10.G2, 1802.3716.12D6.B1.E3 and 1802.3717, 5F11.A11.E7 was sequenced and further analyzed.

Example 2

Obtaining fully human antibodies to huCD40.

Fully human anti-huCD40 monoclonal antibodies that bind to the same epitope and/or cross-block binding of the humanized anti-CD40 antibodies described herein may find use in the methods of the present invention. Such antibodies can be generated using transgenic mice that express human antibody genes as described below.

Antigen

The soluble recombinant huCD40 protein is used as an antigen for immunization. The soluble fusion protein consists of the extracellular portion of huCD40 fused at its C-terminus to mouse IgG2a Fc. This fusion protein is referred to herein as the huCD40-muFc fusion protein. The fusion protein is prepared by standard recombinant DNA techniques and expressed in transfected CHO cells which secrete a soluble fusion protein into the culture supernatant. The CHO host cells used for transfection are obtained from Invitrogen (Cat. No. 11619-012). The secreted soluble fusion protein is isolated for use as an immunogen. The sequence of full-length human CD40, including the signal sequence, is shown in SEQ ID NO:1.

Transgenic mice

Fully human anti-human CD40 monoclonal antibodies are generated using CMD ⁺⁺ ;JKD ⁺⁺ ;KCo5(9272) ⁺ ;SC20 ⁺ genotype mice (hereinafter referred to as KM® mice). Individual transgene designations are given in parentheses followed by line numbers for randomly integrated transgenes. The symbols ++ and + indicate homozygotes or hemizygotes; however, since mice were typically screened using a PCR-based assay that did not allow the authors to distinguish between heterozygosity and homozygosity for randomly integrated human Ig transgenes, the + designation can be assigned to mice that are actually homozygous for these elements. In this line, the endogenous mouse kappa light chain gene is homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820, a endogenous mouse heavy chain gene

- 51 040302 homozygous destroyed, as described in example 1 of WO 2001/09187. In addition, this strain of mice carries the human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology

14:845-851, a yeast artificial chromosome (YAC) carrying the majority of the human kappa light chain locus as described in WO 2000/026373.

Immunization of mice

To generate fully human anti-human CD40 monoclonal antibodies, KM® mice are immunized with isolated huCD40-muFc fusion protein. General immunization regimens are described in Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. At the first antigen infusion, the mice are approximately 4 months old. For intraperitoneal or subcutaneous immunization of mice, a preparation of isolated recombinant huCD40-muFc antigen (10 μg isolated from transfected mammalian cells expressing the fusion protein) or 300-19 cells transfected with human CD40 is used. The immunogens are mixed 1:1 with RIBI adjuvant (Sigma, cat. no. M6536).

Mice are immunized five times at intervals of 5-7 days. The first and second immunizations are carried out with the recombinant protein. The third immunization is carried out with cells, the fourth immunization with protein and the fifth immunization with cells. One week after the last immunization, mice are bled for analysis of antigen-specific antibody titers. The immune response is monitored by retroorbital blood sampling. Plasma is screened by FACS analysis using transfected 300-19 cells, and mice with the highest anti-human CD40 human IgG titers are used for fusion. Mice received a final booster injection with intravenous (IV) and intraperitoneal (IP) injection of soluble antigen two days before and with transfected cells three days prior to sacrifice and spleen isolation.

