Anti-CD40 antibodies and uses thereof

Abstract

Provided herein are agonistic antibodies, or antigen binding portions thereof, that bind to human CD40 and comprise improved heavy and light chain variable regions that confer increased yield and reduced aggregation. The invention also provides methods of treating cancer or chronic infection by administering an antibody of the invention to a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 610,642, filed December 27, 2017, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listing submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted Sequence Listing (Name: 3338.112PC01_SeqListing_ST25.txt; Size: 153,067 bytes; Creation Date: December 14, 2018) are incorporated herein by reference in their entirety. Background Art

[0005] Recent studies suggest that human cancer and chronic infections may be treated with drugs that modulate the patient's immune response to malignant or infected cells. See, e.g., Reck & Paz-Ares (2015) Semin. Oncol. 42:402. Agonistic anti-CD40 antibodies such as CP 870893 and dacetuzumab (SGN-40) have been tried to treat cancer on the basis that they can 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 suggest that anti-CD40 antibodies with enhanced specificity for the inhibitory Fc receptor FcγRIIb have enhanced anti-tumor efficacy. See, e.g., WO 2012 / 087928; Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012) Proc. Nat'l Acad. Sci USA 109:10966; Wilson et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754.

[0006] Improved agonistic anti-human CD40 antibodies for treating cancer and chronic infections in human subjects are needed. Such antibodies would preferably be produced in high yield and with low aggregation. Summary of the invention

[0007] The present invention provides improved humanized heavy chain variable domains and light chain variable domains of antibody 12D6, which antibodies exhibit improved yields while retaining substantial affinity for human CD40. Specifically, the present invention provides agonistic anti-huCD40 antibodies comprising improved light chain variable regions L2 to L6 (SEQ ID NOs: 47-51), such as L4 (SEQ ID NO: 49) and / or improved heavy chain variable regions H2 to 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, which are selected from (i) residues 1-119 of SEQ ID NOs: 5, 7, 9, 11, 52, 53 and 54 and SEQ ID NO: 49, respectively; (ii) SEQ ID NO: 54 and residues 1-112 of SEQ ID NOs: 6, 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) residues 1-119 of SEQ ID NO: 11 and SEQ ID NO: 49; and (ii) SEQ ID NO: 54 and SEQ ID NO: 49. In some embodiments, the present invention includes antibodies comprising a heavy chain variable region H2 (SEQ ID NO: 52) and a 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 a light chain kappa constant region of SEQ ID NO: 45. 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.

[0008] In additional embodiments, the anti-huCD40 antibodies of the invention comprise heavy and light chain variable regions that have at least 80%, 85%, 90%, and 95% sequence identity to the heavy and light chain variable regions of any of the antibodies listed in the preceding paragraph. In additional embodiments, the anti-huCD40 antibodies comprise heavy and light chain variable regions that consist essentially of the sequence of the heavy and light chain variable regions of any of the antibodies disclosed herein.

[0009] The present invention further provides nucleic acids encoding the heavy chain variable region and / or light chain variable region or antigen-binding fragments thereof of the first two paragraphs, expression vectors comprising the nucleic acid molecules, cells transformed with the expression vectors, and methods for producing antibodies by expressing antibodies from cells transformed with the expression vectors and recovering the antibodies, as well as pharmaceutical compositions comprising the anti-huCD40 antibodies or antigen-binding fragments thereof of the present invention and vectors.

[0010] The present invention provides a method of enhancing an immune response in a subject, comprising administering to the subject an effective amount of an anti-huCD40 antibody or antigen-binding fragment thereof of the present 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, such as a chronic viral infection, and the anti-viral immune response is enhanced.

[0011] The present invention also provides a method for inhibiting tumor growth in a subject, comprising administering the anti-huCD40 antibody or antigen-binding fragment thereof of the present invention to the subject, thereby inhibiting tumor growth.

[0012] The present invention further provides methods of treating cancer, e.g., by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-huCD40 antibody or antigen-binding fragment thereof of the present 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, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancers. In certain embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0013] The present invention also provides a method of treating a chronic viral infection, such as by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-huCD40 antibody or antigen-binding fragment thereof of the present invention, such as as a pharmaceutical composition, thereby treating the chronic viral infection.

[0014] In certain embodiments, the methods and treatments for modulating immune function described herein include administering an anti-huCD40 antibody of the invention in combination with one or more additional therapeutic agents or as a bispecific agent with one or more additional therapeutic agents, such as, for example, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-CD137 antibody, an anti-CD27 antibody, an anti-CSF-1R antibody, an anti-CTLA-4 antibody, a TLR agonist, or a small molecule antagonist of IDO or TGFβ. In a specific embodiment, anti-huCD40 therapy is combined with anti-PD1 and / or anti-PD-L1 therapy, such as 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.

[0015] In some embodiments, an anti-huCD40 antibody of the invention comprises one or more heavy chains and one or more light chains, such as two heavy chains and two light chains. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 TNF-α expression of immature human dendritic cells titrated with various agonist anti-huCD40 antibodies is shown. Antibody 12D6-24 (x marks) is an unmodified anti-CD40 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 (closed 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 (closed circles) has 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.

[0018] Figure 2A and 2B Activation of human dendritic cells measured by CD83 and CD86 expression when treated with various antibodies of the invention are shown, respectively. Dendritic cells were exposed to the indicated antibodies, stained with fluorescent anti-CD83 and anti-CD86 antibodies, and analyzed by fluorescence activated cell sorting (FACS) as described in Example 3. Signals are expressed as mean fluorescence intensity (MFI). Antibody 12D6 is Figure 1 The antibodies 12D6-24, L4H1 and L4H4 are Figure 1 12D6-24-L4H1 and 12D6-24-L4H4 described in. Antibody 5F11 is another agonist anti-CD40 antibody disclosed herein, and the control is an irrelevant IgG1. The leftmost black bar for each antibody is an unstained sample. The light grey bar in the middle is cells stained with anti-CD83 antibody, and the dark grey bar on the far right is cells stained with anti-CD86 antibody. Figure 2A Data obtained at the excitation wavelength of the anti-CD83 antibody are presented (i.e., staining predominantly showing CD83), whereas Figure 2B Data obtained at the excitation wavelength of the anti-CD86 antibody are presented (ie, staining predominantly showing CD86). DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides isolated antibodies, particularly humanized monoclonal antibodies, that specifically bind to human CD40 ("huCD40") and have agonist activity, and specifically provides improved heavy and light chain variable region sequences that increase yield while still retaining substantial affinity for huCD40.

[0021] Further provided herein are methods of preparing 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 alone or in combination with other immunostimulants (e.g., antibodies) and / or cancer or anti-infective therapies for enhancing immune responses. Thus, the anti-huCD40 antibodies described herein can be used in treatments in a variety of therapeutic applications, including, for example, inhibiting tumor growth and treating chronic viral infections.

[0022] definition

[0023] In order to make this specification easier to understand, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0024] The term "CD40" refers to "TNF receptor superfamily member 5" (TNFRSF5). Unless otherwise specified or clear from the context, references to CD40 herein refer to human CD40 ("huCD40"), and "anti-CD40 antibodies" refer to anti-human CD40 antibodies. Human CD40 is further described as GENE ID NO: 958 and MIM (Mendelian inheritance in man): 109535. The sequence of human CD40 (GenBank Accession No. NP_001241.1), including the 20 amino acid signal sequence, is provided in SEQ ID NO: 1.

[0025] CD40 interacts with CD40 ligand (CD40L), which is also known as TNFSF5, gp39, and CD154. Unless otherwise specified or clear from the context, references herein to "CD40L" refer to human CD40L ("huCD40L"). 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 provided in SEQ ID NO: 2.

[0026] Unless otherwise indicated or clear from the context, the term "antibody" as used herein may include intact antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains thereof. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. 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. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). H and V L It consists of three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0027] Antibodies typically bind specifically to their cognate antigen with high affinity, which is reflected in the 10 -7 Up to 10 -11 M or less dissociation constant (K D ). It is generally considered that any -6 M of K D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and to substantially the same antigen, meaning that it has 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5×10 -9 M or less, most preferably 10 -8 M to 10 -10 M or smaller K D, but 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 to the sequence of the given antigen. For example, an antibody that specifically binds to 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 CD40.

