阅读说明：本技术 抗cd40抗体及其用途 (anti-CD 40 antibodies and uses thereof ) 是由 B·C·巴恩哈特 B·德沃瓦 A·P·杨纽克 S·L·奥卡达 B·L·斯蒂芬斯 J·W·韦 于 2018-12-27 设计创作，主要内容包括：本文提供了激动性抗体或其抗原结合部分,其结合人CD40并包含赋予提高的产率和减少的聚集的改进的重链和轻链可变区。本发明还提供了通过向有此需要的受试者施用本发明的抗体来治疗癌症或慢性感染的方法。(Provided herein are agonistic antibodies, or antigen-binding portions thereof, that bind human CD40 and comprise improved heavy and light chain variable regions conferring increased yield and reduced aggregation. The invention also provides methods of treating cancer or chronic infection by administering the antibodies of the invention to a subject in need thereof.)

1. An isolated antibody, or antigen-binding portion thereof, that specifically binds to human CD40, comprising:

a) a heavy chain variable region having at least 95% sequence identity to a heavy chain variable region selected from the group consisting of residues 1-119 of SEQ ID NOs 5,7, 9,11, and 52-54; and

b) a light chain variable region having at least 95% sequence identity to the light chain variable region of SEQ ID NO. 49.

2. The isolated antibody or antigen binding portion thereof of claim 1, comprising:

a) a heavy chain variable region sequence selected from residues 1-119 of SEQ ID NOs 5,7, 9,11, and 52-54; and

b) the light chain variable region sequence of SEQ ID NO. 49.

3. The isolated antibody or antigen binding portion thereof of claim 2, comprising:

a) 11, residues 1-119 of SEQ ID NO; and

b) the light chain variable region sequence of SEQ ID NO. 49.

4. The isolated antibody or antigen binding portion thereof of claim 2, comprising:

a) 54, the heavy chain variable region sequence of SEQ ID NO; and

b) the light chain variable region sequence of SEQ ID NO. 49.

5. An isolated antibody, or antigen-binding portion thereof, that specifically binds to human CD40, comprising:

a) a heavy chain variable region having at least 95% sequence identity to the heavy chain variable region of SEQ ID NO: 54; and

b) a light chain variable region having at least 95% sequence identity to a heavy chain variable region selected from the group consisting of residues 1-112 of SEQ ID NOs 6,8 and 10.

6. The isolated antibody or antigen-binding portion thereof of claim 5, comprising:

a) 54, the heavy chain variable region sequence of SEQ ID NO; and

b) a light chain variable region sequence selected from residues 1-112 of SEQ ID NOS 6,8 and 10.

7. A nucleic acid encoding the heavy chain variable region and/or the light chain variable region of the antibody or antigen-binding portion thereof of any one of claims 1-6.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A cell transformed with the expression vector of claim 8.

10. A method of making an anti-human CD40 antibody or antigen-binding portion thereof, comprising:

a) expressing the antibody or antigen-binding portion thereof in the cell of claim 9; and

b) isolating the antibody or antigen-binding portion thereof from the cell.

11. A pharmaceutical composition comprising:

a) the antibody or antigen binding portion thereof of any one of claims 1-6; and

b) and (3) a carrier.

12. A method of stimulating an immune response in a subject comprising administering to the subject the pharmaceutical composition of claim 11.

13. The method of claim 12, wherein the subject has a tumor and an immune response against the tumor is stimulated.

14. The method of claim 12, wherein the subject has a chronic viral infection and an immune response against the viral infection is stimulated.

15. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 11.

16. The method of claim 15, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer.

17. A method of treating a chronic viral infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 11.

Background

Recent studies have shown that human cancers and chronic infections can be treated with drugs that modulate the immune response of patients to malignant or infected cells. See, e.g., Reck & Paz-Ares (2015) semini. 402. Agonistic anti-CD 40 antibodies such as CP 870893 and dacetuzumab (SGN-40) have been tried to treat cancer based on their ability to enhance this immune response. See, e.g., Kirkwood et al (2012) CA cancer j. clin.62: 309; vanderheide & Glennie (2013) clin. cancer res.19: 1035. recent experiments in mice have shown that anti-CD 40 antibodies with enhanced specificity for the inhibitory Fc receptor fcyriib have enhanced anti-tumor efficacy. See, for example, WO 2012/087928; li & ravatch (2011) Science 333: 1030; li & ravatch (2012) proc.nat' l acad.sci USA 109: 10966, respectively; wilson et al (2011) Cancer Cell 19: 101, a first electrode and a second electrode; white et al (2011) j.immunol.187: 1754.

there is a need for improved agonistic anti-human CD40 antibodies for use in the treatment of cancer and chronic infections in human subjects. Such antibodies will preferably be produced in high yield and low aggregation.

Disclosure of Invention

The present invention provides improved humanized heavy and light chain variable domains of antibody 12D6 that exhibit improved yields while retaining substantial affinity for human CD 40. In particular, the invention provides agonistic anti-huCD 40 antibodies comprising a modified light chain variable region L2 through L6(SEQ ID NOS: 47-51), such as L4(SEQ ID NO: 49) and/or a modified heavy chain variable region H2 through H4(SEQ ID NOS: 52-54), such as heavy chain H4(SEQ ID NO:54, respectively). In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region pair selected from (i) SEQ ID NOs: 5. 7,9, 11, 52, 53 and 54 and residues 1-119 of SEQ ID NO: 49; (ii) SEQ ID NO:54 and SEQ ID NO: 6. residues 1-112 of 8 or 10. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region pair, respectively, selected from (i) seq id NOs: 11 and residues 1-119 of SEQ ID NO: 49; and (ii) SEQ ID NO:54 and SEQ ID NO: 49. in some embodiments, the invention includes an antibody comprising the heavy chain variable region H2(SEQ ID NO: 52) and the light chain variable region L4(SEQ ID NO: 49). Any of these antibodies may optionally further comprise a heavy chain constant region comprising IgG1f (SEQ ID NO: 44) and/or the amino acid sequence of SEQ ID NO: 45 light chain kappa constant region. Alternatively, any of these antibodies may optionally further comprise a heavy chain constant region comprising one or more amino acid substitutions to enhance agonist activity.

In further embodiments, the anti-huCD 40 antibodies of the invention comprise heavy and light chain variable regions having at least 80%, 85%, 90%, and 95% sequence identity to the heavy and light chain variable regions of any one of the antibodies listed in the preceding paragraph. In further embodiments, the anti-huCD 40 antibody comprises a heavy chain variable region and a light chain variable region consisting essentially of the sequences of the heavy chain variable region and the light chain variable region of any one of the antibodies disclosed herein.

The invention further provides nucleic acids encoding the heavy chain variable region and/or the light chain variable region of the first two paragraphs or antigen binding fragments thereof, expression vectors comprising the nucleic acid molecules, cells transformed with the expression vectors, and methods of producing antibodies by expressing antibodies from cells transformed with the expression vectors and recovering the antibodies, as well as pharmaceutical compositions comprising the anti-huCD 40 antibodies of the invention or antigen binding fragments thereof and a vector.

The invention provides a method of enhancing an immune response in a subject comprising administering to the subject an effective amount of an anti-huCD 40 antibody or antigen-binding fragment thereof of the invention, such that the immune response in the subject is enhanced. In certain embodiments, the subject has a tumor and the immune response against the tumor is enhanced. In another embodiment, the subject has a viral infection, e.g., a chronic viral infection, and the antiviral immune response is enhanced.

The invention also provides a method of inhibiting tumor growth in a subject, comprising administering to the subject an anti-huCD 40 antibody, or antigen-binding fragment thereof, of the invention, thereby inhibiting tumor growth.

The invention further provides a method of treating cancer, e.g., by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-huCD 40 antibody or antigen-binding fragment thereof of the invention, e.g., as a pharmaceutical composition, thereby treating 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, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphomas, leukemias, myelomas, sarcomas, and virus-related cancers. In certain embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

The invention also provides a method of treating a chronic viral infection, e.g., by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-huCD 40 antibody or antigen-binding fragment thereof of the invention, e.g., as a pharmaceutical composition, thereby treating the chronic viral infection.

In certain embodiments, the methods of modulating immune function and methods of treatment described herein comprise administering the anti-huCD 40 antibodies of the invention in combination with or as a bispecific agent with one or more additional therapeutic agents, e.g., an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-LAG 3 antibody, an anti-GITR antibody, an anti-OX 40 antibody, an anti-CD 73 antibody, an anti-TIGIT antibody, an anti-CD 137 antibody, an anti-CD 27 antibody, an anti-CSF-1R antibody, an anti-CTLA-4 antibody, a TLR agonist, or a small molecule antagonist of IDO or TGF β. In particular embodiments, anti-huCD 40 therapy is combined with anti-PD 1 and/or anti-PD-L1 therapy, 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.

In some embodiments, the anti-huCD 40 antibodies of the invention comprise one or more heavy chains and one or more light chains, e.g., two heavy chains and two light chains.

Brief Description of Drawings

FIG. 1 shows TNF- α expression of immature human dendritic cells titrated with various agonist anti-huCD 40 antibodies. Antibody 12D6-24 (labeled x) is an unmodified anti-CD 40 antibody (heavy and light chain sequences: SEQ ID NOS: 11 and 8, respectively). Antibodies 12D6-24-L4H1-6P1 (open squares) and 12D6-24-L4H1-6P2 (filled squares) have a modified light chain variable domain consisting of the L4 sequence (SEQ ID NO: 49) and an unmodified heavy chain variable domain (i.e., SEQ ID NO: 11). 6P1 and 6P2 refer to dendritic cells from two separate human donors. Antibody 12D6-24-L4H4 (filled circles) had a modified light chain variable domain consisting of the L4 sequence (SEQ ID NO: 49) and a modified heavy chain variable domain consisting of the H4 sequence (SEQ ID NO: 54). See example 3.

Fig. 2A and 2B show the activation of human dendritic cells as measured by CD83 and CD86 expression, respectively, when treated with various antibodies of the invention. Dendritic cells were exposed to the indicated antibodies, stained with fluorescent anti-CD 83 and anti-CD 86 antibodies, and analyzed by Fluorescence Activated Cell Sorting (FACS) as described in example 3. The signal is expressed as Mean Fluorescence Intensity (MFI). Antibody 12D6 is antibody 12D6-24 as described in FIG. 1, and antibodies L4H1 and L4H4 are antibody 12D6-24-L4H1 and antibody 12D6-24-L4H4 as described in FIG. 1, respectively. Antibody 5F11 is another agonist anti-CD 40 antibody disclosed herein, while the control is an unrelated IgG 1. The left-most black bar of each antibody is an unstained sample. The middle light gray bar is cells stained with anti-CD 83 antibody, while the right-most dark gray bar is cells stained with anti-CD 86 antibody. Fig. 2A provides data obtained at the excitation wavelength of the anti-CD 83 antibody (i.e., showing staining primarily of CD 83), while fig. 2B provides data obtained at the excitation wavelength of the anti-CD 86 antibody (i.e., showing staining primarily of CD 86).

Detailed Description

The present invention provides isolated antibodies, particularly humanized monoclonal antibodies, that specifically bind human CD40 ("huCD 40") and have agonist activity, and in particular provides improved heavy and light chain variable region sequences that improve yield and still retain substantial affinity for huCD 40.

Further provided herein are methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated to contain the antibodies or fragments. Also provided herein are methods of using the antibodies for enhancing immune responses, alone or in combination with other immunostimulants (e.g., antibodies) and/or cancer or anti-infection therapy. Thus, the anti-huCD 40 antibodies described herein can be used in therapy in a variety of therapeutic applications, including, for example, inhibiting tumor growth and treating chronic viral infections.

Definition of

In order to make the present specification more comprehensible, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "CD 40" refers to "TNF receptor superfamily member 5" (TNFRSF 5). Unless otherwise indicated or clear from context, reference herein to CD40 refers to human CD40 ("huCD 40") and "anti-CD 40 antibody" refers to anti-human CD40 antibody. Human CD40 is further described as GENE ID NO: 958 and MIM (mendelian inheritance in humans): 109535. the sequence of human CD40 (GenBank accession No. NP _001241.1) comprising a 20 amino acid signal sequence is as set forth in SEQ ID NO:1 is provided.

CD40 interacts with CD40 ligand (CD40L), also known as TNFSF5, gp39 and CD 154. Unless otherwise stated or clear from context, reference herein to "CD 40L" refers to human CD40L ("huCD 40L"). Human CD40L is further described as GENE ID NO: 959 and MIM: 300386. the sequence of human CD40L (GenBank accession No. NP _000065.1) is as set forth in SEQ ID NO: 2 is provided.

The term "antibody" as used herein may include whole antibodies and any antigen-binding fragment thereof (i.e., "antigen-binding portion") or single chains thereof, unless otherwise indicated or clear from the context. In one embodiment, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains, CH1, CH2 and CH 3. In some naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated herein as V)_L) And a light chain constant region. The light chain constant region consists of one domain CL. V_HAnd V_LThe regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each V_HAnd V_LConsists of three CDRs and four Framework Regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

Antibodies typically specifically bind their cognate antigen with high affinity, which is reflected by 10^-7To 10^-11Dissociation constant (K) of M or less_D). Generally any is considered to be greater than about 10^-6K of M_DIndicating non-specific binding. As used herein, "specific binding to an antigen"antibody" refers to an antibody that binds an antigen and substantially the same antigen with high affinity, meaning having 10^-7M or less, preferably 10^-8M or less, even more preferably 5 × 10^-9M or less, most preferably 10^-8M to 10^-10K of M or less_DBut does not bind unrelated antigens with high affinity. 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 the sequence of the given antigen. For example, an antibody that specifically binds human CD40 may also cross-react with CD40 from certain non-human primate species (e.g., cynomolgus monkeys), but may not cross-react with CD40 from other species or antigens other than CD 40.