Obtaining hybridomas producing human monoclonal antibodies to human CD40

Mouse splenocytes isolated from high titer KM® mice and a mouse myeloma fusion partner are fused using electric field-based electrofusion using a Cyto Pulse large cell fusion chamber electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD) . Single cell suspensions of immunized mouse spleen lymphocytes were fused with an equal number of non-secreting mouse P3X63 Ag8.6.53 myeloma cells (ATCC CRL 1580) (fusion number: 2541). The obtained cells are seeded at 2.0x10 ⁴ cells/well in flat-bottomed microtiter plates in selective DMEM medium containing high concentration of glucose (Cellgro, no. 10-013-CM) and 10% fetal bovine serum (Hyclone, no. SH30071.03) and supplemented with betamercaptoethanol (1000χ, Gibco, no. 21985-023), 7 mM HEPES (Cellgro 25-060-C1), additional 2 mM L-glutamine (Cellgro 25-005-C1), HAT (50χ, Sigma, no. H-0262 ), 5% hybridoma cloning factor (BioVeris, no. 210001), 10% P388DI conditioned medium (AtCc, no. CRL TIB-63), and penicillinstreptomycin (100χ, Cellgro, no. 30-002-CI). Approximately 7 days later, the portion of the medium containing HAT was replaced with medium containing HT (Cellgro, #25-047-CI).

After a period of 10 to 12 days, individual wells are screened for the presence of human IgG/human kappa light chain antibodies using a homogeneous HTRF assay. In this assay, supernatants from 96-well fusion plates were mixed with europium cryptate labeled goat anti-human IgG (Fc fragment specific), biotinylated goat anti-human kappa light chain (Bethyl, #A80-115B), streptavidin-XLent and incubated within 1 hour. Then the plates are read on the RUBYstar scanner.

Then, hybridoma cells from wells positive for human IgG/human kappa light chain or human IgG/human lambda light chain antibodies are screened by FACS using 300-19 cells transfected with human CD40 and untransfected 30019 cells as controls. FACS-positive baselines are transferred to 24-well plates. A few days later, cell supernatants from individual wells were re-screened by FACS to confirm IgG specificity for human CD40.

Hybridomas are cloned by serial dilution and re-screened by FACS.

Example 3

Humanization of antibodies to huCD40.

The parent antibodies (mouse) of the present invention have been humanized for potential use as human therapeutics. For each mouse variable domain, the most closely matching human germline region sequence was selected and these human framework regions were used to replace mouse frameworks in the CDR-grafted versions of the antibodies. Additional amino acid substitutions, such as backbone backmutations, were then made as needed to restore the binding affinity of the humanized antibody. Amino acid substitutions were also made to eliminate sequence mobilities. Correspondence of sequences of various forms of antibodies according to the present invention is presented in table. 6. Link

The expression of various anti-huCD40 agonist antibodies with human soluble CD40 was determined by biolayer interferometry (BLI) analysis using a labelless Fortebio Octet RED (Rapid system-Extended Detection) interaction analysis device. All studies were carried out in 10 mM Na _x PO ₄ , 130 mM NaCl, 0.05% surfactant P20 (pH 7.1) at 25°C. Briefly, anti-huCD40 antibody supernatants (diluted to 10 μg/mL or captured undiluted if the supernatant concentration was less than 10 μg/mL) were captured on protein A-coated biosensors (Pall Fortebio, no. 18-5010) using a loading time of 90 s and stirring speed 1000 rpm. Supernatants were first screened for binding to 1 μM recombinant human CD40 monomer (huCD40 monomer) using an association and dissociation time of 180 s, at 1000 rpm, with two 15 s conditioning steps using 10 mM glycine pH 1.5 between binding cycles. . Then, all supernatants that showed a binding signal in the 1 µM huCD40 monomer experiment were tested for binding to seven different concentrations of huCD40 monomer in a series of three-fold dilutions, where the highest concentration was 10 µM huCD40 monomer or 1 µM huCD40 monomer, depending on the strength of the binding signal in screening experiment with 1 µM. The results for selected antibodies and variants of their sequences are shown in table. 6.