[0028] Unless otherwise indicated, immunoglobulins 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: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, exist in several allotypes that differ from each other in a maximum of a few amino acids. Unless otherwise indicated, antibodies of the present invention comprise an IgG1f constant domain (SEQ ID NO: 44). Unless otherwise indicated, "antibodies" may include, for example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0029] 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 to an antigen (e.g., human CD40). Examples of binding fragments encompassed by the term "antigen-binding portion / fragment" of an antibody include: (i) Fab fragments - consisting of V L 、V H , CL and CH1 domains; (ii) F(ab′) 2 Fragment - a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a fragment consisting of V H and CH1 domains; (iv) a V fragment consisting of a single arm of the antibody L and V H The Fv fragment consists of a domain, and (v) a V H An isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs linked by a synthetic linker may comprise an antigen binding domain of an antibody if capable of binding to an antigen.

[0030] Single-chain antibody constructs are also included in the present invention. L and VH They are encoded by different genes, but they can be linked using recombinant methods with synthetic linkers to form a single protein chain, where V L and V H The domains are paired to form a monovalent molecule called a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242: 423-426 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 described 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 intact antibodies. Antigen-binding portions / fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0031] Unless otherwise indicated, when used in reference to an antibody, such as in the claims, the word "fragment" refers to the antigen-binding fragment of an antibody, so that "antibody or fragment" has the same meaning as "antibody or antigen-binding fragment thereof."

[0032] "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies with two different heavy chain / light chain pairs that produce two antigen binding sites with specificity for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79: 315-321; Kostelny et al. (1992) J. Immunol. 148, 1547-1553.

[0033] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a specific epitope, or an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a specific epitope. Typically, such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody, and will be bred without intentionally introducing any sequence changes. Therefore, the term "human monoclonal antibody" refers to a monoclonal antibody with a variable region and an optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, human monoclonal antibodies are produced by hybridomas, for example, by fusing B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice with a genome comprising human heavy chain transgenes and light chain transgenes) to immortalized cells to obtain hybridomas.

[0034] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) or a hybridoma prepared from the animal, which is transgenic or transchromosomal for human immunoglobulin genes, (b) antibodies isolated from a host cell transformed to express the antibody, such as antibodies isolated from a transfectoma, (c) antibodies isolated from a recombinant combinatorial human antibody library, and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations, such as those that occur during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region comprises an antigen binding domain, which is encoded by a variety of genes that are rearranged to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called 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 rearranged and somatically mutated nucleic acid sequences encoding the light and heavy chain immunoglobulin polypeptides that respond to the antigen may differ from the original germline sequences, but may be substantially identical or similar (i.e., having at least 80% identity).

[0035] "HuMAb" refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody of the present 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 mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted to human framework sequences. The terms "human" antibody and "fully human" antibody are synonyms.

[0036] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most or all of the amino acids in one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0037] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. A "hybrid" antibody refers to an antibody with different types of heavy and light chains, e.g., a mouse (parent) heavy chain and a humanized light chain, or vice versa.

[0038] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies).

[0039] "Allotype" refers to naturally occurring variants within a particular isotype group that differ by one or a few amino acids. See, e.g., Jefferis et al. (2009) mAbs 1:1.

[0040] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0041] 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 CD40). However, an isolated antibody that specifically binds an epitope of CD40 may have cross-reactivity with other CD40 proteins from other species.

[0042] "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, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with an antigen binding domain (e.g., antibody variable domain).

[0043] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternative splice forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIb or equivalently FcγRIIB). Table 1 summarizes the various properties of human FcγRs. Most innate effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIb, while natural killer (NK) cells in mice and humans selectively express an activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express inhibitory FcγRIIb. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor it binds.

[0044] Table 1

[0045] Characteristics of human FcγR

[0046]

[0047] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises CH1 in each of the two heavy chains of the antibody. 2 and CH 3 Constant domains: IgM and IgE Fc regions contain three heavy chain constant domains (C HDomains 2-4). For IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. 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 an amino acid residue at position C226 or P230 of the heavy chain (or an amino acid between these two amino acids) to the carboxyl terminus, where numbering is according to the EU index in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of U.S. Patent Application Publication No. 2008 / 0248028. C of the human IgG Fc region H2 The domain extends from about amino acid 231 to about amino acid 340, while the C H3 The domain is located in the Fc region H2 The C-terminal side of the domain, i.e., it extends from about amino acid 341 of IgG to about amino acid 447 (including the C-terminal lysine). As used herein, the Fc region can be a native sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in an isolate or in the context of a protein polypeptide comprising Fc (such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin)).

[0048] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; 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.

[0049] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to an antibody binding to an epitope on a predetermined antigen without binding to other antigens. Generally, the equilibrium dissociation constant (K) of antibody (i) binding is D ) is approximately less than 10 -7 M, for example, less than about 10 -8 M, 10 -9 M or 10 -10 M or even lower, when determined by, for example, using a predetermined antigen (e.g., recombinant human CD40) as an analyte and an antibody as a ligand 2000 surface plasmon resonance (SPR) technology, or Scatchard analysis of antibody binding to antigen-positive cells; and (ii) the affinity of binding to the predetermined antigen is at least two times greater than its affinity for binding to non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. Therefore, an antibody that "specifically binds to human CD40" refers to an antibody that binds to human CD40 with a binding affinity of at least 10 -7 M or less, for example, less than about 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to soluble or cell-bound human CD40. Antibodies that "cross-react with cynomolgus monkey CD40" are antibodies that cross-react with 10 -7 M or less, for example, less than about 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibody that binds to cynomolgus monkey CD40.

[0050] As used herein, the term " assoc " or "k a ” refers to the association rate constant for a specific antibody-antigen interaction, and the term “k dis " or "k d " refers to the dissociation rate constant for a specific antibody-antigen interaction. As used herein, the term "K D ” refers to the equilibrium dissociation constant, which is given by k d With k a The ratio (i.e. k d / k a ) and expressed as molar concentration (M). D The K value can be determined using methods well known in the art. D Preferred methods are biolayer interferometry (BLI) analysis, preferably using a ForteBio Octet RED instrument, surface plasmon resonance, preferably using a biosensor system (e.g. surface plasmon resonance system), or flow cytometry and Scatchard analysis.

[0051] In the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof, the term "EC50" refers to the concentration of an antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal response (ie, halfway between the maximal response and baseline).

[0052] The term "binds to immobilized CD40" refers to the ability of an antibody described herein to bind to CD40 (eg, to CD40 expressed on the surface of a cell or attached to a solid support).

[0053] As used herein, the term "cross-reactivity" refers to the ability of an antibody described herein to bind to CD40 from a different species. For example, an antibody described herein that binds human CD40 may also bind to CD40 from another species (e.g., cynomolgus CD40). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA) or binding to or otherwise functionally interacting with cells that physiologically express CD40. Methods for determining cross-reactivity include standard binding assays described herein, for example, using 2000SPR instrument (Biacore AB, Uppsala, Sweden) was used Surface plasmon resonance (SPR) analysis or flow cytometry.

[0054] As used herein, the term "naturally occurring" applies to an object and refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that is isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

[0055] A "polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as but not limited to glycosylation, phosphorylation or disulfide bonds. A "protein" may contain one or more polypeptides.

[0056] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be a cDNA.

[0057] Also provided are "conservative sequence modifications" of the antibody sequences provided herein, i.e., modifications of the nucleotide and amino acid sequences that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. For example, modifications can be 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, in which amino acid residues are replaced by amino acid residues with similar side chains. 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a predicted nonessential amino acid residue in an anti-CD40 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 abolish antigen binding are well known in the art. See, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997).

[0058] Alternatively, in another embodiment, mutations can be randomly introduced along all or a portion of the anti-CD40 antibody coding sequence, such as by saturation mutagenesis, and the resultant modified anti-CD40 antibodies can be screened for improved binding activity.