Unless otherwise specified, the immunoglobulin may be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: human IgG1, IgG2, IgG3 and IgG4, and mouse IgG1, IgG2a, IgG2b and IgG 3. Immunoglobulins, such as human IgG1, exist as several allotypes that differ from each other by a maximum of a few amino acids. Unless otherwise indicated, the antibodies of the invention comprise an IgG1f constant domain (SEQ ID NO: 44). Unless otherwise indicated, "antibody" may include, for example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthesizing an antibody; and single chain antibodies.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., human CD 40). Examples of binding fragments encompassed by the term "antigen binding portion/fragment" of an antibody include: (i) fab fragment-from V_L、V_HA CL and CH1 domain; (ii) f (ab')₂Fragment-a bivalent fragment comprising two Fab fragments linked by disulfide bonds of the hinge region; (iii) from V_HAnd a CH1 domain; (iv) v from one arm of an antibody_LAnd V_H(iv) an Fv fragment consisting of domain, and (V) a V_HdAb fragments consisting of domains (Ward et al (1989) Nature 341: 544-546). An isolated Complementarity Determining Region (CDR), or a combination of two or more isolated CDRs linked by a synthetic linker, can comprise the antigen binding domain of an antibody if capable of binding an antigen.

Single chain antibody constructs are also encompassed by the invention. Although two domains of the Fv fragment V_LAnd V_HEncoded by different genes, but can be joined by synthetic linkers using recombinant methods, making them a single protein chain, where V_LAnd V_HThe region pairs form monovalent molecules known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423 Asonic 426 and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion/fragment" of an antibody. These and other potential configurations are found in Chan&Carter (2010) nat. rev. immunol.10: 301. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for utility in the same manner as are intact antibodies. Antigen binding portions/fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

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

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs, which produces two antigen-binding sites with specificity for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990) clin. exp. immunol.79: 315- > 321; kostelny et al (1992) J.Immunol.148, 1547-1553.

As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be propagated without intentionally introducing any sequence changes. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and optionally constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma, e.g., a hybridoma obtained by fusing a B cell obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain transgene) to an immortalized cell.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or from a hybridoma prepared from the animal, (b) antibodies isolated from a host cell transformed to express the antibody, such as an antibody isolated from a transfectoma, (c) antibodies isolated from a library of recombinant combinatorial human antibodies, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize particular human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations, such as those that occur during antibody maturation. The variable region comprises an antigen-binding domain encoded by a variety of genes that are rearranged to form antibodies specific for foreign antigens, as is known in the art (see, e.g., Lonberg (2005) Nature Biotech.23 (9): 1117-1125). In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (known as somatic mutations or hypermutations) to increase the affinity of the antibody for the foreign antigen. The constant region will change in another response to the antigen (i.e., isotype switching). Thus, the nucleic acid sequences encoding the rearrangements and somatic mutations of the light and heavy chain immunoglobulin polypeptides that respond to an antigen may be different from the original germline sequences, but may be substantially identical or similar (i.e., at least 80% identical).

"human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are synonymous.

"humanized" antibodies refer to antibodies in which some, most, or all of the amino acids outside of the CDR domains of a non-human antibody (e.g., a mouse antibody) are substituted with corresponding amino acids derived from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside of the CDR domains have been substituted with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions have not been altered. Small additions, deletions, insertions, substitutions or modifications of amino acids may be permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody.

"chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, for example, an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody. "hybrid" antibody refers to an antibody having different types of heavy and light chains, such as a mouse (parental) heavy chain and a humanized light chain, and vice versa.

As used herein, "isotype" refers to the class of antibodies encoded by heavy chain constant region genes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

"allotype" refers to naturally occurring variants within a particular isotype panel that differ in one or more amino acids. See, e.g., Jefferis et al (2009) mAbs 1: 1.

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

As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD40 is substantially free of antibodies that specifically bind antigens other than CD 40). However, an isolated antibody that specifically binds to an epitope of CD40 may have cross-reactivity with other CD40 proteins from other species.

"effector functions" derived from the interaction of an antibody Fc region with certain Fc receptors include, but are not necessarily limited to, Clq binding, Complement Dependent Cytotoxicity (CDC), Fc receptor binding, Fc γ R-mediated effector functions such as ADCC and antibody dependent cell mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptors; BCR). Such effector functions typically require binding of the Fc region to an antigen binding domain (e.g., an antibody variable domain).

An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcR binding to IgG antibodies comprise receptors of the Fc γ R family, including allelic variants and alternatively spliced forms of these receptors. The Fc γ R family consists of three activating receptors (Fc γ RI, Fc γ RIII and Fc γ RIV in mice; Fc γ RIA, Fc γ RIIA and Fc γ RIIIA in humans) and one inhibiting receptor (Fc γ RIIb or equivalent Fc γ RIIb). Table 1 summarizes various properties of human Fc γ R. Most innate effector cell types co-express one or more activating Fc γ rs and inhibitory Fc γ RIIb, while Natural Killer (NK) cells selectively express one activating Fc receptor (Fc γ RIII in mice and Fc γ RIIIA in humans) but not inhibitory Fc γ RIIb in mice and humans. Human IgG1 binds to most human Fc receptors, and human IgG1 is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor that it binds.

TABLE 1

Characterization of human Fc γ R

"Fc region" (fragment crystallizable 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 to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (C1 q). Thus, the Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises CH in each of the two heavy chains of an antibody₂And CH₃A constant domain; the IgM and IgE Fc regions comprise three heavy chain constant domains (C) per polypeptide chain_HDomains 2-4). For IgG, the Fc region comprises the immunoglobulin domains C γ 2 and C γ 3 and the hinge between C γ 1 and C γ 2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position C226 or P230 of the heavy chain (or the amino acid between these two amino acids) to the carboxy terminus, with numbering according to the EU index as in Kabat. Kabat et al (1991) Sequences of proteins of Immunological Interest, National Institutes of Health, Bethesda, Md; see also fig. 3c-3f of U.S. patent application publication No. 2008/0248028. C of human IgG Fc region_H2The domain extends from about amino acid 231 to about amino acid 340, and C_H3Domain located in the Fc region C_H2The C-terminal side of the domain, i.e., it extends from about amino acid 341 to about amino acid 447 (including the C-terminal lysine) of the IgG. As used herein, an Fc region can be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to the isolation of a protein polypeptide comprising Fc, such as a "binding protein comprising an Fc region," also known as an "Fc fusion protein" (e.g., an antibody or immunoadhesin)The area in the object or in its background.

A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc regions and naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc. See, e.g., Jefferis et al (2009) mAbs 1: 1.

as used herein, the terms "specifically binds," "selectively binds," and "specifically binds" refer to an antibody that binds an epitope on a predetermined antigen and does not bind other antigens. In general, the equilibrium dissociation constant (K) for antibody (i) binding_D) Is less than about 10^-7M, e.g. less than about 10^-8M、10^-9M or 10^-10M or even lower, as determined by, for example, using a predetermined antigen (e.g., recombinant human CD40) as the analyte and an antibody as the ligand

Surface Plasmon Resonance (SPR) technique in 2000 surface plasmon resonance instrument, or Scatchard analysis of antibody binding to antigen positive cells; and (ii) binds to the predetermined antigen with at least two-fold greater affinity than it binds to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Thus, an "antibody that specifically binds human CD 40" is referred to as 10^-7M or less, e.g. less than about 10^-8M、10^-9M or 10^-10M or even lower K_DAn antibody that binds soluble or cell-bound human CD 40. An antibody that "cross-reacts with cynomolgus monkey CD 40" is 10^-7M or less, e.g. less than about 10^-8M、10^-9M or 10^-10M or even lower K_DAn antibody that binds cynomolgus monkey CD 40.

The term "k" as used herein_assoc"or" k_a"refers to the association rate constant for a particular antibody-antigen interaction, and the presentThe term "k" as used herein_dis"or" k_d"refers to the dissociation rate constant for the interaction of a particular antibody with an antigen. As used herein, the term "K_D"refers to the equilibrium dissociation constant, which is defined by k_dAnd k is_aRatio of (i.e. k)_d/k_a) Obtained and expressed as molarity (M). K of antibody_DThe values may be determined using methods well known in the art. Determination of K of antibodies_DPreferred methods of (3) are bio-layer interferometry (BLI) analysis, preferably using a ForteBio Octet RED device, surface plasmon resonance, preferably using a biosensor system (e.g.using a ForteBio Octet RED device)

Surface plasmon resonance system), or flow cytometry and Scatchard analysis.

In the context of in vitro or in vivo assays using antibodies or antigen-binding fragments thereof, the term "EC 50" refers to the concentration of antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal response (i.e., half-way between the maximal response and baseline).

The term "binds to immobilized CD 40" refers to the ability of an antibody described herein to bind to CD40 (e.g., bind to CD40 expressed on the surface of a cell or attached to a solid support).

As used herein, the term "cross-reactive" refers to the ability of an antibody described herein to bind CD40 from a different species. For example, an antibody described herein that binds human CD40 can also bind CD40 from another species (e.g., cynomolgus monkey CD 40). As used herein, cross-reactivity can be measured by: specific reactivity with purified antigens or binding to or otherwise functionally interacting with cells physiologically expressing CD40 is detected in binding assays (e.g., SPR, ELISA). Methods for determining cross-reactivity include standard binding assays as described herein, e.g., using2000SPR instruments (Biacore AB, Uppsala, Sweden)Surface Plasmon Resonance (SPR) analysis or flow cytometry.

As used herein, the term "naturally occurring" applies to an object that refers to the fact that the object may be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that is isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

"polypeptide" refers to a chain comprising at least two amino acid residues joined in series, the length of the chain having no upper limit. One or more amino acid residues in a protein may comprise modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may comprise 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-stranded or double-stranded, and may be a cDNA.

Also provided are "conservative sequence modifications" of the antibody sequences provided herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of an antibody encoded by or containing the nucleotide sequence to an antigen. For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, wherein an amino acid residue is substituted with an amino acid residue having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a predicted nonessential amino acid residue in an anti-CD 40 antibody with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not abrogate antigen binding are well known in the art. See, e.g., Brummell et al, biochem.32: 1180-; kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al proc.natl.acad.sci.usa 94: 412-417(1997).

Alternatively, in another embodiment, mutations may be introduced randomly along all or a portion of the anti-CD 40 antibody coding sequence, for example by saturation mutagenesis, and the resulting modified anti-CD 40 antibody may be screened to increase binding activity.

The term "substantial homology," with respect to nucleic acids, means that two nucleic acids, or designated sequences thereof, are identical in 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 the nucleotides) and have appropriate nucleotide insertions or deletions when optimally aligned and compared. Alternatively, substantial homology exists when the segments will hybridize to the complementary sequence of the strand under selective hybridization conditions.

The term "substantial homology" with respect to polypeptides means that two polypeptides or designated sequences thereof are identical in at least about 80% of the amino acids (typically at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids) and have appropriate amino acid insertions or deletions when optimally aligned and compared.

The percent identity between two sequences is a function of the number of gaps that need to be introduced in order to perform an optimal alignment of the two sequences, the length of each gap, and the number of identical positions that the sequences share when optimally aligned (i.e.,% homology-number of identical positions/total number of positions x 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the nwsgapdna. cmp matrix and GAP weights of 40, 50, 60, 70 or 80 and length weights of 1, 2,3, 4,5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of e.meyers and w.miller (cabaos, 4: 11-17(1989)), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.mol.biol. (48): 444-453(1970)) algorithm, which has been incorporated into the GAP program in the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights of 16, 14, 12, 10, 8,6, or 4, and length weights of 1, 2,3, 4,5, or 6.

The nucleic acid and protein sequences described herein can further be used as "query sequences" to search public databases to, for example, identify related sequences. Altschul et al (1990) j.mol.biol.215: the NBLAST and XBLAST programs (version 2.0) of 403-10 perform such searches. A BLAST nucleotide search can be performed using the NBLAST program (score 100, word length 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program (score 50, word length 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain a gap alignment for comparison purposes, one can use, for example, Altschul et al (1997) Nucleic acids sRes.25 (17): 3389 by using Gapped BLAST as described in 3402. When BLAST and Gapped BLAST programs are used, the default parameters for the respective programs (e.g., XBLAST and NBLAST) can be used.

The nucleic acid may be present in the intact cell, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of the chromosome) or proteins, are "isolated" or "rendered substantially pure" by standard techniques, including alkaline/SDS treatment, CsCl bands, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See Ausubel et al, eds., Current protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, are also included.