Table 6. Binding affinity of antibodies to CD40

Antibody Type Vh V _L K _D (nM) 12D6 Initial mouse mouse 3.6 12D6-03 humanized Grafted CDR VH1-18 vaccinated CDR VKII 011 4.8 12D6-22 humanized Grafted CDR VH1-18 VKII 011 G29A 3.6 12D6-23 humanized M100bLVHl-18 vaccinated CDR VKII oil 4.3 12D6-24 humanized MIOObL VH1-18 VKII on G29A 5.6 5F11 initial mouse mouse 4.7 5F11-03 humanized Grafted CDR VHl-e vaccinated >5000

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CDR VKIV VZ 5F11-17 humanized VHl-e G27Y S30T V37I R38K A40R M48I R66K V67T I69L vaccinated CDR VKIV VZ 8.4 5F11-23 humanized VHl-e G27Y S30T V37I R38K A40R M48I R66K V67T I69L VKIV VZ M4L N22S 4.9 5F11-45 humanized VHl-e G27Y S30T R66K V67T I69L VKIV VZ M4L 12.3* 19G3 original mouse mouse 870 19G3-03 humanized Grafted CDR VH3-21 vaccinated CDR VKII 011 absence binding 19G3-11 humanized VH3-21S94R vaccinated CDR VKII 011 850 19G3-22 humanized VH3-21S94R VKII Oil N28Q 99 19G3-23 humanized VH3-21S94R VKII Oil G29A 1300 5G7 initial mouse mouse 89 5G7-03 humanized Grafted CDR VH1-18 vaccinated CDR VKI A20 340 5G7-22 humanized VH1-18M69L A93V vaccinated CDR VKI A20 50 5G7-25 humanized VH1-18 M69L T71V A93V G55A vaccinated CDR VKI A20 69 5G7-28 humanized VH1-18 M69L T71V A93VG55AM96L vaccinated CDR VKI A20 230 8E8 original mouse mouse 85 8E8-05 humanized Grafted CDR VHl-e vaccinated CDR VKII A19 absence binding

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8E8-56 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L vaccinated CDR VKII A19 82 8E8-62 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L D99E vaccinated CDR VKII A19 31 8E8-66 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L VKII Al 9 G29A 82 8E8-67 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L D99E VKII Al 9 G29A 28 8E8-70 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L D99E_G55S vaccinated CDR VKII A19 low signal 8E8-71 humanized VHl-e L4F G27Y S30T R66K V67A T68L I69L D99E_G55S VKII Al 9 G29A low signal

* = for comparison purposes, the original 5F11, when run in the same assay in parallel with the 5F11-45 antibody, showed a K _{D of} 8.2 nM (not 4.7 nM)

For humanized antibodies, columns Vh and _Vl show the germline human variable domains on which the framework regions are based. Grafted CDRs refers to variable domains containing unmodified parent CDRs (mouse) grafted directly onto said human framework sequences. The numbering of residues for all variants of sequences in Table. 6 is given by Kabat, and thus the positions of the sequence changes in Table. 6 do not correspond to the numbering of antibody sequence residues in the sequence listing. Underscores are used to indicate sequence modifications that are intended to correct for potential sequence mobilities, such as residues that undergo chemical modification and potential disruption, leading to product heterogeneity. Other sequence modifications are designed to restore affinity, typically back-mutation of the framework (recovery from the germline of a human framework sequence to the original mouse framework residue). The modification of M69L in the heavy chain of the 5G7 antibody is a back-mutation of the framework and a mobility correction.

Example 4

Anti-CD40 antibody epitope sorting experiments.