[0059] For nucleic acids, the term "substantial homology" means that two nucleic acids, or designated sequences thereof, are identical in at least about 80% of the nucleotides (usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides) with appropriate insertions or deletions of nucleotides when optimally aligned and compared. Alternatively, substantial homology exists when these segments will hybridize under selective hybridization conditions to the complement of a strand.

[0060] For polypeptides, the term "substantial homology" means that two polypeptides or designated sequences thereof are identical in at least about 80% of the amino acids (usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids) with appropriate amino acid insertions or deletions when optimally aligned and compared.

[0061] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences when they are optimally aligned, taking into account the number of gaps that need to be introduced in order to optimally align the two sequences, the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0062] 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 a gap weight of 40, 50, 60, 70 or 80 and a length weight 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 (CABIOS, 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 a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0063] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to search public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, wordlength = 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, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0064] Nucleic acids can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids purified from other cellular components or other contaminants (e.g., other cellular nucleic acids (e.g., other parts of chromosomes)) or proteins by standard techniques (including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art) are "isolated" or "appear substantially pure." See Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0065] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0066] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of that cell. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0067] "Immune response" refers to a biological response in vertebrates to foreign factors that protects the organism from these foreign factors and the 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 (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected with pathogens, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the vertebrate body. The immune response includes, for example, activation or inhibition of T cells, such as effector T cells or Th cells, such as CD4 + or CD8 + T cells, or inhibit or deplete T reg Cells. "T effect" ("T eff”) cells refer to T cells with cytolytic activity (e.g. CD4 + and CD8 + T cells) and T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but not regulatory T cells (T reg cell).

[0068] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0069] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mainly induced by CD8 + T cell mediated.

[0070] "Immunomodulator" or "immunomodulator" refers to such an agent, for example, a component of a signal transduction pathway that can participate in regulating, controlling or modifying an immune response. "Regulating", "regulating" or "modifying" an immune response refers to any change in the cells of the immune system or any change in the activity of such cells (e.g., effector T cells). This regulation 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 changes that may occur in the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced functions in the tumor microenvironment. In a preferred embodiment, the immunomodulator is located on the surface of a T cell. "Immunomodulatory target" or "immunomodulatory target" is an immunomodulator targeted for binding, and its activity is changed by the combination of a substance, an agent, a part, a compound or a molecule. Immunomodulatory targets include, for example, receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands") on the cell surface.

[0071] "Immunotherapy" refers to the treatment of a subject having a disease, or at risk of developing a disease or recurrence of a disease, by methods that include inducing, enhancing, suppressing or otherwise modifying an immune response.

[0072] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to a therapy that results in an increase (induce, enhance, or stimulate, all used interchangeably) an immune response in a subject, such as for the treatment of cancer.

[0073] "Enhancing an endogenous immune response" refers to increasing the effectiveness or potency of an existing immune response in a subject. For example, the increase in effectiveness and potency can be achieved by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0074] As used herein, the term "linked" refers to the association of two or more molecules. The connection can be covalent or non-covalent. The connection can also be genetic (i.e., recombinant fusion). Such connection can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0075] As used herein, "administering" refers to the use of any of a variety of methods and delivery systems known to those skilled in the art to introduce a composition comprising a therapeutic agent into the body. Preferred routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. Alternatively, antibodies described herein may be administered by non-parenteral routes, such as local, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. It may also be administered, for example, once, multiple times and / or in one or more extended periods.

[0076] As used herein, the terms "inhibit" or "block" are used interchangeably and encompass partial and complete inhibition / blocking of at least about 50%, such as at least about 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0077] As used herein, "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body through the lymphatic system or bloodstream.

[0078] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or procedure performed on a subject or the administration of an active agent to a subject with the goal of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease. Prevention refers to administration to subjects who do not have a disease to prevent the occurrence of the disease or minimize the effects of the disease, if it occurs.

[0079] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering from a recurrence of the disease, inhibits the development or recurrence of the disease. 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, such as in human subjects during clinical trials, in animal model systems that are predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0080] For example, an anticancer agent is a drug that slows down cancer progression or promotes cancer regression in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes cancer regression to the extent of eliminating cancer. "Promoting cancer regression" refers to the administration of an effective amount of a drug alone or in combination with an anti-tumor agent to cause a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, the prevention of damage or disability caused by disease torture, or otherwise improves the patient's disease symptoms. Pharmacological efficacy refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to an acceptable low level of toxicity or other adverse physiological effects (side effects) at the cell, organ and / or organism level due to the administration of a drug.

[0081] For example, for treating tumors, the therapeutically effective amount or dosage of the drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to untreated subjects. In the most preferred embodiment, the therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, that is, preferably inhibits cell growth or tumor growth by 100%. The ability of the compound to inhibit tumor growth can be evaluated using the assay described below. Inhibition of tumor growth may not occur immediately after treatment, and may only occur after a period of time or after repeated administration. Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell growth, and this inhibitory effect can be measured in vitro by assays known to skilled technicians. In other preferred embodiments described herein, tumor regression can be observed and can last for at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

[0082] As used herein, unless the context clearly indicates otherwise, "combination" therapy is intended to encompass the administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, simultaneous administration. Specifically, combination therapy encompasses co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, so long as the administration of one therapeutic agent is conditioned in some way on the administration of the other therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a specified period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0083] The terms "patient" and "subject" refer to any person receiving prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat a subject having cancer.

[0084] Various aspects described herein are described in further detail in the following subsections.

[0085] I. Anti-CD40 Antibodies

[0086] The present application discloses agonistic anti-huCD40 antibodies with desirable properties for use as therapeutic agents for treating diseases such as cancer and chronic viral infections. These properties 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-CD40 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., mAb 12D6-24 (comprising SEQ ID NOs: 11 and 8 or variable domains thereof). The improvement may be measured by any property including yield and percentage of monomeric antibody, or by the lack of multimers and other high molecular weight species.

[0087] Anti-CD40 antibody sequence variants

[0088] Some variability in the antibody sequences disclosed herein can be tolerated and still retain the desired properties of the antibody. The CDR regions are delineated using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Accordingly, the present invention provides an anti-huCD40 antibody comprising a heavy chain 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 chain and / or light chain variable domain sequence of an antibody disclosed herein (e.g., the 12D6 antibody comprising the heavy chain 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).

[0089] II. Engineered and Modified Antibodies

[0090] Targeted antigen binding

[0091] In various embodiments, the antibodies of the present invention are modified to selectively block antigen binding in tissues and environments where antigen binding is harmful, but allow antigen binding in beneficial situations. In one embodiment, a blocking peptide "mask" is produced that specifically binds to the antigen binding surface of the antibody and interferes with antigen binding, and the mask is connected 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 can be used to treat cancer in which protease levels are greatly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment dissociates the masking / blocking peptide, thereby selectively binding to the antigen in the tumor, rather than selectively binding to the antigen in peripheral tissues where antigen binding may cause unwanted side effects.

[0092] Alternatively, in a related embodiment, a bivalent binding compound ("masking ligand") comprising two antigen binding domains is developed that binds to the two antigen binding surfaces of a (bivalent) antibody and interferes with antigen binding, wherein the two binding domain masks are connected to each other (but not to the antibody) by a cleavable linker (e.g., cleavable by a peptidase). See, e.g., International Patent Application Publication No. WO2010 / 077643 of Tegopharm. The masking ligand may comprise or be derived from an antigen that is intended to bind to the antibody or may be generated independently. Such a masking ligand may be used to treat cancer, wherein the level of proteases in the tumor microenvironment is greatly increased compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment dissociates the two binding domains from each other, thereby reducing the affinity of the antigen binding surface of the antibody. The resulting dissociation of the masking ligand from the antibody allows selective binding of the antigen in the tumor, rather than in peripheral tissues where antigen binding may cause unwanted side effects.