As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that comprises a nucleic acid not naturally occurring in that cell, and may be a cell into which a recombinant expression vector has been introduced. It is understood that such terms are not intended to refer to the particular subject cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

An "immune response" refers to a biological response in a vertebrate against a foreign factor that protects an organism from such foreign factorFactors and diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., 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 the liver, including antibodies, cytokines, and complements, which results in the selective targeting, binding, damaging, destroying, and/or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells from the vertebrate body, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. Immune responses include, for example, activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4⁺Or CD8⁺T cells, or inhibiting or depleting T_regA cell. "T Effect" ("T)_eff") cells refer to T cells having cytolytic activity (e.g., CD 4)⁺And CD8⁺T cells) and T helper (Th) cells that secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (T cells)_regA cell).

As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD 8)⁺Cells) and helper T cells (e.g., CD 4)⁺A cell). T cell mediated responses include, for example, cytotoxicity and proliferation of T cells.

As used herein, the term "Cytotoxic T Lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses were mainly characterized by CD8⁺T cell mediation.

An "immunomodulator" or "immunomodulator" refers to an agent, for example, that may be involved in modulating, regulating or modifying a component of a signaling pathway of an immune response. By "modulating", "regulating" or "modifying" an immune response is meant any alteration of a cell of the immune system or of the activity of such a cell (e.g., an effector T cell). Such modulation includes stimulation or inhibition of the immune system, which is manifested as an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In a preferred embodiment, the immunomodulator is located on the surface of a T cell. An "immunomodulatory target" or "immunomodulatory target" is an immunomodulatory agent targeted for binding, the activity of which is altered by the binding of a substance, agent, moiety, 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 a subject having a disease, or at risk of developing a disease or suffering from a relapse of a disease, by methods that include inducing, enhancing, suppressing or otherwise modifying an immune response.

"immunostimulatory therapy" or "immunostimulatory therapy" refers to a therapy that results in an increase (induction, enhancement, or stimulation, all of which are used interchangeably) of an immune response in a subject, e.g., for the treatment of cancer.

By "enhancing an endogenous immune response" is meant increasing the effectiveness or potency of an existing immune response in a subject. For example, an increase in effectiveness and potency may be achieved by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

As used herein, the term "linked" refers to the association of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetic (i.e., a recombinant fusion). Such linkage can be accomplished using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

As used herein, "administering" refers to introducing a composition comprising a therapeutic agent into the body using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

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

As used herein, "cancer" refers to a broad class of diseases characterized by uncontrolled growth of abnormal cells in vivo. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may migrate to distal parts of the body through the lymphatic system or the bloodstream.

As used herein, the terms "treatment" and "treatment" refer to any type of intervention or process performed on a subject or administration of an active agent to a subject with the goal of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease. Prevention is directed to administration to a subject not suffering from a disease to prevent the onset of the disease or to minimize the effects of the disease, if any.

The term "effective dose" or "effective dose" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. By "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is meant any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by: reduced severity of disease symptoms, increased frequency and duration of disease-free symptomatic periods, or prevention of injury or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that inhibits the development or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from disease recurrence. The ability of a therapeutic or prophylactic agent to promote regression of a disease or inhibit the development or recurrence of a disease can be assessed using a variety of methods known to those skilled in the art, for example in human subjects during clinical trials, in animal model systems where efficacy in humans can be predicted, or by assaying the activity of the agent in an in vitro assay.

For example, an anti-cancer agent is a drug that slows cancer progression or promotes cancer regression in a subject. In a preferred embodiment, the therapeutically effective amount of the drug promotes regression of the cancer to the extent that the cancer is eliminated. By "promoting cancer regression" is meant that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease-symptom-free periods, prevention of injury or disability due to disease affliction, or otherwise ameliorates the disease symptoms in a patient. Pharmacological efficacy refers to the ability of a drug to promote regression of a patient's cancer. Physiological safety refers to an acceptably low level of toxicity or other adverse physiological effects (side effects) at the cellular, organ and/or organism level as a result of administration of the drug.

For example, for treating a tumor, a therapeutically effective amount or dose of the drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, still more preferably by at least about 80%, relative to an untreated subject. In a most preferred embodiment, the therapeutically effective amount or dose of the drug completely inhibits cell growth or tumor growth, i.e., 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, and may occur only after a period of time or after repeated administrations. Alternatively, such properties of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to the skilled artisan. In other preferred embodiments described herein, tumor regression may be observed and may 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, "combination" therapy is intended to encompass administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, simultaneous administration, unless the context clearly indicates otherwise. In particular, combination therapy encompasses co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that administration of one therapeutic agent is somehow conditioned to administration of another therapeutic agent. For example, a therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt et al (2011) Blood 117: 2423.

the terms "patient" and "subject" refer to any person who receives prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat a subject having cancer.

Various aspects described herein are described in further detail in the following subsections.

I. anti-CD 40 antibodies

The present application discloses agonistic anti-huCD 40 antibodies with desirable properties for use as therapeutic agents for the treatment of diseases such as cancer and chronic viral infections. These characteristics include one or more of the following: the ability to bind human CD40 with high affinity, acceptably low immunogenicity in human subjects, and acceptably high levels of antibody production and low aggregation when expressed in mammalian cells (e.g., CHO). The anti-CD 40 antibodies of the invention may be referred to as improved antibodies, in which case the improvement may be measured relative to the original unmodified form of the antibody, e.g., mAb12D6-24 (comprising SEQ ID NOs 11 and 8 or variable domains thereof). Improvement can be measured by any property including yield and percentage of monomeric antibody, or by the absence of multimers and other high molecular weight species.

anti-CD 40 antibody sequence variants

Certain variability in the antibody sequences disclosed herein can be tolerated and still retain the desired properties of the antibody. CDR regions were delineated 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, NIHPublication No. 91-3242). Accordingly, the invention provides anti-huCD 40 antibodies comprising a heavy and/or light chain variable domain sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy and/or light chain variable domain sequence of an antibody disclosed herein (e.g., a 12D6 antibody comprising the heavy and light chain variable domains of SEQ ID NOS: 54 and 49, SEQ ID NOS: 11 and 49, or SEQ ID NOS: 52 and 49).

Engineered and modified antibodies

Targeted antigen binding

In various embodiments, the antibodies of the invention are modified to selectively block antigen binding in tissues and environments in which antigen binding is detrimental, but to allow antigen binding where beneficial. In one embodiment, a blocking peptide "mask" is generated that specifically binds to and interferes with antigen binding to the antigen binding surface of the antibody, the mask being attached to each binding arm of the antibody by a peptidase-cleavable linker. See, for example, U.S. patent No. 8,518,404 to CytomX. Such constructs are useful for treating cancers in which protease levels are greatly increased in the tumor microenvironment as compared to non-tumor tissue. Selective cleavage of the cleavable linker in the tumor microenvironment causes dissociation of the masking/blocking peptide, thereby allowing selective antigen binding in the tumor, rather than in peripheral tissues where antigen binding may cause unwanted side effects.

Alternatively, in related embodiments, a bivalent binding compound ("masking ligand") comprising two antigen binding domains is developed that binds to two antigen binding surfaces of a (bivalent) antibody and interferes with antigen binding, wherein the two binding domain masks are linked to each other (but not to the antibody) by a cleavable linker (e.g. cleavable by a peptidase). See, for example, international patent application publication No. WO2010/077643 to Tegopharm corporation. The masking ligand may comprise or be derived from an antigen which is intended to bind to the antibody or which may be generated separately. Such masked ligands are useful for treating cancer, where protease levels are greatly increased in the tumor microenvironment compared to non-tumor tissue. Selective cleavage of the cleavable linker in the tumor microenvironment causes the two binding domains to dissociate from each other, thereby reducing the avidity of the antigen-binding surface of the antibody. Dissociation of the resulting masking ligand from the antibody allows the antigen to bind selectively in the tumor, rather than in peripheral tissues where antigen binding may cause unwanted side effects.

Fc and modified Fc

The antibody of the invention may comprise a variable domain of the invention in combination with constant domains comprising different Fc regions, the constant domains being selected based on the biological activity, if any, of the antibody for its intended use. Salfeld (2007) nat. biotechnol.25: 1369. for example, human IgG can be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, wherein each of these subclasses comprises a uniquely contoured Fc region with binding to one or more of the Fc γ receptors (activating receptor Fc γ RI (CD64), Fc γ RIIA, Fc γ RIIC (CD32a, C); Fc γ RIIIA and Fc γ RIIIB (CD16a, b) and the inhibitory receptor Fc γ RIIB (CD32b) and binding to the first component of complement (C1 q); human IgG1 and IgG3 bind to all Fc γ receptors; IgG2 binds to Fc γ RIIAH₁₃₁Bind and are specific for Fc γ RIIA_R131FcγRIIIA_V158Has lower affinity; IgG4 was compared with Fc γ RI, Fc γ RIIA, Fc γ RIIB, Fc γ RIIC and Fc γ RIIIA_V158Combining; and the inhibitory receptor Fc γ RIIB has lower affinity than all other Fc γ receptors for IgG1, IgG2, and IgG 3. Bruhns et alHuman (2009) Blood 113: 3716. studies showed that Fc γ RI does not bind IgG2, and Fc γ RIIIB does not bind IgG2 or IgG 4. As above. Generally, with respect to ADCC activity, human IgG 1. gtoreq.IgG 3> IgG 4. gtoreq.IgG 2. As a result, for example, IgG1 constant domains, rather than IgG2 or IgG4, may be selected for use in drugs that require ADCC; IgG3 can be selected if NK cells expressing Fc γ RIIIA, monocytes of macrophages are activated; if the antibody is to be used to desensitize an allergic patient, IgG4 may be selected. IgG4 may also be selected if the desired antibody lacks all effector functions.

The anti-huCD 40 variable regions described herein may be linked (e.g., covalently linked or fused) to an Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc, which may be any allotype or isoallotype, for example, for IgG 1: g1m, G1m1(a), G1m2(x), G1m3(f), G1m17 (z); for IgG 2: g2m, G2m23 (n); for IgG 3: 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, e.g., Jefferis et al (2009) mAbs 1: 1). The choice of allotype may be affected by potential immunogenicity issues, such as minimizing the formation of anti-drug antibodies.

In some embodiments, the anti-CD 40 antibodies of the invention have Fc binding to Fc γ RIIb or have enhanced Fc binding to Fc γ RIIb, which can provide enhanced agonism. See, for example, WO 2012/087928; and Li&Ravech (2011) Science 333: 1030; wilson et al (2011) cancer cell 19: 101, a first electrode and a second electrode; white et al (2011) j.immunol.187: 1754. the variable regions described herein may be linked to Fc variants that enhance affinity for the inhibitory receptor Fc γ RIIb, for example to enhance apoptosis induction or adjuvant activity. Li&Ravech (2012) proc.nat' l acad.sci.usa109: 10966, respectively; U.S. patent application publication 2014/0010812. Such variants may be provided having Fc γ RIIb⁺Antibodies to immunomodulatory activity associated with cells, including, for example, B cells and monocytes. In one embodiment, the Fc variant provides selectively enhanced affinity for Fc γ RIIb relative to one or more activating receptors. Such variants may also exhibit enhanced FcR-mediated cross-linkingResulting in enhanced therapeutic efficacy. The modifications to alter binding to Fc γ RIIb include one or more modifications at positions selected from 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328 and 332 according to the EU index. Exemplary substitutions for enhancing Fc γ RIIb affinity include, but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to Fc γ RIIb include 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E, and 267E-328F. In particular, the S267E, G236D, S239D, L328F and I332E variants, including the S267E-L328F double variants, of human IgG1 have particular values in specifically enhancing affinity for the inhibitory Fc γ RIIb receptor. Chu et al (2008) mol. immunol.45: 3926; U.S. patent application publication 2006/024298; WO 2012/087928. Substitutions and other mutations can be made by adding P238D (Mimoto et al (2013) protein.&Selection 26: 589; WO2012/1152410) and V262E and V264E (Yu et al (2013) j.am.chem.soc.135: 9723 and WO2014/184545) to obtain a para-Fc γ RIIb (distinct from Fc γ RIIa_R131) Enhanced specificity. See WO 2017/004006.

Half-life extension

In certain embodiments, the antibody is modified to increase its biological half-life. A variety of approaches are possible. This can be done, for example, by increasing the binding affinity of the Fc region for FcRn. In one embodiment, the antibody is altered within the CH1 or CL region to include a salvage receptor binding epitope taken from the two loops of the CH2 domain of the Fc region of IgG as described by Presta et al in U.S. patent nos. 5,869,046 and 6,121,022. Other exemplary Fc variants that increase binding to FcRn 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): 6213A 6216, Hinton et al (2006) Journal of Immunology 176: 346-. See U.S. patent No. 8,367,805.

Modifications to certain conserved residues (I253, H310, Q311, H433, N434) in IgG Fc have been proposed, such as the N434A variant (Yeung et al, (2009) j. immunol.182: 7663) as a means of increasing FcRn affinity, thereby increasing the half-life of the antibody in circulation. See WO 98/023289. A combination Fc variant comprising M428L and N434S has been shown to increase FcRn binding and increase serum half-life by up to five-fold. Zalevsky et al (2010) nat. biotechnol.28: 157. the combinatorial Fc variants comprising T307A, E380A, and N434A modifications also extended the half-life of IgG1 antibody. Petkova et al (2006) int.immunol.18: 1759. in addition, combinatorial Fc variants comprising M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S variants are shown to increase half-life. See WO 2009/086320.