Epitope sorting experiments can be performed to determine which anti-human CD40 antibodies compete with each other for binding to huCD40 and thus bind to similar epitopes. Pairwise competition between huCD40 antibodies is defined as follows, wherein the control antibody is bound to the surface of the sensor chip, the test antibody is pre-incubated with the huCD40 polypeptide construct in the mixture, and the pre-incubated mixture is passed over the sensor chip to determine the extent to which the test antibody interferes with binding of the polypeptide construct huCD40 with a control antibody on the surface of the chip. Such competition experiments can be carried out using the SPR BIACORE® device. Briefly, a control anti-huCD40 antibody is immobilized on the surface of a CM5 sensor chip (Series S, GE Healthcare, cat. no. BR-1005-30), flowcell2, flowcell3 and flowcell4 (5000 PE) and flowcelll are used as a negative control. The antibody to be tested is diluted to 120 μg/ml (2x) at initial concentration. A series of dilutions of the test antibody is carried out by diluting the antibody concentration with buffer 1:3 for seven different concentrations and a control sample (with 0 μg/ml) to obtain a titration curve. Each series of antibody concentrations is divided into two halves. In the first half of the concentration series, 40 nM (2x) human CD40 antigen (e.g., huCD40/Fc) is added to give a final concentration series (60 μg/mL - 0.0 μg/mL) and 20 nM final antigen concentration per well. In the second half of the concentration series, a buffer is added in place of the antigen so as to obtain an antibody diluted to the same concentration, and this half is treated as a control. Anti-CD40 and huCD40/Fc test antibody complexes are incubated for 2 hours. 40 μl of the complexes are injected onto the control antibody-coated surface at 30 μl/min. A BIACORE® T200 surface plasmon resonance device is used, and the movable

-55 040302 Buffer is HBE-EP, GE Healthcare, ref. No. BR-1001-88, filtered, degassed, 0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% P20 surfactant. The surface is regenerated with 25 mM NaOH (order code: BR-1003-58, GE Healthcare) at 100 µl/min for 5 s. The data is analyzed using Microsoft Excel, where the test antibody concentration is plotted against the respective response units to obtain titration curves.

The results of such epitope sorting experiments with anti-CD40 antibodies of the present invention are shown in FIG. 2. Antibodies 5F11 and 8E8 block ligand binding (CD40L). The five anti-CD40 antibodies of the present invention fall into three epitope groups, 12D6/5G7/19G3, 5F11/5G7/19G3 and 8E8.

Example 5

Epitope mapping by HDX.

The epitopes of the anti-huCD40 antibodies 12D6, 5G7, 19G3 and 5F11 of the present invention were determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Using HDX-MS, it probes protein conformation and conformational dynamics in solution by monitoring the rate and degree of exchange of hydrogen atoms of the framework amides for deuterium. Huang & Chen (2014) Anal. Bioanalytical Chem. 406:6541; Wei et al. (2014) Drug Disc. Today 19:95. The level of HDX depends on the availability of the hydrogen atoms of the backbone amides and the hydrogen bonds of the proteins to the solvent. The increase in protein mass at HDX can be accurately determined by MS. When coupled with enzymatic digestion, structural characterization at the peptide level can be resolved, allowing surface-exposed peptides to be distinguished from internally folded peptides or from peptides that are isolated at the interface of the protein-protein complex. Typically, deuterium labeling and subsequent quenching experiments followed by enzymatic digestion, peptide separation and MS analysis are carried out.

Prior to epitope mapping experiments, deuterium-free experiments were performed to list common peptides for recombinant human CD40 monomer (10 μM) and CD40 protein complexes with mAbs 12D6, 5G7, 19G3, and 5F11 (molar ratio 1:1). In the HDX-MS experiment, 5 µl of each sample (CD40 or CD40 with mAb 12D6, 5G7, 19G3 and 5F11, respectively) was diluted in 55 µl of D2O buffer (10 mM phosphate buffer, D2O, pH 7.0) to start the labeling reactions. The reactions were carried out for different time periods: 20 s, 1 min, 10 min and 240 min. At the end of each labeling reaction period, the reaction was quenched by adding quench buffer (100 mM phosphate buffer with 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v/v) and 50 µl of the quenched sample was injected into the Waters system. HDX-MS for analysis. Deuterium uptake of total peptic peptides was monitored in the absence/presence of the anti-CD40 mAb. The resulting sequence coverage was 82%.

HDX epitopes for anti-CD40 mAbs 12D6, 5G7, 19G3, and 5F11 are shown in Table 1. 7. Antibody 12D6 protected two peptides, one of which (residues 11-28) included part of the signal sequence and thus with low probability is physiologically significant in itself, but indicates that 12D6 makes contacts in the region 21-28 , which mAbs 5G7 and 19G3 do not.