[0093] Fc and modified Fc

[0094] The antibodies of the present invention may comprise the variable domains of the present invention combined with constant domains comprising different Fc regions, which are selected based on the biological activity (if any) of the antibodies for the intended use. Salfeld (2007) Nat. Biotechnol. 25: 1369. For example, human IgG can be divided into four subclasses, namely IgG1, IgG2, IgG3 and IgG4, each of which comprises an Fc region having a unique profile of binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32a, c); FcγRIIIA and FcγRIIIB (CD16a, b) and inhibitory receptor FcγRIIB (CD32b) and binding to the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIAH 131 Binds to FcγRIIA R131 FcγRIIIA V158 Has lower affinity; IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 Binding; and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Bruhns et al. (2009) Blood 113: 3716. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Same as above. Generally, with respect to ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2. As a result, for example, the IgG1 constant domain, rather than IgG2 or IgG4, can be selected for use in drugs requiring ADCC; if NK cells, macrophages, and monocytes expressing FcγRIIIA are activated, IgG3 can be selected; if the antibody is to be used to desensitize allergy patients, IgG4 can be selected. IgG4 can also be selected if the antibody is expected to lack all effector functions.

[0095] The anti-huCD40 variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, such as an IgG1, IgG2, IgG3 or IgG4 Fc, which can be of any allotype or isoallotype, e.g., for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v). See, e.g., Jefferis et al. (2009) mAbs 1:1). The choice of allotype may be influenced by potential immunogenicity issues, such as minimizing the formation of anti-drug antibodies.

[0096] In some embodiments, the anti-CD40 antibodies of the invention have an Fc that binds to FcγRIIb or has enhanced binding to FcγRIIb, which can provide enhanced agonism. See, e.g., WO 2012 / 087928; and Li & Ravetch (2011) Science 333:1030; Wilson et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754. The variable regions described herein can be linked to Fc variants that enhance affinity for the inhibitory receptor FcγRIIb, for example to enhance apoptosis induction or adjuvant activity. Li & Ravetch (2012) Proc. Nat′l Acad. Sci. USA 109:10966; U.S. Patent Application Publication 2014 / 0010812. Such variants can provide Fc variants that have binding to FcγRIIb. +In one embodiment, the Fc variants provide an enhanced affinity for the selective FcR-mediated cross-linking of FcRs, resulting in enhanced therapeutic efficacy. The modification used to change the binding to FcγRIIb is included in one or more modifications selected from the positions of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328 and 332 according to the EU index. Exemplary substitutions for enhancing affinity of FcγRIIb 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 FcγRIIb include 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E and 267E-328F. Specifically, the S267E, G236D, S239D, L328F and I332E variants of human IgG1, including the S267E-L328F double variant, have particular value in specifically enhancing affinity for the inhibitory FcγRIIb receptor. Chu et al. (2008) Mol. Immunol. 45: 3926; U.S. Patent Application Publication 2006 / 024298; WO2012 / 087928. The addition of the P238D substitution and other mutations (Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; WO2012 / 1152410) as well as V262E and V264E (Yu et al. (2013) J. Am. Chem. Soc. 135:9723 and WO2014 / 184545) to FcγRIIb (distinguished from FcγRIIa) can be achieved. R131 ) Enhanced specificity. See WO2017 / 004006.

[0097] Half-life extension

[0098] In certain embodiments, the antibody is modified to increase its biological half-life. Various methods are possible. For example, this can be accomplished by increasing the binding affinity of the Fc region to FcRn. In one embodiment, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al., antibodies are changed in CH1 or CL regions to include rescue receptor binding epitopes obtained from two loops of the CH2 domain of the Fc region of IgG. 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):6213-6216, Hinton et al. (2006) Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al. (2001) Journal of Biological Chemistry 276 (9): 6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) Journal of Immunology 169: 5171-5180, Dall'Acqua et al. (2006) Journal of Biological Chemistry 281: 23514-23524). See U.S. Pat. No. 8,367,805.

[0099] Modification of certain conserved residues in IgG Fc (I253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al., (2009) J. Immunol. 182:7663), has been proposed as a way to increase FcRn affinity, thereby increasing the half-life of the antibody in circulation. See WO 98 / 023289. Combination Fc variants comprising M428L and N434S have been shown to increase FcRn binding and increase serum half-life by up to five-fold. Zalevsky et al. (2010) Nat. Biotechnol. 28:157. Combination Fc variants comprising T307A, E380A and N434A modifications also extend the half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol. 18:1759. In addition, combined Fc variants comprising M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S variants have also been shown to extend half-life. See WO 2009 / 086320.

[0100] Furthermore, a combined Fc variant comprising M252Y, S254T and T256E increased half-life by nearly 4-fold. Dall'Acqua et al. J. Biol. Chem. 281: 23514. A related IgG1 modification (M252Y-S254T-T256E-H433K-N434F) that provides enhanced FcRn affinity but reduced pH dependence has been used to create an IgG1 construct ("MST-HNAbdeg") that acts as a competitor to prevent binding of other antibodies to FcRn, which results in increased clearance of another antibody (either endogenous IgG (e.g., in an autoimmune setting) or another exogenous (therapeutic) mAb). Vaccaro et al. (2005) Nat. Biotechnol. 23: 1283; WO 2006 / 130834.

[0101] Other modifications for increasing FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6,277,375; 6,821,505; WO 97 / 34631; and WO 2002 / 060919.

[0102] In certain embodiments, hybrid IgG isotypes can be used to increase FcRn binding, and potentially increase half-life. For example, IgG1 / IgG3 hybrid variants can be constructed by using the IgG1 position in the CH2 and / or CH3 districts from the amino acid substitutions of IgG3 at positions where two isotypes are different. Therefore, hybrid variant IgG antibodies comprising one or more substitutions such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R and 436F can be constructed. In other embodiments described herein, IgG1 / IgG2 hybrid variants can be constructed by using the IgG2 position in the CH2 and / or CH3 districts from the amino acid substitutions of IgG1 at positions where two isotypes are different. Therefore, hybrid variant IgG antibodies comprising one or more substitutions such as one or more of the following amino acid substitutions can be constructed: 233E, 234L, 235L, -236G (which refers to inserting glycine at position 236) and 327A. See US Patent No. 8,629,113. Hybrids of IgG1 / IgG2 / IgG4 sequences have been generated and are said to increase serum half-life and improve expression. US Patent No. 7,867,491 (SEQ ID NO: 18 therein).

[0103] The serum half-life of the antibody of the present invention can also be increased by pegylation. Antibodies can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. For pegylated antibodies, antibodies or their fragments are reacted with polyethylene glycol (PEG) reagents (e.g., reactive esters or aldehyde derivatives of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment, usually. Preferably, pegylation is carried out by acylation or alkylation with reactive PEG molecules (or similar reactive water-soluble polymers). 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 antibody without glycosylation. Methods for pegylated proteins are known in the art and can be applied to antibodies as described herein. See, for example, EP 0154316 to Nishimura et al. and EP 0401384 to Ishikawa et al.

[0104] Alternatively, in some cases, 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. Nucl. Med. 41: 355) and H435A / R, I253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29: 2819) can reduce FcRn binding, thereby reducing half-life (increasing clearance rate) for use in situations where rapid clearance is preferred, such as medical imaging. See also Kenanova et al. (2005) Cancer Res. 65: 622. Other ways to enhance clearance include formatting the antigen-binding domain of the invention into an antibody fragment, such as a Fab fragment, that lacks the ability to bind to FcRn. This modification can reduce the circulating half-life of the antibody from several weeks to several hours. Then, if necessary, selective PEGylation of the antibody fragment can be used to fine-tune (increase) the half-life of the antibody fragment. Chapman et al. (1999) Nat. Biotechnol. 17: 780. Antibody fragments can also be fused to human serum albumin (e.g., in a fusion protein construct) to increase half-life. Yeh et al. (1992) Proc. Nat'l Acad. Sci. USA 89: 1904. Alternatively, bispecific antibodies can be constructed with 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 patent references cited therein. Alternatively, specialized polypeptide sequences can be added to antibody fragments to increase half-life, for example, "XTEN" polypeptide sequences. Schellenberger et al. (2009) Nat. Biotechnol. 27: 1186; International Patent Application Publication WO 2010 / 091122.