Furthermore, a combination Fc variant comprising M252Y, S254T, and T256E increased half-life by nearly 4-fold. Dall' Acqua et al j.biol.chem.281: 23514. related IgG1 modifications (M252Y-S254T-T256E-H433K-N434F) that provide enhanced FcRn affinity but reduced pH dependence have been used to create IgG1 constructs ("MST-hnabddeg"), used as competitors to prevent binding of other antibodies to FcRn, which results in increased clearance of another antibody (either endogenous IgG (e.g. in the autoimmune environment) or another exogenous (therapeutic) mAb). Vaccarao et al (2005) nat. biotechnol.23: 1283. about.; WO 2006/130834.

Other modifications for increasing FcRn binding are described in Yeung et al (2010) j.immunol.182: 7663-; 6,277,375; 6,821,505, respectively; 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 hybrid variant can be constructed by substituting an amino acid from IgG3 for the IgG1 position in the CH2 and/or CH3 regions at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies comprising one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F, may be constructed. In other embodiments described herein, an IgG1/IgG2 hybrid variant can be constructed by substituting an amino acid from IgG1 for position IgG2 in the CH2 and/or CH3 regions at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that comprise one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (which refers to the insertion of a glycine at position 236), and 327A. See U.S. patent No. 8,629,113. Hybrids of the IgG1/IgG2/IgG4 sequences have been generated, purportedly to increase serum half-life and improve expression. U.S. patent No.7,867,491 (serial No. 18 therein).

The serum half-life of the antibodies of the invention may also be increased by pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is reacted with a polyethylene glycol (PEG) reagent (e.g., a reactive ester or aldehyde derivative of PEG), typically under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed by acylation or alkylation with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0154316 to Nishimura et al and EP 0401384 to Ishikawa et al.

Alternatively, in certain circumstances, it may be desirable to reduce, rather than increase, the half-life of the antibodies of the invention. Modifications in the Fc of human IgG1, such as I253A (Homick et al (2000) j.nuclear.med.41: 355) and H435A/R, I253A or H310A (Kim et al (2000) eur.j.immunol.29: 2819) can reduce FcRn binding and thereby reduce half-life (increase clearance) for use in situations where rapid clearance is preferred, such as medical imaging. See also kenannova et al (2005) cancer res.65: 622. other means of enhancing clearance include formatting the antigen binding domains of the invention as antibody fragments, e.g., Fab fragments, that lack the ability to bind FcRn. This modification can reduce the circulating half-life of the antibody from weeks to hours. Selective pegylation of the antibody fragment can then be used to fine-tune (increase) the half-life of the antibody fragment, if necessary. Chapman et al (1999) nat. biotechnol.17: 780. antibody fragments may also be fused to human serum albumin (e.g., in a fusion protein construct) to increase half-life. Yeh et al (1992) Proc.nat' lAcad.Sci.USA 89: 1904. alternatively, bispecific antibodies can be constructed using a first antigen-binding domain of the 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 can be added to antibody fragments to increase half-life, e.g., "XTEN" polypeptide sequences. Schellenberger et al (2009) nat. biotechnol.27: 1186 of a mixture of; international patent application publication WO 2010/091122.

Additional Fc variants

When using an IgG4 constant domain, it is generally preferred to include the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule, e.g., reduces Fab arm exchange between therapeutic antibodies and endogenous IgG4 in the treated patient. Labrijn et al (2009) nat biotechnol.27: 767; reddy et al (2000) j. immunol.164: 1925.

potential protease cleavage sites in the hinge of the IgG1 construct could be eliminated by D221G and K222S modifications, thereby increasing antibody stability. WO 2014/043344.

The affinity and binding properties of an Fc variant to its ligand (Fc receptor) can be determined by a variety of in vitro assays known in the art (based onBiochemical or immunological assays) including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assays (ELISA) or Radioimmunoassays (RIA)) or kinetics (e.g.SPR analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, Fluorescence Resonance Energy Transfer (FRET), gel electrophoresis, and chromatographic analysis (e.g., gel filtration). These and other methods may use labels on one or more of the components being examined and/or employ a variety of detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, W.E, editors, Fundamental Immunology, 4 th edition, 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, aglycosylated antibodies lacking all effector functions may be prepared by mutating a conserved asparagine residue at position 297 (e.g., N297A), thereby abolishing complement and Fc γ RI binding. Bolt et al (1993) eur.j.immunol.23: 403. see also Tao & Morrison (1989) j.immunol.143: 2595 (using N297Q in IgG1 to eliminate glycosylation at position 297).

Although aglycosylated antibodies generally lack effector function, mutations may be introduced to restore this function. Aglycosylated antibodies, such as produced by the N297A/C/D/or H mutation or produced in systems that do not glycosylate proteins (e.g., E. coli), may be further mutated to restore Fc γ R binding, such as S298G and/or T299A/G/or H (WO 2009/079242), or E382V and M428I (Jung et al (2010) proc. nat' l acad. sci. usa 107: 604).

Glycoengineering can also be used to modify the anti-inflammatory properties of IgG constructs by altering the α 2, 6 sialic acid content of the carbohydrate chain attached at Asn297 of the Fc region, where an increased ratio of α 2, 6 sialylated forms results in enhanced anti-inflammatory effects. See Nimmerjahn et al (2008) ann. rev. immunol.26: 513. conversely, where anti-inflammatory properties are not required, it may be useful to reduce the proportion of antibodies having α 2, 6 sialylated saccharides. Methods of modifying the α 2, 6 sialylation content of an antibody, for example by selective purification of the α 2, 6 sialylation form or by enzymatic modification, are provided in U.S. patent application publication No. 2008/0206246. In other embodiments, the amino acid sequence of the Fc region may be modified to mimic the effects of α 2, 6 sialylation, for example, by including F241A modifications. WO 2013/095966.

Physical Properties of antibodies

The antibodies described herein may comprise one or more glycosylation sites in the light or heavy chain variable region. Such glycosylation sites can result in increased immunogenicity of the antibody or altered pK of the antibody due to altered antigen binding (Marshall et al (1972) Ann. Rev. biochem.41: 673-. Glycosylation has been known to occur on motifs containing N-X-S/T sequences. In some cases, it is preferred to have an anti-huCD 40 antibody that does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues in the glycosylation region.

In certain embodiments, the antibodies described herein do not comprise an asparagine isomerization site. Deamidation of asparagine can occur at either the N-G or D-G sequences and result in the production of isoaspartic acid residues which introduce kinks into the polypeptide chain and reduce its stability (isoaspartic acid action).

Each antibody will have a unique isoelectric point (pI) which typically falls within a pH range of 6 to 9.5. The pI of the IgG1 antibody typically falls within a pH range of 7-9.5, and the pI of the IgG4 antibody typically falls within a pH range of 6-8. It is speculated that antibodies with pI values outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, an anti-CD 40 antibody having a pI value falling within the normal range is preferred. This can be achieved by selecting antibodies with pI in the normal range or mutating charged surface residues.

Each antibody will have a characteristic melting temperature, with higher melting temperatures indicating greater overall stability in vivo (Krishnhamurthy)&Manning (2002) curr. pharm. biotechnol.3: 361-71). Generally, T is preferred_M1(temperature of initial unfolding) greater than 60 ℃, preferably greater than 65 ℃, even more preferably greater than 70 ℃. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20: 1952-60; Ghirland 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 do not degrade rapidly. Degradation of antibodies can be achieved using Capillary Electrophoresis (CE) and MALDI-MS (Alexander)&Hughes (1995) Ahal chem.67: 3626-32).

In another preferred embodiment, antibodies are selected that have minimal aggregation effects that may result in triggering of unwanted immune responses and/or altered or unfavorable pharmacokinetic properties. Typically, antibodies with an aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, even more preferably 5% or less are acceptable. The degree of aggregation can be measured by several techniques, including Size Exclusion Columns (SEC), High Performance Liquid Chromatography (HPLC), and light scattering.

Nucleic acid molecules

Another aspect described herein relates to a nucleic acid molecule encoding an antibody described herein. The nucleic acid may be present in the intact cell, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., naturally linked to the isolated DNA) or proteins, by standard techniques, including alkali/SDS treatment, CsCl bands, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See, e.g., Ausubel et al, eds (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not comprise intron sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cdnas encoding the light and heavy chains of antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the libraries.

Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein (e.g., an antibody constant region or a flexible linker). The term "operably linked" as used herein refers to the joining together of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments are maintained in frame.

Isolated DNA encoding a VH region can be converted to a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and/or CH 3). The sequence of the Human heavy chain constant region gene is known in the art (see, e.g., Kabat, EA et al Sequences of Proteins of immunological Interest, 5 th edition, U.S. department of Health and Human Services, NIHPublication No.91-3242) and DNA fragments comprising 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, e.g., an IgG1 region. For a Fab fragment heavy chain gene, the DNA encoding the VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as the Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequence of the Human light chain constant region gene is known in the art (see, e.g., Kabat, e.a., et al (1991) Sequences of Proteins of immunological Interest, 5 th edition, u.s.department of Health and Human Services, NIHPublication No.91-3242) and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To generate the scFv genes, DNA fragments encoding VH and VL are ligated with a linker encoding a flexible linker (e.g., encoding the amino acid sequence (Gly)₄-Ser)₃) Such that the VH and VL sequences are expressed as a continuous single chain protein in which the VL and VH regions are linked together by a flexible linker (see, e.g., 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).

Antibody production

Generation of transfectomas producing monoclonal antibodies against CD40

Antibodies of the invention, including antibodies specific for the provided sequences and other related anti-CD 40 antibodies, can be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, to express an antibody or antibody fragment thereof, DNA 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 hybridomas expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" refers to the linkage of an antibody gene into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. Antibody light chain genes and antibody heavy chain genesEither by insertion into separate vectors or by insertion of both genes into the same expression vector. The antibody gene is inserted into one or more expression vectors by standard methods (e.g., linking complementary restriction sites to the antibody gene fragment and vector, or blunt-ended if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to construct full-length antibody genes of any antibody isotype by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype, such that V_HOne or more C's in segments and carriers_HThe segments are operably connected and V_LC in sections and carriers_LThe segments are operably connected. 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 amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain gene, the recombinant expression vector may also carry regulatory sequences that control the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the 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, Calif. (1990)). One skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct the expression of high levels of proteins in mammalian cells, such as promoters and/or enhancers derived from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and polyoma viruses. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the beta-globin promoter. Furthermore, the regulatory elements consist of sequences from different sources, such as the SR α promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia virus type 1 (Takebe, Y., et al (1988) mol.cell.biol.8: 466-472).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may also carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 to Axel et al). For example, typically, a selectable marker gene confers resistance to a drug such as G418, hygromycin or methotrexate on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" of various forms is intended to cover a wide variety of commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although it is theoretically possible to express the antibodies described herein in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells is most preferred, and most preferred is expression of the antibodies in mammalian host cells, because such eukaryotic cells, and particularly mammalian cells, are more likely to assemble and secrete a correctly folded and immunologically active antibody than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibodies (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6: 12-13). The antibodies of the invention may also be produced in glycoengineered strains of pichia pastoris. Li et al (2006) nat biotechnol.24: 210.

preferred mammalian host cells for expression of the recombinant antibodies described herein include Chinese hamster ovary (CHO cells) (including DHFR-CHO cells such as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-. In particular, another preferred expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or, more preferably, the antibody to be secreted into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

The N-and C-termini of the antibody polypeptide chains of the invention may differ from the expected sequences due to commonly observed post-translational modifications. For example, antibody heavy chains typically lack the C-terminal lysine residue. Dick et al (2008) biotechnol.bioeng.100: 1132. the N-terminal glutamine residues and a small number of glutamic acid residues are often converted to pyroglutamic acid residues on both the light and heavy chains of therapeutic antibodies. Dick et al (2007) biotechnol. bioeng.97: 544; liu et al (2011) j.biol.chem.286: 11211.

the amino acid sequences of various agonist anti-huCD 40 antibodies of the invention are provided in the sequence listing, which are summarized in table 7. For the reasons described above, the C-terminal lysine is not included in any sequence of the heavy chain or heavy chain constant domain in the sequence listing. However, in alternative embodiments, each heavy chain of the anti-huCD 40 antibodies of the invention and/or the genetic construct encoding such antibodies or the heavy or light chains thereof comprises the additional lysine residue at the C-terminus of one or both heavy chains.

VI. determination

The antibodies described herein can be tested for binding to CD40 by, for example, a standard ELISA. Briefly, microtiter plates were coated with 1-2. mu.g/ml purified CD40 in PBS, followed by blocking with 5% bovine serum albumin in PBS. Dilutions of antibodies (e.g., dilutions of plasma from CD 40-immunized mice) were added to each well and incubated at 37 ℃ for 1-2 hours. The plates were washed with PBS/tween and then incubated with a second reagent conjugated to horseradish peroxidase (HRP) (e.g., antibody for human antibodies or otherwise having a human heavy chain constant region, goat anti-human IgG Fc specific polyclonal reagent) for 1 hour at 37 ℃. After washing, the plates were developed with ABTS substrate (Moss Inc., product: ABTS-1000) and analyzed by spectrophotometer at OD 415-. Sera from immunized mice that bound to the cell line expressing human CD40, but not to the control cell line not expressing CD40, were then further screened by flow cytometry. Briefly, a CHO cell expressing CD40 was generated by contacting a CHO cell with an anti-CD 40 antibody at a 1: 20 dilutions were incubated together to assess binding of the anti-CD 40 antibody. Cells were washed and binding was detected with PE-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.). Preferably, the mice that produced the highest titers are used for fusion. If a mouse anti-huCD 40 antibody is to be detected, a similar experiment can be performed using an anti-mouse detection antibody.