Table 7 HDX Epitopes

clone CD40 residues (SEQ ID NO: 1) Subsequence 12D6 11-28 WGCLLTAVHPEPPTACRE 12D6 21-35 EPPTACREKQYLINS 5G7 21-35 EPPTACREKQYLINS 19G3 21-35 EPPTACREKQYLINS 5F11 58-66 ECLPCGESE

Example 6

Epitope mapping by yeast display.

Epitopes for selected chimeric or humanized anti-huCD40 antibodies of the present invention are determined by displaying randomly mutated huCD40 extracellular region variants in yeast and sorting the yeast based on their inability to bind to particular antibodies. Selected yeast cells unable to bind are expanded and subjected to additional selection steps based on their inability to bind to particular chimeric or humanized forms of the antibodies of the present invention. See, for example, Chao et al. (2004) J. Mol. Biol. 342:539. For the obtained yeast, sequences for huCD40 variants are determined and analyzed for the effect of each residue on antibody binding. Binding epitopes for antibodies of the present invention are defined as loci in the huCD40 sequence where single amino acid mutations disrupt binding to the anti-huCD40 antibodies of the present invention.

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Briefly, reduced fidelity PCR is used to clone human CD40-encoding DNA in a construct, allowing expression of huCD40 variants as N-terminal portions of fusion proteins additionally containing the c-myc tag sequence and the yeast cell wall protein Aga1p. Such constructs, when expressed in yeast (Saccharomyces cerevisiae), exhibit huCD40 polypeptide variants on the yeast cell surface anchored to the cell surface by the Agalp polypeptide. The c-myc tag can optionally be used as a positive control for displaying the huCD40 fusion proteins on a given yeast cell. Yeast cells are sorted by FACS, and cells that express correctly folded huCD40 fusion proteins (as determined by binding of a control mouse anti-huCD40 antibody detected by allophycocyanin (APC) labeled goat anti-mouse IgG secondary antibodies) but do not bind to the antibodies of the present invention (as determined by detection of phycoerythrin (PE) labeled goat anti-human IgG antibodies as secondary antibodies) are pooled, propagated and used in subsequent selection steps. For the yeast constructs remaining after several selection steps, the huCD40 sequence is determined. To confirm good mutant coverage at each position of the huCD40 sequence, control experiments without selection were performed with anti-huCD40 antibodies and a baseline was provided to normalize the results obtained with the selected libraries.

Example 7

Agonistic activity of antibodies to CD40.

The effect of Fc sequence variation on the agonist activity of selected anti-CD40 antibodies of the present invention was assessed by determining immature dendritic cell (DC) activation. Experiments were performed with anti-CD40 mAb 12D6-24 constructs with sequences of human IgG1f (control), Fc SE, SELF, P238D, V4, V8 and V12. Human monocytes (CD14+) were isolated from healthy normal donors using plastic adhesion or human CD14 microbeads (Miltenyi Biotec). Monocytes were cultured with 100 ng/ml GM-CSF (Miltenyi Biotec) and 100 ng/ml IL-4 (Miltenyi Biotec). On day 2 and day 5, half of the medium was removed and reconstituted. Immature dendritic cells were harvested on days 6-7. DC two donors were incubated with the indicated concentrations of antibodies overnight at 37°C. Cell culture supernatants were harvested and analyzed for human IL-6 production (Cisbio). See fig. 3A and B.

Control human IgG1f antibodies did not induce IL-6 secretion when used at concentrations up to 100 nM, and the P238D variant provided only a weak induction. In contrast, all SE, SELF, V9 and V12 variants significantly increased IL-6 secretion, while V8 and V4 showed an intermediate effect. These results roughly correlate with binding affinity for FcyRIIb and confirm that using the correct Fc sequence variants can result in anti-CD40 antibodies with increased agonist activity.