[0105] Additional Fc variants

[0106] 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., reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in a treated patient. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925.

[0107] Potential protease cleavage sites in the hinge of IgG1 constructs can be eliminated by D221G and K222S modifications, thereby increasing the stability of the antibody. WO 2014 / 043344.

[0108] The affinity and binding properties of an Fc variant to its ligand (Fc receptor) can be determined by various in vitro assays known in the art (biochemical or immunological based assays), including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., The present invention relates to the method for detecting the binding affinity of the antibody of the present invention. The present invention relates to the method for detecting the binding affinity of the antibody of the present invention. The present invention relates to the method for detecting the binding affinity of the antibody of the present invention. The present invention relates to the method for detecting the binding affinity of the antibody of the present invention. The present invention relates to the method for detecting the binding affinity of the antibody of the present invention. The present invention relates to the method for detecting the binding affinity of the antibody of the present invention.

[0109] In other embodiments, the glycosylation of the antibody is modified to increase or decrease effector function. For example, an aglycosylated antibody lacking all effector function can be prepared by mutating the conserved asparagine residue at position 297 (e.g., N297A), thereby eliminating complement and FcγRI 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).

[0110] Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, such as aglycosylated antibodies produced by N297A / C / D / or H mutations or aglycosylated antibodies produced in a system that does not glycosylate proteins (e.g., E. coli), can be further mutated to restore FcγR 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).

[0111] Glycoengineering can also be used to modify the anti-inflammatory properties of IgG constructs by changing the α2,6 sialic acid content of the carbohydrate chain attached to Asn297 of the Fc region, where an increase in the proportion of α2,6 sialylated forms leads to an enhanced anti-inflammatory effect. 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 with α2,6 sialylated carbohydrates. Methods for modifying the α2,6 sialylation content of antibodies, such as by selectively purifying the α2,6 sialylated 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 can be modified to mimic the effects of α2,6 sialylation, for example by including a F241A modification. WO 2013 / 095966.

[0112] III. Antibody Physical Properties

[0113] The antibodies described herein may comprise one or more glycosylation sites in the light chain or heavy chain variable region. Such glycosylation sites may 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: 673-702; Gala and Morrison (2004) J. Immunol. 172: 5489-94; Wallick et al. (1988) J. Exp. Med. 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al. (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some cases, it is preferred to have an anti-huCD40 antibody that does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues in the glycosylation region.

[0114] In certain embodiments, the antibodies described herein do not comprise an asparagine isomerism site. Deamidation of asparagine can occur on NG or DG sequences and result in the generation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and reduces its stability (isoaspartic acid effect).

[0115] Each antibody will have a unique isoelectric point (pi), which typically falls within the pH range of 6 to 9.5. The pI of an IgG1 antibody typically falls within the pH range of 7-9.5, and the pI of an IgG4 antibody typically falls within the 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, it is preferred to have an anti-CD40 antibody with a pI value within the normal range. This can be achieved by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0116] Each antibody will have a characteristic melting temperature, wherein a higher melting temperature indicates greater overall stability in vivo (Krishnamurthy & Manning (2002) Curr. Pharm. Biotechnol. 3:361-71). In general, a T M1 (Temperature of initial unfolding) greater than 60°C, preferably greater than 65°C, even more preferably greater than 70°C. The melting point of the antibody can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm Res 20: 1952-60; Ghirlando et al. (1999) Immunol Lett. 68: 47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr. Sci. 40: 343-9). In a preferred embodiment, an antibody is selected that does not degrade rapidly. The degradation of the antibody can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander & Hughes (1995) Ahal Chem. 67: 3626-32).

[0117] In another preferred embodiment, antibodies with minimal aggregation effects are selected, which can lead to triggering of unwanted immune responses and / or altered or unfavorable pharmacokinetic properties. Typically, antibodies with an aggregation degree 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. Aggregation can be measured by several techniques, including size exclusion columns (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0118] IV. Nucleic Acid Molecules

[0119] Another aspect described herein relates to nucleic acid molecules encoding antibodies described herein. Nucleic acids can exist in intact cells, in cell lysates, or in partially purified or substantially pure forms. When purified from other cell components or other contaminants (e.g., other cellular nucleic acids (e.g., other chromosomal DNA, such as chromosomal DNA naturally connected to 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, nucleic acids are "isolated" or "present as substantially pure". See F.Ausubel et al., editors (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids described herein can be, for example, DNA or RNA, and may or may not include intron sequences. In certain embodiments, nucleic acids are cDNA molecules.

[0120] 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 further described below), cDNAs encoding the light and heavy chains of the 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.

[0121] 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 operations, the DNA fragment encoding 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 linking two DNA fragments together so that the amino acid sequence encoded by the two DNA fragments remains in the reading frame.

[0122] By operably linking the DNA encoding VH to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2 and / or CH3), the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al. Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, e.g., an IgG1 region. For Fab fragment heavy chain genes, the DNA encoding VH can be operably linked to another DNA molecule encoding only a heavy chain CH1 constant region.

[0123] By operably connecting the DNA encoding VL to another DNA molecule encoding the constant region CL of the light chain, the DNA encoding the separation of the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and the DNA fragments comprising these regions can be obtained by standard PCR amplification. The constant region of the light chain can be a kappa or lambda constant region.

[0124] To generate scFv genes, DNA fragments encoding VH and VL are combined with a DNA fragment encoding a flexible linker (e.g., a DNA fragment encoding an amino acid sequence (Gly 4 -Ser) 3 ) are operably linked to another fragment such that the VH and VL sequences are expressed as a continuous single-chain protein in which the VL and VH regions are connected 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).

[0125] V. Antibody Production

[0126] Generation of transfectomas producing monoclonal antibodies against CD40

[0127] Antibodies of the invention, including sequence-specific antibodies and other related anti-CD40 antibodies provided herein, can be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229: 1202).

[0128] For example, in order to express an antibody or its antibody fragment, DNA encoding part or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning performed using a hybridoma expressing an antibody of interest), and the DNA can be inserted into an expression vector so that the gene is operably linked to a transcription and translation control sequence. In this context, the term "operably linked" refers to connecting the antibody gene to a vector so that the transcription and translation control sequences in the vector exert their intended functions of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cells used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or the two genes can be inserted into the same expression vector. The antibody gene is inserted into one or more expression vectors by standard methods (e.g., complementary restriction sites are connected to the antibody gene fragment and the vector, or flat-end connection is performed if there is no restriction site). By inserting the light chain and heavy chain variable regions of the antibodies described herein into expression vectors encoding the heavy chain constant region and the light chain constant region of the desired isotype, it can be used to construct a full-length antibody gene of any antibody isotype, so that V H The segment and one or more C H segments are operably connected, and V L C in the segment and vector L The segments are operably connected. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0129] In addition to the antibody chain gene, the recombinant expression vector may also carry a regulatory sequence that controls 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 the antibody chain gene. Such regulatory sequences are described in, for example, Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)) and polyomavirus. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0130] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector may also carry other sequences, such as sequences (such as replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene helps to select the host cell into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017 of Axel et al.). For example, usually, the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate on the host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) gene (for dhfr-host cells with methotrexate selection / amplification) and neo gene (for G418 selection).

[0131] To express the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to cover a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies described herein in prokaryotic or eukaryotic host cells, it is most preferred to express the antibodies in eukaryotic cells, and it is most preferred to express the antibodies in mammalian host cells because such eukaryotic cells, especially mammalian cells, are more likely to assemble and secrete correctly folded and immunologically active antibodies than prokaryotic cells. Prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today 6: 12-13). The antibodies of the present invention can also be produced in glycoengineered strains of the yeast Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24: 210.