The ELISA described above can be used to screen for antibodies and thus hybridomas that produce antibodies that show positive reactivity with CD40 immunogen. Hybridomas that produce antibodies that bind CD40 (preferably with high affinity) can then be subcloned and further characterized. One clone that retains the reactivity of the parental cell can then be selected from each hybridoma (by ELISA) to prepare a cell bank and used to purify the antibody.

To purify the anti-CD 40 antibody, selected hybridomas were cultured in two-liter spinner flasks for monoclonal antibody purification. The supernatant may be filtered and concentrated, followed by affinity chromatography on protein A-Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, possibly by OD₂₈₀The concentration was determined using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at-80 ℃.

To determine whether the selected anti-CD 40 monoclonal antibodies bind a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected with streptavidin-labeled probes. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CD 40-coated ELISA plates, as described above.

To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for the particular isotype of antibody. For example, to determine the isotype of human monoclonal antibodies, wells of microtiter plates can be coated overnight with 1. mu.g/ml of anti-human immunoglobulin at 4 ℃. After blocking with 1% BSA, the plates were allowed to react with 1. mu.g/ml or less of the test monoclonal antibody or purified isotype control for one to two hours at ambient temperature. The wells can then be reacted with human IgG1 or human IgM specific alkaline phosphatase conjugated probes. The plates were developed and analyzed as described above.

To test monoclonal antibodies for binding to live cells expressing CD40, flow cytometry may be used. Briefly, cell lines expressing membrane bound CD40 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibody in PBS containing 0.1% BSA for 1 hour at 4 ℃. After washing, the cells were reacted with Phycoerythrin (PE) -labeled anti-IgG antibody under the same conditions as the primary antibody staining. The sample can be analyzed by a FACScan instrument using light and side scatter properties to gate individual cells and determine the binding of labeled antibodies. In addition to or instead of flow cytometry assays, alternative assays using fluorescence microscopy may be used. Cells can be suitably stained as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but its sensitivity may be reduced depending on the density of the antigen.

The reactivity of the anti-huCD 40 antibody with CD40 antigen can be further tested by western blotting. Briefly, cell extracts from CD40 expressing cells can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens were 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 alkaline phosphatase and visualized with a BCIP/NBT substrate sheet (sigmachem. co., st.louis, MO).

Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-CD 40 antibodies include standard assays known in the art, e.g., biolayer interferometry (BLI) analysis and use

Of 2000SPR instruments (Biacore AB, Uppsala, Sweden)Surface Plasmon Resonance (SPR).

In one embodiment, the antibody specifically binds to the extracellular region of human CD 40. The antibody can specifically bind to a particular domain (e.g., a functional domain) within the extracellular domain of CD 40. In certain embodiments, the antibody specifically binds to an extracellular region of human CD40 and an extracellular region of cynomolgus monkey CD 40. Preferably, the antibody binds human CD40 with high affinity.

Bispecific molecules

The antibodies described herein can be used to form bispecific molecules. The anti-CD 40 antibody or antigen-binding fragment thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, the antibodies described herein may 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 encompassed by the term "bispecific molecule" as used herein. To produce a bispecific molecule described herein, an antibody described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule is produced.

Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for CD40 and a second binding specificity for a second target epitope. In embodiments where the bispecific molecule described herein is multispecific, the molecule may further comprise a third binding specificity.

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

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

Bispecific molecules described herein can be prepared by conjugating multiple component binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, covalent conjugation can be performed using a variety of coupling or crosslinking agents. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5' -dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-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 Paulus (1985) Behring Ins.Mitt.No.78, 118-; brennan et al (1985) Science 229: 81-83) and Glennie et al (1987) J.Immunol.139: 2367-. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical co. (Rockford, IL).

When the binding specificities are antibodies, they may be conjugated by thiol bonding 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 thiol residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. When the bispecific molecule is mAb x mAb, mAb x Fab, Fab x F (ab')₂Or ligand x Fab fusion proteins, the method is particularly useful. The bispecific molecules described herein may be single chain molecules comprising one single chain antibody and a binding determinant, or may be single chain bispecific molecules comprising two binding determinants. A bispecific molecule can comprise at least two single chain molecules. Methods for making bispecific molecules are described, for example, in U.S. Pat. nos. 5,260,203; 5,455,030, respectively; 4,881,175, respectively; 5,132, 405; 5,091,513; 5,476,786, respectively; 5,013,653, respectively; 5,258,498 and 5,482,858.

Binding of a bispecific molecule to its specific target can be confirmed using art-recognized methods, such as enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or western blot analysis. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

VIII. composition

Also provided are compositions, e.g., pharmaceutical compositions, containing one or more of the anti-CD 40 antibodies or antigen-binding fragments thereof described herein formulated with a pharmaceutically acceptable carrier. Such compositions can comprise one or a combination of (e.g., two or more different) antibodies or immunoconjugates or bispecific molecules described herein. For example, the pharmaceutical compositions described herein may comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on a target antigen or have complementary activity.

In certain embodiments, the compositions comprise anti-CD 40 antibody at a concentration of at least 1mg/ml, 5mg/ml, 10mg/ml, 50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml, or 1-300mg/ml or 100-300 mg/ml.

The pharmaceutical compositions described herein may also be administered in combination therapy, i.e. in combination with other agents. For example, a combination therapy may include an anti-CD 40 antibody described herein in combination with at least one other anti-cancer and/or T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies described herein.

In some embodiments, the therapeutic compositions disclosed herein may include other compounds, drugs, and/or agents useful for treating cancer. Such compounds, drugs and/or agents may include, for example, chemotherapeutic drugs, small molecule drugs or antibodies that stimulate an immune response against a given cancer. In some cases, a therapeutic composition can include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-OX 40 (also referred to as CD134, TNFRSF4, ACT35, and/or TXGP1L) antibody, an anti-LAG-3 antibody, an anti-CD 73 antibody, an anti-CD 137 antibody, an anti-CD 27 antibody, an anti-CSF-1R antibody, a TLR agonist, or a small molecule antagonist of IDO or TGF β.

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

The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects (see, e.g., Berge, SM 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 those derived from non-toxic inorganic acids (e.g., hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like), as well as those derived from non-toxic organic acids (e.g., 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 those derived from alkaline earth metals (e.g., sodium, potassium, magnesium, calcium, etc.), as well as those derived from non-toxic organic amines (e.g., N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

The pharmaceutical compositions described herein may further comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, α -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 nonaqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., 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. For example, proper fluidity can be maintained, for example, by the use of a coating material such as lecithin in the case of dispersion, by the maintenance of the required particle size, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures as well as by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. Additionally, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, it is contemplated that it will be used in the pharmaceutical compositions described herein. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or more of the ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% active ingredient, in combination with a pharmaceutically acceptable carrier, in one hundred percent.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms described herein are determined by and directly depend on the following factors: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the art of compounding such active compounds with respect to sensitivity to treat an individual.

For administration of the antibody, the dosage will range from about 0.0001 to 100mg/kg of body weight of the host, more usually 0.01 to 5 mg/kg. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight or 10mg/kg body weight or in the range of 1-10mg/kg body weight. An exemplary treatment regimen entails administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated range. Therapeutic antibodies are typically administered on multiple occasions. The interval between single doses may be, for example, weekly, monthly, every three months, or yearly. The intervals may also be irregular, as indicated by measuring blood levels of antibodies to the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1 to 1000. mu.g/ml, and in some methods, about 25 to 300. mu.g/ml.

The antibody may be administered as a slow release formulation, in which case less frequent administration is required. The dose and frequency depend on the half-life of the antibody in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the lifetime. In therapeutic applications, it is sometimes desirable to use relatively high doses at relatively short intervals until progression of the disease is reduced or terminated, and preferably until the patient exhibits partial or complete remission of the disease symptoms. Thereafter, although continued treatment is not required in many immunooncological indications, a prophylactic regimen may optionally be administered to the patient.

The actual dosage level of the active ingredient in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, while being non-toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition described herein or ester, salt or amide thereof employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, body weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dose" of an anti-CD 40 antibody described herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, and prevention of damage or disability due to disease affliction. In the case of cancer, a therapeutically effective dose preferably prevents further worsening of the physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, abnormal nevus characteristics, changes in the appearance of the nevus (including asymmetry, borders, color and/or diameter, newly pigmented skin regions, abnormal nevus), areas of darkening beneath the nails, breast bumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, dyspnea, hemoptysis, hematuria, hematochezia, nausea, vomiting, liver metastases, lung metastases, abdominal fullness, abdominal distension, abdominal dropsy, vaginal bleeding, constipation, abdominal fullness, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, lung metastases, Bladder metastasis, liver metastasis, bone metastasis, kidney metastasis and pancreas metastasis, dysphagia, and the like. The therapeutic effect may be observed immediately after the first administration of the agonistic anti-huCD 40mAb of the present invention, or may only be observed after a period of time and/or a series of doses. This delayed effect is only observed after several months of treatment, up to 6, 9 or 12 months. Given the delayed effect exhibited by certain immunotumoral agents, it is crucial that the lack of therapeutic efficacy of the agonistic anti-huCD 40mAb of the present invention cannot be determined prematurely.

A therapeutically effective dose can prevent or delay the onset of cancer, for example a therapeutically effective dose may be desirable when there is early or preliminary signs of disease. Laboratory tests for diagnosing Cancer include chemical methods (including measurement of soluble CD40 or CD40L levels) (Hock et al (2006) Cancer 106: 2148; Chung & Lim (2014) j. trans. med.12: 102), hematology, serology, and radiology. Thus, any clinical or biochemical assay that monitors any of the foregoing can be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. One of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The compositions described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration for the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

Alternatively, the antibodies described herein may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration.

The active compounds can be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations have been patented or are generally known to those skilled in the art. See, e.g., Sustainated and controlled Release Drug Delivery Systems, J.R. Robinson, eds., Marcel Dekker, Inc., New York, 1978.

The therapeutic composition can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions described herein can be administered using a needleless hypodermic injection device, such as those described in U.S. Pat. nos. 5,399,163; 5,383,851, respectively; 5,312,335, respectively; 5,064,413, respectively; 4,941,880, respectively; 4,790,824, respectively; or 4,596,556. Examples of well-known implants and modules for use with the anti-huCD 40 antibodies described herein include: U.S. patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent No. 4,486,194, which discloses a therapeutic device for administering a drug through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multiple chambers; and U.S. patent No. 4,475,196, which discloses 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-huCD 40 antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the Blood Brain Barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, U.S. Pat. nos. 4,522,811; 5,374,548, respectively; and 5,399,331. Liposomes can comprise one or more moieties that are selectively transported into a particular cell or organ, thereby enhancing targeted drug delivery (see, e.g., v.v. ranade (1989) j.clin.pharmacol.29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannoside (Umezawa et al (1988) biochem. Biophys. Res. Commun.153: 1038); antibodies (P.G.Blueman et al (1995) FEBSLett.357: 140; M.Owais et al (1995) antibiotic. Agents Chemother.39: 180); the surfactant protein a receptor (Briscoe et al (1995) am.j. physiol.1233: 134); p120(Schreier et al (1995) am. J. Physiol.1233: 134); and p120(Schreier et al (1994) J.biol.chem.269: 9090); see also k.keinanen; m.l. laukkanen (1994) FEBS lett.346: 123; j.j.killion; fidler (1994) Immunomethods 4: 273.

IX. use and method

The antibodies, antibody compositions, and methods described herein have a number of in vitro and in vivo utilities, relating to enhancing immune responses, for example, by agonizing CD40 signaling. In a preferred embodiment, the antibodies described herein are human or humanized antibodies. For example, the anti-huCD 40 antibodies described herein can be administered to cells in culture in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of diseases. Accordingly, provided herein are methods of altering an immune response in a subject comprising administering to the subject an antibody or antigen-binding fragment thereof described herein, thereby enhancing, stimulating, or up-regulating the immune response in the subject.

Preferred subjects include human patients in whom an enhanced immune response is desired. The methods are particularly useful for treating human patients suffering from diseases that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In a particular embodiment, the method is particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-huCD 40 antibodies described herein can be administered with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject). When the antibody to CD40 is administered with another agent, the two may be administered separately or simultaneously.

Also included are methods for detecting the presence of human CD40 antigen or measuring the amount of human CD40 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody or antigen-binding fragment thereof that specifically binds human CD40 under conditions that allow for the formation of a complex between the antibody or fragment thereof and human CD 40. The formation of complexes is then detected, wherein a difference in complex formation between the samples as compared to the control sample indicates the presence of human CD40 antigen in the sample. In addition, the anti-CD 40 antibodies described herein can be used to purify human CD40 by immunoaffinity purification.