In addition, differences in agonist activity between antibodies with variable regions 8E8, 5G7, 12D6, 19G3 and 5F11 were evaluated in experiments using chimeric anti-CD40 mAb constructs containing a common human IgG1f V12 constant domain. Activation was determined in vitro immature dendritic cells (isolated as described in the previous paragraph) by seeding the cells in a 96-well plate, adding antibodies as indicated, and incubating overnight at 37°C. The cells were then harvested and stained with a fluorescent anti-CD54 antibody that was detected by fluorescence-activated cell sorting (FACS). See fig. 4.

The control human antibody IgG1f did not cause a significant increase in CD54 levels, while 12D6 caused a significant increase in CD54 levels. Antibodies 19G3 and 8G8 were somewhat less effective than 12D6, and antibodies 5G7 and 5F11 were similar to each other and showed relatively low stimulation. Activation results do not necessarily correlate with CD40 binding affinity, as the 5F11 antibody has almost the highest binding affinity and is a weak agonist, while the 19G3 antibody is the opposite. Regardless of the reasons for the difference, these results confirm that in order to obtain anti-CD40 antibodies with increased agonistic activity, it is important to use the correct sequence of the antigen-binding domain.

Example 8

Elimination of DG isomerization.

Antibodies of the present invention containing V4 and V9 Fc sequence variants exhibit unacceptable levels of DG isomerization at position D270. Such isomerization is undesirable as it results in product heterogeneity and potentially reduces efficiency. To reduce this isomerization, the aspartic acid residue at position 270 of Fc V4 is replaced with glutamic acid (D270E), and position 270 of Fc V9 is replaced with alanine, glutamic acid, glutamine, serine and threonine (D270A, D270E, D270Q, D270S, D270T). Supernatants were obtained from cells expressing these antibodies and analyzed for binding to hCD32/FcyRII. Selected isolated antibodies without mutations at position 270 were used as controls. These experiments were performed on mAb 12D6-24 and 5F11-45 variable domain antibodies. The results are shown in FIG. 5.

For Fc variants V4 and V9, the D270E substitution resulted in a slight decrease in receptor binding. Other V9 Fc variants tested (D270A, D270Q, D270S, D270T) resulted in greater reduction in receptor binding. The results were independent of whether the antibody was 12D6 or 5F11. In all cases, the tested relative binding (by rank) to the three receptors (FcyRIIaH131, FcyRIIa-R131 and FcyRIIb) remained similar with approximately equivalent binding to FcYRIIa-R131 and FcyRIIb and significantly weaker binding to FcyRIIa-H131.

For both variants, V4 and V9, the D270E substitution retained acceptably high affinity for FcyR and retained specificity for FcyRIIb, which is desirable to increase the agonist activity of the anti-CD40 antibodies of the present invention.

Table 8. Brief description of the sequence listing

SEQ GO Description 1 Human CD40 (NP_001241) 2 Human CD40L-gp39 (NP_000065.1) 3 Chimeric heavy chain 12D6 4 Chimeric light chain 12D6 5 Heavy chain 12D6-03 6 Light chain 12D6-03 7 Heavy chain 12D6-22 8 Heavy chain 12D6-22 V9 9 Light chain 12D6-22/12D6-24 10 Heavy chain 12D6-23 AND Light chain 12D6-23 12 Heavy chain 12D6-24 13 Heavy chain 12D6-24 P238D 14 Heavy chain 12D6-24 SE 15 Heavy chain 12D6-24 SELF 16 Heavy chain 12D6-24 V4 17 Heavy chain 12D6-24 V4 D270E 18 Heavy chain 12D6-24 V8 19 Heavy chain 12D6-24 V9 20 Heavy chain 12D6-24 V9 D270E 21 Heavy chain 12D6-24 VII 22 Heavy chain 12D6-24 VI2 23 Chimeric heavy chain 5F11 24 Chimeric light chain 5F11 25 Heavy chain 5F11-17 26 Light chain 5F11-17 27 Heavy chain 5F11-23 28 Light chain 5F11-23 29 Heavy chain 5F11-45