[0132] Preferred mammalian host cells for expressing the recombinant antibodies described herein include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, which are used with a DHFR selection marker, for example as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system 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 expression of the antibody in the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0133] Due to commonly observed post-translational modifications, the N-termini and C-termini of the antibody polypeptide chains of the present invention may differ from the expected sequence. For example, antibody heavy chains often lack a C-terminal lysine residue. Dick et al. (2008) Biotechnol. Bioeng. 100: 1132. The N-terminal glutamine residue and a small amount 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.

[0134] The amino acid sequences of various agonist anti-huCD40 antibodies of the invention are provided in the sequence listing and are summarized in Table 7. For the reasons described above, the C-terminal lysine is not included in any of the sequences of the heavy chains or heavy chain constant domains in the sequence listing. However, in alternative embodiments, each heavy chain of an anti-huCD40 antibody of the invention and / or a genetic construct encoding such an antibody or its heavy or light chain comprises this additional lysine residue at the C-terminus of one or both heavy chains.

[0135] VI. Determination

[0136] The antibodies described herein can be tested for binding to CD40 by, for example, a standard ELISA. Briefly, microtiter plates are coated with 1-2 μg / ml of purified CD40 in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (e.g., dilutions of plasma from CD40-immunized mice) are added to each well and incubated at 37°C for 1-2 hours. The plates are washed with PBS / Tween and then incubated with a secondary reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies or antibodies otherwise having a human heavy chain constant region, goat anti-human IgG Fc-specific polyclonal reagent) at 37°C for 1 hour. After washing, the plates are developed with ABTS substrate (Moss Inc., product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Sera from immunized mice that bind to cell lines expressing human CD40 but not to control cell lines that do not express CD40 are then further screened by flow cytometry. Briefly, the binding of anti-CD40 antibodies was assessed by incubating CD40-expressing CHO cells with anti-CD40 antibodies at a 1:20 dilution. The cells were washed and binding was detected with PE-labeled anti-human IgG Ab. Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Preferably, mice producing the highest titers were used for fusion. If mouse anti-huCD40 antibodies are to be tested, similar experiments can be performed using anti-mouse detection antibodies.

[0137] ELISA as described above can be used to screen for antibodies and, therefore, hybridomas that produce antibodies that show positive reactivity with the CD40 immunogen. Hybridomas that produce antibodies that bind CD40 (preferably with high affinity) can then be subcloned and further characterized. From each hybridoma, a clone that retains the reactivity of the parental cells (by ELISA) can then be selected to make a cell bank and used to purify antibodies.

[0138] To purify anti-CD40 antibodies, selected hybridomas can be grown in two-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated, then affinity chromatographed with protein A-agarose (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, and the OD 280 Determine concentration using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored at -80°C.

[0139] To determine whether the selected anti-CD40 monoclonal antibodies bind to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using a streptavidin-labeled probe. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using CD40-coated ELISA plates as described above.

[0140] To determine the isotype of the purified antibody, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated overnight at 4°C with 1 μg / ml anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with 1 μg / ml or less of the test monoclonal antibody or purified isotype control at ambient temperature for one to two hours. The wells can then be reacted with a probe conjugated to alkaline phosphatase specific for human IgG1 or human IgM. The plate is developed and analyzed as described above.

[0141] To test the binding of monoclonal antibodies to live cells expressing CD40, flow cytometry can be used. Briefly, cell lines expressing membrane-bound CD40 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, the cells are reacted with phycoerythrin (PE)-labeled anti-IgG antibodies under the same conditions as primary antibody staining. Samples can be analyzed by a FACScan instrument using light and side scatter properties to gate single cells and determine the binding of labeled antibodies. In addition to or instead of flow cytometry assays, alternative assays using fluorescence microscopy can be used. Cells can be appropriately stained as described above and examined by fluorescence microscopy. This method allows visualization of single cells, but its sensitivity may be reduced depending on the density of the antigen.

[0142] Anti-huCD40 antibodies can be further tested for reactivity with the CD40 antigen by Western blotting. Briefly, cell extracts from cells expressing CD40 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0143] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-CD40 antibodies include standard assays known in the art, for example, biolayer interferometry (BLI) analysis and use of 2000SPR instrument (Biacore AB, Uppsala, Sweden) Surface plasmon resonance (SPR).

[0144] In one embodiment, the antibody specifically binds to the extracellular region of human CD40. The antibody can specifically bind to a specific domain (e.g., a functional domain) within the extracellular domain of CD40. In certain embodiments, the antibody specifically binds to the extracellular region of human CD40 and the extracellular region of cynomolgus monkey CD40. Preferably, the antibody binds to human CD40 with high affinity.

[0145] VII. Bispecific Molecules

[0146] The antibodies described herein can be used to form bispecific molecules. Anti-CD40 antibodies or antigen-binding fragments thereof can be derived or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. In fact, the antibodies described herein can be derived 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 covered by the term "bispecific molecule" used herein. In order to produce the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, so that a bispecific molecule is produced.

[0147] Thus, 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 molecules described herein are multispecific, the molecules may further comprise a third binding specificity.

[0148] In one embodiment, the bispecific molecules described herein comprise at least one antibody or antibody fragment thereof as a binding specificity, such as Fab, Fab′, F(ab′), 2 , Fv or single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or 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.

[0149] 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.

[0150] The 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 the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimalimidide (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 those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0151] When the binding specificities are antibodies, they can be conjugated via sulfhydryl 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 sulfhydryl residues, preferably one, prior to conjugation.

[0152] 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′) 2 This method is particularly useful when the bispecific molecule is a single chain antibody or a ligand x Fab fusion protein. The bispecific molecules described herein can be single chain molecules comprising one single chain antibody and a binding determinant, or can be single chain bispecific molecules comprising two binding determinants. The bispecific molecule can comprise at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498 and 5,482,858.

[0153] Binding of a bispecific molecule to its specific target can be confirmed using methods recognized in the art, 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., antibody) specific for the complex of interest.

[0154] VIII. Composition

[0155] Compositions, such as pharmaceutical compositions, are also provided, which contain one or more anti-CD40 antibodies or antigen-binding fragments thereof described herein, formulated with a pharmaceutically acceptable carrier. Such compositions may contain 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 contain a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or have complementary activities.

[0156] In certain embodiments, the composition comprises an anti-CD40 antibody at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, or 1-300 mg / ml or 100-300 mg / ml.

[0157] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy can include an anti-CD40 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 uses of the antibodies described herein.

[0158] In some embodiments, the therapeutic compositions disclosed herein may include other compounds, drugs and / or agents for treating cancer. Such compounds, drugs and / or agents may include, for example, chemotherapy drugs, small molecule drugs or antibodies that stimulate an immune response to a given cancer. In some cases, the therapeutic composition may include, for example, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibodies, anti-LAG-3 antibodies, anti-CD73 antibodies, anti-CD 137 antibodies, anti-CD27 antibodies, anti-CSF-1R antibodies, TLR agonists or small molecule antagonists of IDO or TGFβ.

[0159] 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.

[0160] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do 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 acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.), and 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, etc.). 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,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0161] The pharmaceutical compositions described herein may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0162] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0163] These compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents in the composition, such as sugar, sodium chloride, etc. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption such as aluminum monostearate and gelatin.

[0164] 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. Unless any conventional media or agents are incompatible with the active compound, they may be considered for use in the pharmaceutical compositions described herein. Supplementary active compounds may also be incorporated into the composition.

[0165] Therapeutic compositions must generally be sterile and stable under production and storage conditions. The composition can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion and by using a surfactant.

[0166] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent as required with one or more combinations of the above-listed ingredients, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier comprising a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which produce powders of active ingredients and any additional desired ingredients from their previously sterile filtered solutions.

[0167] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on 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 that produces a therapeutic effect. Typically, out of one hundred percent, this amount ranges from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% active ingredient, in combination with a pharmaceutically acceptable carrier.

[0168] The dosage regimen is adjusted to provide the best desired response (e.g., a 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 therapeutic situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. The specifications of 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 specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of compounding such active compounds for treating individual sensitivity.

[0169] For administration of antibodies, dosages range from about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of the host's body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1-10 mg / kg. Exemplary treatment regimens require 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.