Given the ability of the anti-huCD 40 antibodies described herein to enhance co-stimulation of T cell responses (e.g., antigen-specific T cell responses), provided herein are in vitro and in vivo methods of stimulating, enhancing, or up-regulating antigen-specific T cell responses, e.g., anti-tumor T cell responses, using the antibodies described herein. anti-CD 40 antibodies can be used to enhance CD4⁺And CD8⁺T cell response. The T cell may be T_effCells, e.g. CD4+ T_effCell, CD8+ T_effCells, T helper cells (T)_h) And T cell toxicity (T)_c) A cell.

Further included are methods of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an anti-huCD 40 antibody described herein, thereby enhancing the immune response (e.g., an antigen-specific T cell response) in the subject. In a preferred embodiment, the subject is a tumor-bearing subject and the immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative. The subject may also be a subject carrying a virus and the immune response to the virus is enhanced.

Further provided are methods of inhibiting tumor cell growth in a subject comprising administering to the subject an anti-huCD 40 antibody described herein, thereby inhibiting the growth of a tumor in the subject. Also provided are methods of treating a chronic viral infection in a subject, comprising administering to the subject an anti-huCD 40 antibody described herein, thereby treating the chronic viral infection in the subject.

In certain embodiments, the anti-huCD 40 antibody is administered to the subject as an adjunct therapy. Treatment of subjects with cancer with anti-huCD 40 antibodies can result in long-lasting responses relative to current standard of care; a long-term survival of at least 1, 2,3, 4,5, 10 or more years, a relapse-free survival of at least 1, 2,3, 4,5, or 10 or more years. In certain embodiments, treatment of a subject with cancer with an anti-huCD 40 antibody prevents or delays the recurrence of cancer, e.g., for 1, 2,3, 4,5, or 10 years or more. anti-CD 40 treatment may be used as the first or second line of treatment.

These and other methods described herein are discussed in further detail below.

Cancer treatment

Provided herein are methods for treating a subject having cancer, comprising administering to the subject an anti-huCD 40 antibody described herein, thereby treating the subject, e.g., such that the growth of a cancerous tumor is inhibited or reduced and/or the tumor regresses. The anti-huCD 40 antibody alone can be used to inhibit the growth of cancerous tumors. Alternatively, the anti-huCD 40 antibody can be used in combination with another agent, such as other immunogenic agents, standard cancer therapy, or other antibodies, as described below. Also provided are combinations with inhibitors of PD-1, such as anti-PD-1 or anti-PD-L1 antibodies. See, e.g., ellmarrk et al (2015) OncoImmunology 4: 7e 1011484.

Accordingly, provided herein are methods of treating cancer in a subject, e.g., by inhibiting tumor cell growth, comprising administering to the subject a therapeutically effective amount of an anti-huCD 40 antibody described herein, e.g., a humanized form of 12D6, 5F11, 8E8, 5G7, or 19G3, or an antigen-binding fragment thereof. The antibody can be a humanized anti-huCD 40 antibody (e.g., any humanized anti-huCD 40 antibody described herein), a human chimeric anti-huCD 40 antibody, or a humanized non-human anti-huCD 40 antibody, e.g., a human, chimeric or humanized anti-huCD 40 antibody that competes for binding with at least one anti-huCD 40 antibody described specifically herein, or binds to the same epitope as at least one anti-huCD 40 antibody described specifically herein.

Cancers whose growth can be inhibited using the antibodies of the present invention include cancers that are generally responsive to immunotherapy. Non-limiting examples of cancers to be treated include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (e.g., Renal Cell Carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or carcinomas), gastric cancer, germ cell tumor, pediatric sarcoma, sinus natural killer (sinonasal natural) cell, melanoma (e.g., metastatic malignant tumors such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cervical cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, solid tumors of childhood, carcinoma of the ureter, carcinoma of the renal pelvis, neoplasms of the Central Nervous System (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, virus-related cancers (e.g. Human Papillomavirus (HPV) -related tumors), and cells derived from either of two major blood cell lineages (that produce granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lines (that produce B, t, NK and plasma cells)), such as ALL types of leukemia, lymphoma and myeloma (e.g., acute, chronic, lymphocytic and/or myelogenous leukemia, such as acute leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), and Chronic Myelogenous Leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; 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), solitary granulocytic sarcoma, and chloroma; lymphomas such as Hodgkin's Lymphoma (HL), non-hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunocytoma T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disease, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic lymphoid lineage tumors, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, follicular lymphoma, Diffuse Histiocytic Lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, Chronic Lymphocytic Leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); seminomas, teratomas, central and peripheral nerve tumors (including astrocytomas, schwannomas); tumors of mesenchymal origin (including fibrosarcoma, rhabdomyoma, and osteosarcoma); and other tumors (including melanoma, pigmented xeroderma, stratum corneum acanthoma, seminoma, thyroid follicular cancer, and teratoma), hematopoietic tumors of lymphoid lineage, such as T cell and B cell tumors, including but not limited to T cell diseases (e.g., T prolymphocytic leukemia (T-PLL), which includes small cell and brain cell types); large granular lymphocytic leukemia (LGL), preferably of the T cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head or neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The methods described herein can also be used to treat metastatic cancer, refractory cancer (e.g., cancer refractory to prior immunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody), and recurrent cancer.

Nevertheless, the agonist anti-huCD 40 antibodies of the invention would not be useful in the treatment of hematological cancers with CD40 expression, which can be exacerbated by treatment with CD40 agonists. Certain cancers are known to express CD40 and therefore experience such exacerbations and can therefore be absolutely excluded. In other embodiments, based on the test results, a particular tumor sample is tested for CD40 expression and excluded from the therapy of the agonist anti-huCD 40 antibodies of the invention.

The anti-huCD 40 antibody can be administered as monotherapy or as the sole immunostimulatory therapy, or can be administered in a cancer vaccine strategy in combination with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, 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 peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. A number of experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO equivalent Book Spring: 60-62; Lothoothis, C., 2000, ASCO equivalent Book Spring: 300-. In one of these strategies, autologous or allogeneic tumor cells are used to prepare vaccines. These cell vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective antigen presentation activator for tumor vaccination. Dranoff et al (1993) proc.natl.acad.sci.usa 90: 3539-43.

The study of gene expression and large-scale gene expression patterns in various tumors has led to the definition of so-called tumor-specific antigens. Rosenberg, SA (1999) Immunity 10: 281-7. In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and cells from which tumors arise, such as the melanocyte antigens gp100, MAGE antigens and Trp-2. More importantly, many of these antigens can be shown to be targets for tumor-specific T cells found in the host. CD40 agonists can be used in conjunction with a collection of recombinant proteins and/or peptides expressed in a tumor to generate an immune response against these proteins. These proteins are generally considered by the immune system as self-antigens and are therefore tolerated by the immune system. Tumor antigens may include the protein telomerase, which is essential for chromosomal telomere synthesis and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al (1994) Science 266: 2011-2013). The tumor antigen can also be a "neoantigen" expressed in cancer cells as a result of a somatic mutation that alters the protein sequence or produces a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or an idiotype from a B cell tumor.

Other tumor vaccines can include proteins from viruses associated with human cancers, such as Human Papilloma Virus (HPV), hepatitis virus (HBV and HCV), and Kaposi's Herpes Sarcoma 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 tumor tissue itself. These heat shock proteins comprise fragments of proteins from tumor cells, and these HSPs are highly efficient in delivery to antigen presenting cells to elicit tumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al (1997) Science 278: 117-120).

Dendritic Cells (DCs) are potent antigen presenting cells that can be used to elicit antigen-specific responses. DCs can be produced ex vivo and loaded with a variety of protein and peptide antigens as well as tumor cell extracts (Nestle et al (1998) Nature medicine 4: 328-332). DCs can also be transduced by genetic means to express these tumor antigens. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al (2000) Nature medicine 6: 332-336). As a method of vaccination, DC immunization can be effectively combined with a CD40 agonist to activate (release) a more effective anti-tumor response.

Agonism of CD40 may also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). Agonism of CD40 may be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of chemotherapeutic agent administered (Mokyr et al (1998) Cancer Research 58: 5301-5304). An example of such a combination is the anti-huCD 40 antibody in combination with dacarbazine for the treatment of melanoma. Another example of such a combination is the combination of an anti-huCD 40 antibody with interleukin-2 (IL-2) for the treatment of melanoma. The scientific basis behind the use of CD40 agonists in combination with chemotherapy is that cell death is the result of the cytotoxic effects of most chemotherapeutic compounds, which should result in elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that can act synergistically with CD40 agonists through cell death are radiation, surgery and hormone deprivation. Each of these protocols produces a source of tumor antigens in a host. Angiogenesis inhibitors may also be used in combination with CD40 agonists. Inhibition of angiogenesis results in tumor cell death, which can provide tumor antigens into host antigen presentation pathways.

The anti-huCD 40 antibodies described herein can also be used in combination with bispecific antibodies that target Fc α or Fc γ receptor expressing effector cells to tumor cells (see, e.g., U.S. patent nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecific antibodies have been used to target macrophages to tumor sites. Such targeting may be more effective in activating tumor-specific responses. Agonism by CD40 enhances the T cell arm of these responses. Alternatively, the antigen can be delivered directly to the DCs by using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating the immunosuppressive proteins expressed by the tumor. These include TGF-. beta. (Kehrl et al (1986) J. exp. Med. 163: 1037-. Antibodies to each of these entities may be used in combination with anti-huCD 40 antibodies to counteract the effect of immunosuppressive agents and to promote a tumor immune response in the host.

anti-CD 40 antibodies are able to effectively replace T cell helper activity. Ridge et al (1998) Nature 393: 474-478. Activating antibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40(Weinberg et al (2000) Immunol.164: 2160-.

There are also several experimental treatment protocols involving ex vivo activation and expansion of antigen-specific T cells, and adoptive transfer of these cells into recipients to stimulate antigen-specific T cells against tumors (Greenberg & Riddell (1999) Science 285: 546-51). These methods may also be used to activate a T cell response to a pathogenic agent such as CMV. Ex vivo activation in the presence of anti-CD 40 antibodies can increase the frequency and activity of adoptively transferred T cells.

Chronic viral infection

In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, the method comprising administering to the subject an anti-huCD 40 antibody or antigen-binding fragment thereof, thereby treating the infectious disease in the subject.

Similar to its use in tumors as discussed above, antibody-mediated CD40 agonism may be used alone or in combination with vaccines as adjuvants to enhance immune responses to pathogens, toxins, and autoantigens. Examples of pathogens for which such treatment may be particularly useful include those for which no effective vaccine is currently available, or for which conventional vaccines are not fully effective. These include, but are not limited to, HIV, hepatitis (A, B and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (pseudomonas aeruginosa). CD40 agonism is particularly useful against infection by pathogens such as HIV that present altered antigens during the course of infection. These new epitopes were considered foreign when anti-human CD40 antibodies were administered, thus eliciting strong T cell responses.

Some examples of pathogenic viruses that cause an infection that can be treated by the methods described herein include HIV, hepatitis virus (A, B or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV), epstein-barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus, and arbovirus encephalitis (arbovirus) virus.

Some examples of pathogenic bacteria that cause infections that can be treated by the methods described herein include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia (serratia), pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulinum, anthrax, plague, leptospirosis, and lyme disease bacteria.

Some examples of infection-causing pathogenic fungi that can be treated by the methods described herein include candida (candida albicans, candida krusei, candida glabrata (glabrata), candida tropicalis (tropicalis), etc.), Cryptococcus neoformans (Cryptococcus neoformans), Aspergillus (Aspergillus) (Aspergillus fumigatus), Aspergillus niger (Aspergillus niger), etc.), mucor (mucor), Absidia (aspergillis), rhizopus (rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis (Paracoccus brasiliensis), Coccidioides (Coccidioides smitii), and Histoplasma capsulatum (Histosporium).

Some examples of pathogenic parasites that cause infection that may be treated by the methods described herein include amebic dysentery (Entamoeba histolytica), Giardia coli (Balantidium coli), proteus fortunei (naegleriafareri), proteus spinuloides (Acanthamoeba sp.), Giardia lamblia (Giardia lambia), Cryptosporidium sp (Cryptosporidium sp.), Pneumocystis carinii (pneumosystis carinii), Plasmodium vivax (Plasmodium vivax), Babesia mica (Babesia), Trypanosoma brucei (Trypanosoma cruzi), Trypanosoma cruzi (Trypanosoma cruzi), Leishmania donovani (Leishmania donovani), Toxoplasma gondii (Toxoplasma gondii), Trypanosoma brasilii (neospora japonica).

In all of the above methods, CD40 agonism may be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced presentation of tumor antigens. See, e.g., Holliger (1993) Holliger proc.natl.acad.sci.usa 90: 6444- > 6448; poljak (1994) Structure 2: 1121-1123.

Vaccine adjuvant

The anti-huCD 40 antibodies described herein can be used to enhance antigen-specific immune responses by co-administering the anti-huCD 40 antibodies with an antigen of interest, such as a vaccine. Accordingly, provided herein is a method of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) an anti-huCD 40 antibody or antigen-binding fragment thereof, thereby enhancing an immune response to the antigen in the subject. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include the antigens discussed in the section above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria, or other pathogens described above.