- 58 040302

thirty Light chain 5F11-45 31 Heavy chain 5F11-45 SE 32 Heavy chain 5F11-45 SELF 33 Heavy chain 5F11-45 V4 34 Heavy chain 5F11-45 D270E V4 35 Heavy chain 5F11-45 V8 36 Heavy chain 5F11-45 V9 37 Heavy chain 5F11-45 V9 D270E 38 Heavy chain 5F11-45 VII 39 Heavy chain 5F11-45 VI2 40 Chimeric heavy chain 8E8 41 Chimeric light chain 8E8 42 Heavy chain 8E8-56 43 Light chain 8E8-56 44 Heavy chain 8E8-62 45 Light chain 8E8-62 46 Heavy chain 8E8-67 47 Light chain 8E8-67 48 Heavy chain 8E8-70 49 Light chain 8E8-70 50 Heavy chain 8E8-71 51 Light chain 8E8-71 52 Chimeric heavy chain 5G7 53 Chimeric light chain 5G7 54 Heavy chain 5G7-22 55 Light chain 5G7-22 56 Heavy chain 5G7-25 57 Light chain 5G7-25 58 Chimeric heavy chain 19G3 59 Chimeric light chain 19G3 60 Heavy chain 19G3-11 61 V9 19G3-11

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Claims (9)

62 Light chain 19G3-11 63 Heavy chain 19G3-22 64 Light chain 19G3-22 65 Human IgGlf constant domain 66 SE constant domain 67 SELF constant domain 68 P23 constant domain 8D 69 Constant Domain V4 70 Constant domain V4 D270E 71 V7 constant domain 72 V8 constant domain 73 V9 constant domain 74 Constant domain V9 D270E 75 Constant Domain VII 76 V12 constant domain 77 Kappa light chain constant domain 78 Signal sequence The sequence listing provides mature heavy and light chain sequences (ie, the sequences do not contain signal peptides). The signal sequence for producing antibodies of the present invention, for example in human cells, is shown in SEQ ID NO: 78. Equivalents Those skilled in the art will recognize, or use no more than routine experimentation, to determine many equivalents to the specific embodiments described herein. Such equivalents are intended to be included by the following claims. CLAIM 1. An isolated antibody or antigen-binding portion thereof that specifically binds to human CD40 containing a heavy chain and a light chain, wherein a) the heavy chain contains the sequences CDRH1, CDRH2 and CDRH3 containing residues 3135, 50-66 and 99-106 respectively of SEQ ID NO: 29; And b) the light chain contains the sequences CDRL1, CDRL2 and CDRL3 containing residues 24-38, 54-60 and 93-101 respectively of SEQ ID NO: 30. 2. The isolated antibody or antigen-binding portion thereof according to claim 1, containing the sequences of the heavy and light chain variable domains containing residues 1-117 and 1-111 of SEQ ID NO:29 and SEQ ID NO: 30, respectively. 3. Nucleic acid encoding the variable region of the heavy chain of the antibody or antigen-binding portion of claim 2. 4. Nucleic acid encoding the variable region of the light chain of the antibody or antigen-binding portion of claim 2. 5. A nucleic acid encoding the variable region of both the heavy chain and the light chain of an antibody or antigen-binding portion thereof according to claim 2. 6. An expression vector containing a nucleic acid molecule according to any one of claims 3-5. 7. Host cell transformed with the expression vector of claim 6. 8. A method for producing an antibody to human CD40 or its antigen-binding part, comprising: a) expression of the antibody or antigen-binding portion thereof in a cell transformed with an expression vector containing nucleic acids encoding the heavy and light chain variable regions of the antibody or antigen-binding portion thereof, according to any one of claims 1 to 6; And b) isolating the antibody or its antigen-binding portion from the cell. 9. Pharmaceutical composition containing: a) the antibody or antigen-binding portion of claim 1; b) carrier. -