[0170] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the range shown. The therapeutic antibody is usually administered on multiple occasions. The intervals between single doses may be, for example, weekly, monthly, every three months or annually. Intervals may also be irregular as indicated by measuring the blood levels of antibodies to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

[0171] Antibodies can be administered as sustained release formulations, in which case less frequent administration is required. Dosage and frequency depend on the half-life of the antibody in the patient. Typically, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment throughout their lives. In therapeutic applications, relatively high doses are sometimes required to be used at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete relief of symptoms of the disease. Thereafter, although continuous treatment is not required in many immuno-oncology indications, preventive regimens may be optionally administered to patients.

[0172] Actual dosage levels of the active ingredients 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 on a variety of pharmacokinetic factors, including the activity of the particular composition described herein employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in conjunction with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0173] A "therapeutically effective dose" of an anti-CD40 antibody as 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 impairment or disability due to disease affliction. In the case of cancer, a therapeutically effective dose preferably prevents further worsening of physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, abnormal mole features, changes in the appearance of a mole (including asymmetry, borders, color and / or diameter, newly pigmented areas of skin, abnormal moles), darkening areas under the nails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, difficulty breathing, coughing up blood, blood in the urine, blood in the stool, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal fullness, abdominal distension, abdominal effusion, vaginal bleeding, constipation, abdominal fullness, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis and pancreatic metastasis, difficulty swallowing, etc. After the first administration of the agonist anti-huCD40 mAb of the invention, the therapeutic effect may be observed immediately, or only after a period of time and / or a series of doses. Such delayed effects may be observed only after several months of treatment, up to 6, 9 or 12 months. In view of the delayed effects shown by certain immuno-oncology agents, it is important not to prematurely determine that the agonist anti-huCD40 mAb of the invention lacks therapeutic efficacy.

[0174] A therapeutically effective dose can prevent or delay the onset of cancer, for example, a therapeutically effective dose may be desired when there are early or preliminary signs of the 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. Therefore, 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 the amount based on factors such as the subject's stature, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0175] 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, routes of administration and / or modes will vary according to the desired result. Preferred routes of administration of antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0176] Alternatively, the antibodies described herein can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical administration.

[0177] The active compound can be prepared with a carrier that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations have been patented or are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0178] The therapeutic compositions can be administered using medical devices known in the art. For example, in preferred embodiments, the therapeutic compositions described herein can be administered using a needle-free subcutaneous injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use with the anti-huCD40 antibodies described herein include: U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a drug infusion pump for delivering drugs 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 with multiple chambers; and U.S. Pat. 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.

[0179] In certain embodiments, the anti-huCD40 antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of preparing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); 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; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.

[0180] IX. Purpose and Method

[0181] The antibodies, antibody compositions and methods described herein have many in vitro and in vivo utilities, involving, for example, enhancing immune responses by agonizing CD40 signaling. In a preferred embodiment, the antibodies described herein are human or humanized antibodies. For example, the anti-huCD40 antibodies described herein can be administered to cells in culture in vitro or ex vivo, or, for example, administered to human subjects in vivo, to enhance immunity in a variety of diseases. Thus, provided herein are methods of altering an immune response in a subject, comprising administering to a subject an antibody or antigen-binding fragment thereof described herein, thereby enhancing, stimulating or upregulating an immune response in a subject.

[0182] Preferred subjects include human patients in whom an enhanced immune response is desired. The method is particularly suitable 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 specific embodiment, the method is particularly suitable for treating cancer in vivo. In order to achieve antigen-specific enhancement of immunity, the anti-huCD40 antibodies described herein can be administered together 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 an antibody against CD40 is administered together with another agent, the two can be administered separately or simultaneously.

[0183] Also included is a method for detecting the presence of human CD40 antigen in a sample or measuring the amount of human CD40 antigen, comprising contacting a sample and a control sample with a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human CD40 under conditions that allow formation of a complex between the antibody or fragment thereof and human CD40. The formation of the complex is then detected, wherein a difference in complex formation between the samples compared to the control sample indicates the presence of human CD40 antigen in the sample. In addition, the anti-CD40 antibodies described herein can be used to purify human CD40 by immunoaffinity purification.

[0184] Given that the anti-huCD40 antibodies described herein have the ability to enhance co-stimulation of T cell responses (e.g., antigen-specific T cell responses), provided herein are in vitro and in vivo methods for stimulating, enhancing, or upregulating antigen-specific T cell responses, e.g., anti-tumor T cell responses, using the antibodies described herein. Anti-CD40 antibodies can be used to enhance CD4 + and CD8 + T cell response. T cells can be T eff Cells, such as CD4+ T eff cells, CD8+T eff cells, T helper cells (T h ) and T cell cytotoxicity (T c )cell.

[0185] Further included is a method of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an anti-huCD40 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 subject carrying a tumor, and the immune response against the tumor is enhanced. The tumor can 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 can also be a subject carrying a virus, and the immune response to the virus is enhanced.

[0186] Further provided are methods of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an anti-huCD40 antibody described herein, thereby inhibiting the growth of the tumor in the subject. Also provided are methods of treating a chronic viral infection in a subject, comprising administering to the subject an anti-huCD40 antibody described herein, thereby treating the chronic viral infection in the subject.

[0187] In certain embodiments, an anti-huCD40 antibody is administered to a subject as an adjuvant therapy. Treatment of a subject with cancer with an anti-huCD40 antibody can result in a long-term durable response; long-term survival of at least 1, 2, 3, 4, 5, 10 or more years, relapse-free survival of at least 1, 2, 3, 4, 5 or 10 or more years relative to the current standard of care. In certain embodiments, treatment of a subject with cancer with an anti-huCD40 antibody prevents recurrence of the cancer or delays recurrence of the cancer by, for example, 1, 2, 3, 4, 5 or 10 years or more. Anti-CD40 therapy can be used as a first or second line of treatment.

[0188] These and other methods described herein are discussed in further detail below.

[0189] cancer

[0190] Provided herein are methods for treating a subject having cancer, the methods comprising administering to the subject an anti-huCD40 antibody described herein, such that the subject is treated, for example, such that the growth of a cancerous tumor is inhibited or reduced and / or the tumor regresses. Anti-huCD40 antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-huCD40 antibodies can be used in combination with another agent, such as other immunogenic agents, standard cancer treatments, or other antibodies, as described below. Combinations with inhibitors of PD-1, such as anti-PD-1 or anti-PD-L1 antibodies, are also provided. See, for example, Ellmark et al. (2015) OncoImmunology 4:7e1011484.

[0191] Thus, 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-huCD40 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-huCD40 antibody (e.g., any humanized anti-huCD40 antibody described herein), a human chimeric anti-huCD40 antibody, or a humanized non-human anti-huCD40 antibody, e.g., a human, chimeric, or humanized anti-huCD40 antibody that competes for binding with, or binds to the same epitope as, at least one anti-huCD40 antibody specifically described herein.

[0192] Cancers whose growth can be inhibited using the antibodies of the invention include cancers that are typically responsive to immunotherapy. Non-limiting examples of cancers treated include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, 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 carcinoma), gastric cancer, germ cell tumors, pediatric sarcoma, sinonasal natural killer cells (sinonasal natural killer cells ... cervical cancer, cervical cancer, cervical cancer, bladder cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer, cervical cancer killer), melanoma (e.g., metastatic malignancies such as skin or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, ureteral cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, 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 cancers derived from either of the two major blood cell lineages (i.e., myeloid cell lineage (which produces granulocytes, erythrocytes, hematopoiesis, and prostate cancer). hematological malignancies of the myeloid lineage (which produces platelets, macrophages, and mast cells) or lymphoid cell lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas (e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid 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);Lymphoma, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid 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, angioimmunoblastic 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 neoplasms, 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 called mycosis fungoides or Sezary syndrome), and leukemia with Waldenstrom's giant globulin Lymphoplasmacytoid lymphoma (LPL) of leukemia; myelomas, such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called 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); seminoma, teratoma, central and peripheral nervous system tumors (including astrocytoma, schwannoma) ; tumors of mesenchymal origin (including fibrosarcoma, rhabdoid tumor and osteosarcoma); and other tumors (including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma), hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell diseases (such as pre-T lymphocytic leukemia (T-PLL), which includes small cell and brain type cell types); large granular lymphocytic leukemia (LGL), preferably of T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (polymorphic 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 cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, such as with blocking CTLA-4 or PD-1 antibodies), and recurrent cancers. ;

[0193] Nevertheless, the agonist anti-huCD40 antibodies of the invention will not be used to treat hematological cancers with CD40 expression that may be exacerbated by treatment with a CD40 agonist. Certain cancers are known to express CD40 and therefore are subject to such exacerbation and therefore may be absolutely excluded. In other embodiments, specific tumor samples are tested for CD40 expression and excluded from therapy with the agonist anti-huCD40 antibodies of the invention based on the test results.