Suitable routes for administering the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by one of ordinary skill. For example, the antibody composition can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dose of the molecule used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, the anti-huCD 40 antibodies described herein can be co-administered with one or more other therapeutic agents, such as a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody may be linked to the agent (as an immune complex) 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 therapies, such as anti-cancer therapies, e.g., radiation. Such therapeutic agents include, inter alia, antineoplastic agents, such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which are themselves effective only at levels that are toxic or sub-toxic to the patient. Cisplatin is administered intravenously at a dose of 100mg/ml once every four weeks and doxorubicin is administered intravenously at a dose of 60-75mg/ml once every 21 days. Co-administration of the anti-CD 40 antibody or antigen-binding fragment thereof described herein with a chemotherapeutic agent provides two anti-cancer agents that act through different mechanisms that produce cytotoxic effects on human tumor cells. Such co-administration can solve problems caused by the development of resistance to drugs or antigenic changes in tumor cells that would render them unreactive with the antibody.

Also included within the scope described herein are kits comprising an antibody composition (e.g., a human antibody, a bispecific or multispecific molecule, or immunoconjugate) described herein and instructions for use. The kit can further comprise at least one additional agent, or one or more additional human antibodies described herein (e.g., a human antibody with complementary activity that binds to an epitope in a CD40 antigen that is different from the first human antibody). The kit typically includes a label indicating the intended use of the kit contents. The term label includes any written or recorded material provided on or otherwise accompanying the kit.

Combination therapy

In addition to the combination therapies provided above, the anti-CD 40 antibodies described herein can also be used in combination therapies, e.g., for the treatment of cancer, as described below.

The present invention provides methods of combination therapy in which an anti-huCD 40 antibody is co-administered with one or more additional agents, e.g., antibodies, effective to stimulate an immune response, thereby further enhancing, stimulating, or up-regulating the immune response in a subject.

In general, the anti-huCD 40 antibodies described herein can be combined with (i) another agonist of a co-stimulatory receptor and/or (ii) an antagonist of an inhibitory signal on T cells, either of which results in amplification of an antigen-specific T cell response (immune checkpoint modulator). Most co-stimulatory and co-inhibitory molecules are members of the immunoglobulin superfamily (IgSF), and the anti-CD 40 antibodies described herein may be administered with agents that target members of the IgSF family to increase the immune response. An important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, including 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 costimulatory or cosuppression receptors are the family of TNF molecules that bind to members of the homologous TNF receptor family, which includes CD40 and CD40L, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR 68692, TRAILR 8/DR 5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT. beta.R, LIGHT, DcR3, EM, HVGI/TL 1A, TRAMP/DR3, EDAR, EDA1, XEDAR, 2, TNFR 5, lymphotoxin alpha/TNF. beta, TNFR2, TNFR. beta.alpha.alpha.R, TNFR. alpha.beta.23, TNFR.alpha.beta.beta.T, TNFR.beta.Oldh.24, TNFR, TNFR.24, TNFR, see, TRY, TNFR, TRY, see, TRY, etc.

In another aspect, the anti-huCD 40 antibodies can be used in combination with antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF- β, VEGF) or other "immunosuppressive cytokines," or cytokines that stimulate T cell activation, to stimulate an immune response, e.g., for the treatment of proliferative diseases, such as cancer.

In one aspect, T cell responses may be stimulated by combining the anti-huCD 40 mabs of the invention with one or more of the following: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors) (e.g., CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4) and (ii) agonists of proteins that stimulate T cell activation (e.g., B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD 28H).

Exemplary agents that modulate one of the above proteins and may be combined with agonist anti-huCD 40 antibodies (such as those described herein) to treat cancer include:(ii) ipilimumab or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), BMS-936558 (anti-PD-1), pidilizumab/CT-011 (anti-PD-1),pembrolizumab/MK-3475 (anti-PD-1), AMP224 (anti-B7-DC/PD-L2), BMS-936559 (anti-B7-H1), MPDL3280A (anti-B7-H1), MEDI-570 (anti-ICOS), AMG557 (anti-B7H 2), MGA271 (anti-B7H 3-WO11/109400), IMP321 (anti-LAG-3), ureluab/BMS-663513 and PF-05082566 (anti-CD 137/4-1BB), varluumab/CDX-1127 (anti-CD 27), MEDI-6383 and MEDI-6469 (anti-OX 8), RG-7888 (anti-OX 40L-WO06/029879), Aselicept (anti-TACI), Morocitu-CD 3 (anti-CD 3), and IPUMLA-CTLA-4).

Other molecules that may be used in combination with the agonist anti-huCD 40 antibody for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, an agonist anti-huCD 40 antibody can be combined with an antagonist of KIR (e.g., lirilumab).

Other drugs for use in combination therapy 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, WO11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008(WO 11/140249; WO 13/169264; WO 14/036357).

In general, the agonist anti-huCD 40 antibodies described herein can be used with one or more of one or more agonists that link positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1 interaction), deplete or inhibit tregs (e.g., using an anti-CD 25 monoclonal antibody (e.g., darlingumab)) or by ex vivo anti-CD 25 bead depletion), agents that inhibit metabolic enzymes (such as IDO) or reverse/prevent T cell inability or depletion), and agents that cause innate immune activation and/or tumor site inflammation.

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, e.g., a PD-1 antagonist (e.g., an antagonist antibody), a PD-L1 antagonist (e.g., an antagonist antibody), a CTLA-4 antagonist (e.g., an antagonist antibody), and/or a LAG3 antagonist (e.g., an antagonist antibody), thereby stimulating the immune response in the subject, e.g., to inhibit tumor growth or stimulate an antiviral response. In one embodiment, the agonist anti-huCD 40 antibody and the antagonist anti-PD-1 antibody are administered to the subject. In one embodiment, the subject is administered an agonist anti-huCD 40 antibody and an antagonist anti-PD-L1 antibody. In one embodiment, the agonist anti-huCD 40 antibody and antagonist anti-CTLA-4 antibody are administered to the subject. In one embodiment, the at least one additional immunostimulatory antibody (e.g., antagonist anti-PD-1, antagonist anti-PD-L1, antagonist anti-CTLA-4, and/or antagonist anti-LAG 3 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody may be, e.g., a chimeric or humanized antibody (e.g., prepared from mouse anti-PD-1, anti-PD-L1, anti-CTLA-4, and/or anti-LAG 3 antibodies).

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject an agonist anti-huCD 40 antibody and an antagonist PD-1 antibody. In certain embodiments, the agonist anti-huCD 40 antibody is administered at a sub-therapeutic dose, the anti-PD-1 antibody is administered at a sub-therapeutic dose, or both, wherein the sub-therapeutic design is with reference to a monotherapy using the agent in question. Also provided herein are methods for altering adverse events associated with treatment of hyperproliferative diseases with an immunostimulant, comprising administering to a subject an agonist anti-huCD 40 antibody and a sub-therapeutic dose of an anti-PD-1 antibody. In certain embodiments, the subject is a human. In certain embodiments, the anti-PD-1 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD 40 antibody is a humanized monoclonal antibody, e.g., an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

PD-1 antagonists suitable for use in the methods described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein, e.g., AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

Exemplary anti-PD-1 antibodies are

Nastuzumab (BMS-936558) or an antibody comprising the CDRs or variable regions of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 as described in WO 2006/121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (WO 2012/145493)Pembrolizumab/formerly lambrolizumab); AMP-514/MEDI-0680 as described in WO 2012/145493; and CT-011 (pidilizumab; formerly CT-AcTibody or BAT; see, e.g., Rosenblatt et al (2011) J.Immunotherapy 34: 409). Other known PD-1 antibodies and other PD-1 inhibitors include those disclosed in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149, and U.S. Pat. publication No. 2009/0317368Those. Any anti-PD-1 antibody disclosed in WO2013/173223 may also be used. anti-PD-1 antibodies that competitively bind to one of these antibodies, and/or bind to the same epitope on PD-1 as one of these antibodies, may also be used in combination therapy.

In certain embodiments, the anti-PD-1 antibody is 5 × 10^-8K of M or less_DBinds to human PD-1 at 1 × 10^-8K of M or less_DBinds to human PD-1 at 5 × 10^-9K of M or less_DBinding to human PD-1, or at 1 × 10^-8M to 1 × 10^-10K between M or less_DBinds to human PD-1.

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject an agonist anti-huCD 40 antibody and an antagonist PD-L1 antibody. In certain embodiments, the agonist anti-huCD 40 antibody is administered at a sub-therapeutic dose, the anti-PD-L1 antibody is administered at a sub-therapeutic dose, or both are administered at a sub-therapeutic dose. Provided herein are methods for altering adverse events associated with treatment of a hyperproliferative disease with an immunostimulant, comprising administering to a subject an agonist anti-huCD 40 antibody and a sub-therapeutic dose of an anti-PD-L1 antibody. In certain embodiments, the subject is a human. In certain embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD 40 antibody is a humanized monoclonal antibody, e.g., an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as 12a4 in WO 2007/005874 and U.S. patent No.7,943,743), MSB0010718C (WO 2013/79174), or an antibody comprising the CDRs or variable regions of 3G10, 12a4, 10a5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 described in PCT publication WO 07/005874 and U.S. patent No.7,943,743. In certain embodiments, the anti-PD-L1 antibody is MEDI4736 (also known as anti-B7-H1) or MPDL3280A (also known as RG 7446). Any of the anti-PD-L1 antibodies disclosed in WO2013/173223, WO 2011/066389, WO 2012/145493, U.S. patent nos. 7,635,757 and 8,217,149, and U.S. publication No. 2009/145493 may also be used. anti-PD-L1 antibodies that compete with any of these antibodies and/or bind to the same epitope as any of these antibodies can also be used in combination therapy.

In yet another embodiment, the agonist anti-huCD 40 antibodies of the invention are combined with an antagonist of PD-1/PD-L1 signaling (e.g., 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 an OX-40 antagonist, e.g., an anti-GITR or anti-OX 40 antibody disclosed herein.

In another aspect, the immunotumoral agent is a GITR agonist, such as an agonistic GITR antibody. Suitable 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 immunotumoral 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), indoximod or NLG-919(WO 09/73620, WO 09/1156652, WO 11/56652, WO 12/142237).

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject an agonist anti-huCD 40 antibody and a CTLA-4 antagonist antibody described herein. In certain embodiments, the agonist anti-huCD 40 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both, wherein the subtherapeutic design is with reference to a monotherapy using the agent in question. Provided herein are methods for altering adverse events associated with the treatment of hyperproliferative diseases with an immunostimulant, comprising administering to a subject an agonist anti-huCD 40 antibody and a subtherapeutic dose of anti-CTLA-4 antibody. In certain embodiments, the subject is a human. In certain embodiments, the anti-CTLA-4 antibody is an antibody selected from the group consisting of:(ipilimumab or antibody 10D1, described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in the following publications: WO 98/42752; WO 00/37504; U.S. patent nos. 6,207,156; hurwitz et al (1998) proc.natl.acad.sci.usa95 (17): 10067-10071; camacho et al (2004) j.clin.oncology 22 (145): abstract No.2505 (antibody CP-675206); and Mokyr et al (1998) cancer Res.58: 5301-5304. Any anti-CTLA-4 antibody disclosed in WO2013/173223 may also be used.

Provided herein are methods for treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject the agonist anti-huCD 40 antibody and anti-LAG-3 antibody. In further embodiments, the agonist anti-huCD 40 antibody is administered at a sub-therapeutic dose, the anti-LAG-3 antibody is administered at a sub-therapeutic dose, or both are administered at a sub-therapeutic dose. Provided herein are methods for altering adverse events associated with treatment of a hyperproliferative disease with an immunostimulant, comprising administering to a subject an agonist anti-huCD 40 antibody and a sub-therapeutic dose of an anti-LAG-3 antibody. In certain embodiments, the subject is a human. In certain embodiments, the anti-LAG-3 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD 40 antibody is a humanized monoclonal antibody, e.g., an antibody comprising the CDRs or variable regions of an antibody disclosed herein. Examples of anti-LAG 3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in U.S. patent publication nos. US 2011/0150892 and WO 2014/008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-recognized anti-LAG-3 antibodies that may be used include IMP731 described in US 2011/007023. IMP-321 may also be used. anti-LAG-3 antibodies that compete with any of these antibodies and/or bind to the same epitope as any of these antibodies can also be used in combination therapy.

In certain embodiments, the anti-LAG-3 antibody is 5 × 10^-8K of M or less_DBinding human LAG-3 at 1 × 10^-8K of M or less_DBinding human LAG-3 at 5 × 10^-9K of M or less_DCombined with human LAG-3, or 1 × 10^-8M to 1 × 10^-10K between M or less_DBinding human LAG-3.

Administration of an agonist anti-huCD 40 antibody described herein and an antagonist (e.g., an antagonist antibody) against one or more second target antigens, such as LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1, can enhance the immune response to cancer cells in the patient. Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are generally responsive to immunotherapy. Representative examples of cancers treated with the combination therapies of the present disclosure include those cancers specifically listed above in the discussion of monotherapy with the agonist anti-huCD 40 antibody.

In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, a combination of therapeutic antibodies may be administered sequentially. For example, the anti-CTLA-4 antibody and agonist anti-huCD 40 antibody can be administered sequentially, e.g., first the anti-CTLA-4 antibody and then the agonist anti-huCD 40 antibody, or first the agonist anti-huCD 40 antibody and then the anti-CTLA-4 antibody. Additionally or alternatively, the anti-PD-1 antibody and agonist anti-huCD 40 antibody can be administered sequentially, e.g., first the anti-PD-1 antibody is administered, then the agonist anti-huCD 40 antibody is administered; or first the agonist anti-huCD 40 antibody and then the anti-PD-1 antibody. Additionally or alternatively, the anti-PD-L1 antibody and the agonist anti-huCD 40 antibody may be administered sequentially, e.g., first the anti-PD-L1 antibody and then the agonist anti-huCD 40 antibody, or first the agonist anti-huCD 40 antibody and then the anti-PD-L1 antibody. Additionally or alternatively, the anti-LAG-3 antibody and agonist anti-huCD 40 antibody may be administered sequentially, e.g., first the anti-LAG-3 antibody and then the agonist anti-huCD 40 antibody, or first the agonist anti-huCD 40 antibody and then the anti-LAG-3 antibody.

Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of sequential administration may be reversed or the same order maintained at each point in time of administration, sequential administration may be combined with simultaneous administration, or any combination thereof. For example, the first administration of the combination of anti-CTLA-4 antibody and agonist anti-huCD 40 antibody may be simultaneous, the second administration may be sequential to first anti-CTLA-4 antibody and then agonist anti-huCD 40 antibody, the third administration may be sequential to first agonist anti-huCD 40 antibody and then anti-CTLA-4 antibody, and so on. Additionally or alternatively, the first administration of the combination of anti-PD-1 antibody and agonist anti-uCD 40 antibody may be simultaneous, the second administration may be in the order of first anti-PD-1 antibody and then agonist anti-huCD 40 antibody, and the third administration may be in the order of first agonist anti-huCD 40 antibody and then anti-PD-1 antibody, and so forth. Additionally or alternatively, the first administration of the combination of anti-PD-L1 antibody and agonist anti-huCD 40 antibody may be simultaneous, the second administration may be in the order of first anti-PD-L1 antibody and then agonist anti-huCD 40 antibody, and the third administration may be in the order of first agonist anti-huCD 40 antibody and then anti-PD-L1 antibody, and so on. Additionally or alternatively, the first administration of the combination of anti-LAG-3 antibody and agonist anti-huCD 40 antibody may be simultaneous, the second administration may be in the order of first anti-LAG-3 antibody then agonist anti-huCD 40 antibody, and the third administration may be in the order of first agonist anti-huCD 40 antibody then anti-LAG-3 antibody, and so on. Another representative dosing regimen may involve a first administration of the agonist anti-huCD 40 followed by the anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody) in that order, and subsequent administrations may be simultaneous. Optionally, agonist anti-huCD 40, as the only immunotherapeutic agent, or a combination of agonist anti-huCD 40 antibody with one or more additional immunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3) can be further combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, 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 peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). A CD40 agonist and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers) can also be further combined with standard cancer treatments. For example, a CD40 agonist and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers) can be effectively combined with a chemotherapeutic regimen. In these cases, it is possible to reduce the dose of other chemotherapeutic agents administered with the combinations of the present disclosure (Mokyr et al (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of CD40 agonist antibodies with or without additional antibodies (e.g., anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or anti-LAG-3 antibodies), further in combination with dacarbazine for the treatment of melanoma. Another example is the combination of agonist anti-huCD 40 antibodies with or without anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or anti-LAG-3 antibodies, further in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the use of CD40 agonists and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers in combination with chemotherapy is that cell death (which is a result of the cytotoxic effects of most chemotherapeutic compounds) should result in elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may result in a synergistic effect through cell death of a CD40 agonist in combination with or without CDLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers include radiation, surgery or hormone deprivation. Each of these protocols produces a source of tumor antigens in a host. The angiogenesis inhibitor may also be combined with a CD40 agonist and a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker in combination. Inhibition of angiogenesis leads to tumor cell death, which may be the source of tumor antigens entering the host antigen presentation pathway.

The agonist anti-huCD 40 antibody, or the combination of a CD40 agonist with CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocking antibodies, as the sole immunotherapeutic agent, can also be used in combination with bispecific antibodies that target Fc α or Fc γ receptor expressing effector cells to tumor cells. See, for example, U.S. patent nos. 5,922,845 and 5,837,243. Bispecific antibodies can be used to target two separate antigens. The T cell arm of these responses may be enhanced by the use of a CD40 agonist in combination with a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker.

In another example, the combination of an agonistic anti-CD 40 antibody or anti-CD 40 antibody as the sole immunotherapeutic agent with another 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 a LAG-3 agent (e.g., an antibody)) can be combined with an anti-tumor antibody (e.g., an antibody)(rituximab),(trastuzumab),(tositumomab)), (tositumomab))),(ibritumomab) as a carrier,(alemtuzumab),(eprtuzumab)、

(bevacizumab) and

(erlotinib), etc.) in combination. For example, and without wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can lead to cancer cell (e.g., tumor cell) death, which will enhance the immune response mediated by an immunostimulant (e.g., CD40, TIGIT, CTLA-4, PD-1, PD-L1, or LAG-3 agent, such as an antibody). In an exemplary embodiment, treatment of a hyperproliferative disease (e.g., a cancer tumor) can include administration of an agonist anti-huCD 40 antibody and optionally additional antibodies capable of potentiating the hostThe anti-tumor immune response immunostimulant (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, e.g., antibody) in combination with an anti-cancer agent (e.g., antibody), administered simultaneously or sequentially or any combination thereof.

Provided herein are methods for altering adverse events associated with treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulant, comprising administering to a subject an agonist anti-huCD 40 antibody, with or without a subtherapeutic dose of an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent (e.g., antibody). For example, the methods described herein provide a method of 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 metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%. In one embodiment described herein, the non-absorbable steroid is budesonide. Budesonide is a topically acting glucocorticosteroid that is extensively metabolized primarily by the liver following oral administration.(Astra-Zeneca) is a pH and time dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and the entire colon.Has been approved in the united states for the treatment of mild to moderate crohn's disease involving the ileum and/or ascending colon.Conventional oral doses for the treatment of crohn's disease are 6 to 9 mg/day.Released in the intestine before being absorbed and retained in the intestinal mucosa. Once it has passed through the intestinal mucosa to the target tissue,

it is extensively metabolized by the cytochrome P450 system in the liver to negligible glucocorticosteroid-active metabolites. Thus, the bioavailability is low (about 10%). The low bioavailability of budesonide results in improved therapeutic rates compared to other glucocorticosteroids with a smaller first pass metabolic range. Budesonide produces fewer side effects, including hypothalamic-pituitary inhibition, than systemically acting corticosteroids. However, it is taken for a long period of timeResulting in systemic glucocorticosteroid effects such as corticoid overload and adrenal suppression. See PDR 58 th edition 2004; 608-610.

In further embodiments, a CD40 agonist (i.e., an immunostimulatory therapeutic antibody to CD40 and optionally an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody) with or without a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker may be further combined with a salicylate in combination with a nonabsorbable steroid. Salicylates include 5-ASA agents, for example: sulfasalazine (B)

Pharmacia&UpJohn); olsalazine (A) and (B)

Pharmacia&UpJohn); balsalazide (A), (B)Salixpharmaceutics, Inc.); and mesalamine (Procter&Gamble Pharmaceuticals；

Shire US；Axcan Scandipharm，Inc.；

Solvay)。

According to the methods described herein, the salicylate is administered in combination with an agonist anti-huCD 40 antibody with or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodies and a non-absorbable steroid, which may include any overlapping or sequential administration of the salicylate and the non-absorbable steroid, for the purpose of reducing the incidence of colitis induced by immune stimulating antibodies. Thus, for example, a method for reducing the incidence of colitis induced by an immunostimulatory antibody described herein comprises administering a salicylate and a non-absorbable steroid simultaneously or sequentially (e.g., 6 hours after administration of the salicylate after the non-absorbable steroid), or a combination thereof. Furthermore, the salicylate and the non-absorbable steroid may be administered by the same route (e.g., both orally administered) or a different route than the route used to administer the anti-huCD 40 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibody (e.g., the salicylate is administered orally and the non-absorbable steroid is administered rectally).

The agonist anti-huCD 40 antibodies and combination antibody therapies described herein can also be used in combination with other well-known therapies selected for their particular use for the indication being treated (e.g., cancer). The combination of agonist anti-huCD 40 antibodies described herein can be used sequentially with one or more known pharmaceutically acceptable agents.

For example, the agonist anti-huCD 40 antibodies and combination antibody therapies described herein can be used in combination (e.g., simultaneously or separately) with additional therapies, 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 + apo21/TRAIL (6X comb)), one or more proteasome inhibitors (e.g., examples of which are all shown in the figures), and the likeSuch as bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH 3I-2' (Bcl-x1 inhibitor), indoleamine dioxygenase-1 (IDO1) inhibitors (e.g., INCB24360), AT-101(R- (-) -gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax) or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists), iAP (inhibitor of apoptosis proteins) antagonists (e.g., smac7, smac, small molecule smac mimetics, synthetic smac peptides (see Fulda et al, NatMed 2002; 8: 808-15), ISIS23722(LY2181308) or AEG-35156(GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD 20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., VACUMAb), VEGF-and VEGFR targeted anti-angiogenic agents (e.g., VEGFR 640)),) Synthetic triterpenoids (see Hyer et al, Cancer Research 2005; 65: 4799-.

The agonist anti-huCD 40 antibodies and combination antibody therapies described herein can be further used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds useful as antiproliferative cytotoxic agents include, but are not limited to, the following:

alkylating agents (including but not limited to nitrogen mustards, ethylene imine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN)^TM) fosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, and temozolomide.

Antimetabolites (including but not limited to folate 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 agonist anti-huCD 40 antibodies, but are not limited to, taxanes, paclitaxel (paclitaxel may be used as TAXOL)^TMCommercially available), docetaxel, discodermolide (DDM), Dictyostatin (DCT), Peloruside A, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furaezomycin D, desoxyepothilone B1, [17 ]]Dehydrodesoxyepothilone B, [18 ]]Dehydro-desoxyepothilone B, C12, 13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans 9, 10-dehydroepothilone D, cis-9, 10-dehydroepothilone D, 16-demethylepothilone B, epothilone B10, discodermolide (discodermolide), paclitaxel (patupilone) (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA A (discodermolide), TZT-1027 (soladotin), ILX-651(tasidotin hydrochloride), halichondrin B, erigerolin mesylate (E-7389), hemisterlin (HTI-286), E-7974, Cytophilins, Cytophilin-LY 2, maytansine immunoconjugates (maytansine-1), Mbetahistine-1, T-82923, T-38067-T-12, Dexolone-5, Dermacetophenone-12, Dermatopteri-3-12, Dermatopteri-5, Dermatopterin-6-D-6, Dermatoptericin-1-5, Dermatoptericin-6, Dermatoptericin-5, Dermatoptericin-6, Dermatoptericin-5, Dermatoptericin-D-6, Dermatoptericin-D-6, and other isothiophenetidine.

In cases where it is desired to bind the agonist anti-huCD 40 antibody described herein or to quiesce abnormally proliferating cells prior to treatment with the agonist anti-huCD 40 antibody described herein, hormones and steroids (including synthetic analogs) such as 17 a-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drotaandrosterone propionate, testolactone, megestrol acetate, methylprednisolone, methylTestosterone, prednisolone, triamcinolone, clenobuterol ether, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone formate, leuprorelin, flutamide, toremifene, ZOLADEX^TMMay also be used for administration to a patient. Other agents useful in the clinical setting for modulating tumor growth or metastasis, such as anti-mimetic agents, may also be administered as needed when employing the methods or compositions described herein.

Methods for 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), e.g., 1996 edition (Medical Economics Company, Montvale, N.J.07645-1742, USA), the disclosure of which is incorporated herein by Reference.

One or more chemotherapeutic agents and/or radiation therapy may be administered according to treatment regimens well known in the art. It will be apparent to those skilled in the art that the administration of one or more chemotherapeutic agents and/or radiation therapy may vary depending on the disease being treated and the known effects of the one or more chemotherapeutic agents and/or radiation therapy. Also, the treatment regimen (e.g., dose and time of administration) can be varied in view of the observed effect of the administered therapeutic agent on the patient and in view of the observed response of the disease to the administered therapeutic agent, according to the knowledge of the skilled clinician.

X. specific agonist anti-CD 40 antibody

The agonist anti-CD 40 antibodies of the invention with improved humanized heavy and light chain variable region sequences are derived from the anti-CD 40 antibody described in WO 2017/004006. The variable domain and CDR sequence regions of the exemplary antibodies described in WO2017/004006 are provided in the sequence listing and summarized in table 2. The variable domain and CDR region numbering of the improved anti-CD 40 antibody of the invention is the same for all antibodies derived from the same original clone, i.e., the humanized variants provided herein do not include any insertions or deletions, but the amino acid sequence of SEQ ID NO: 47-51 and the light chain variable region sequence of modified mAb12D6 provided in SEQ ID NO: except for the heavy chain variable region sequences provided in 52-54.

TABLE 2

Antibody variable domains and CDRs

The disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference.

Examples