[0194] Anti-huCD40 antibodies can be administered as a monotherapy or as the sole immunostimulatory therapy, or can be administered in combination with immunogenic agents in a cancer vaccine strategy, 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. Many experimental strategies for vaccination against tumors have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043, in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, 5th edition). In one of these strategies, autologous or allogeneic tumor cells are used to prepare vaccines. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective activator of antigen presentation for tumor vaccination. Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-43.

[0195] Studies of gene expression and large-scale gene expression patterns in various tumors have 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 antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. CD40 agonists can be used in combination with a collection of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are generally considered self-antigens by the immune system and are therefore tolerated by the immune system. Tumor antigens can include the protein telomerase, which is required for the synthesis of chromosome telomeres 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). Tumor antigens can also be "neoantigens" expressed in cancer cells due to somatic mutations that alter the protein sequence or create a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or idiotypes from B-cell tumors.

[0196] Other tumor vaccines may include proteins from viruses associated with human cancers, such as human papillomavirus (HPV), hepatitis viruses (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 the tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells, and these HSPs are highly efficient in delivering to antigen presenting cells to elicit tumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120).

[0197] Dendritic cells (DC) are potent antigen presenting cells that can be used to elicit antigen-specific responses. DC can be generated ex vivo and loaded with a variety of protein and peptide antigens and tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DC can also be genetically transduced to express these tumor antigens. For the purpose of immunization, DC has also been directly fused with tumor cells (Kugler et al. (2000) Nature Medicine 6: 332-336). As a vaccination method, DC immunization can be effectively combined with CD40 agonists to activate (release) a more effective anti-tumor response.

[0198] Agonism of CD40 can also be combined with standard cancer treatments such as surgery, radiation, and chemotherapy. Agonism of CD40 can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of the chemotherapeutic agent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of an anti-huCD40 antibody and a dacarbazine for the treatment of melanoma. Another example of such a combination is the combination of an anti-huCD40 antibody and interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale 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 an increase in the levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with CD40 agonists through cell death are radiation, surgery, and hormone deprivation. Each of these regimens produces a source of tumor antigens in the host. Angiogenesis inhibitors can also be used in combination with CD40 agonists. Inhibition of angiogenesis leads to tumor cell death, which can provide tumor antigens into host antigen presentation pathways.

[0199] The anti-huCD40 antibodies described herein can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Pat. 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. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by the agonism of CD40. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0200] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. These include TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these entities can be used in combination with anti-huCD40 antibodies to counteract the effects of the immunosuppressive agents and promote the host's tumor immune response.

[0201] Anti-CD40 antibodies can effectively replace T cell helper activity. Ridge et al. (1998) Nature 393: 474-478. Activating antibodies against T cell co-stimulatory molecules can also provide increased levels of T cell activation, such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol. 164: 2160-2169), CD137 / 4-1BB (Melero et al. Nature Medicine 3: 682-685 (1997)) and ICOS (Hutloff et al. (1999) Nature 397: 262-266).

[0202] There are also several experimental treatment protocols that involve 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 can also be used to activate T cell responses to pathogenic factors such as CMV. Ex vivo activation in the presence of anti-CD40 antibodies can increase the frequency and activity of adoptively transferred T cells.

[0203] Chronic viral infection

[0204] In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, the method comprising administering an anti-huCD40 antibody or antigen-binding fragment thereof to the subject, thereby treating the infectious disease in the subject.

[0205] Similar to its use in tumors discussed above, antibody-mediated CD40 agonism can be used alone or as an adjuvant in combination with vaccines to enhance the immune response to pathogens, toxins and self-antigens. Examples of pathogens for which this treatment approach may be particularly useful include pathogens for which there are currently no effective vaccines, or pathogens 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, Pseudomonas aeruginosa. CD40 agonism is particularly useful against infection by pathogens such as HIV that present altered antigens during infection. When anti-human CD40 antibodies are administered, these new epitopes are recognized as foreign and therefore trigger a strong T cell response.

[0206] Some examples of pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis viruses (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, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0207] Some examples of pathogenic bacteria causing infections that can be treated by the methods described herein include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum, Anthrax, Plague, Leptospirosis, and Lymes disease bacteria.

[0208] Some examples of pathogenic fungi causing infections that can be treated by the methods described herein include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0209] Some examples of pathogenic parasites causing infections that can be treated by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

[0210] In all of the above approaches, CD40 agonism can 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.

[0211] Vaccine adjuvants

[0212] The anti-huCD40 antibodies described herein can be used to enhance an antigen-specific immune response by co-administering the anti-huCD40 antibodies with an antigen of interest, such as a vaccine. Thus, provided herein are methods of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) an anti-huCD40 antibody or an antigen-binding fragment thereof, thereby enhancing an immune response to the antigen in the subject. The antigen can 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 above sections, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria, or other pathogens described above.

[0213] 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 the skilled artisan. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0214] As previously described, the anti-huCD40 antibodies described herein can be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressants. The antibody can be linked to the agent (as an immune complex) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapies, such as anti-cancer therapies such as radiation. Such therapeutic agents include, among others, anti-tumor 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 subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. Co-administration of the anti-CD40 antibodies or antigen-binding fragments thereof described herein with chemotherapeutic agents provides two anti-cancer agents that act by different mechanisms that produce cytotoxic effects on human tumor cells. Such co-administration can solve problems caused by the development of resistance to the drug or changes in the antigenicity of tumor cells that would render them unreactive with the antibody.

[0215] Also included within the scope of what is described herein are kits comprising the antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit may further comprise at least one additional reagent, or one or more additional human antibodies described herein (e.g., human antibodies with complementary activities that bind to an epitope in the CD40 antigen that is different from the first human antibody). The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any text or recorded material on or provided with the kit or otherwise accompanying the kit.

[0216] Combination therapy

[0217] In addition to the combination therapies provided above, the anti-CD40 antibodies described herein can also be used in combination therapies, e.g., for treating cancer, as described below.

[0218] The invention provides methods of combination therapy wherein an anti-huCD40 antibody is co-administered with one or more additional agents, such as antibodies, effective to stimulate an immune response to further enhance, stimulate or upregulate the immune response of a subject.

[0219] Typically, the anti-huCD40 antibodies described herein can be combined with (i) an agonist of another co-stimulatory receptor and / or (ii) an antagonist of an inhibitory signal on a T cell, either of which results in amplification of antigen-specific T cell responses (immune checkpoint regulators). Most co-stimulatory and co-inhibitory molecules are members of the immunoglobulin superfamily (IgSF), and the anti-CD40 antibodies described herein can be administered together with agents targeting members of the IgSF family to increase the immune response. An important family of membrane-bound ligands that bind to co-stimulatory 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 co-stimulatory or co-inhibitory receptors is the family of TNF molecules that bind to cognate TNF receptor family members, which include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14 , TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

[0220] On the other hand, anti-huCD40 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.

[0221] In one aspect, T cell responses can be stimulated by combining an anti-huCD40 mAb of the invention with one or more of: (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, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H).

[0222] Exemplary agents that modulate one of the above proteins and that can be combined with agonist anti-huCD40 antibodies (such as those described herein) to treat cancer include: / ipilizumab 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-B7H2), MGA271 (anti-B7H3 - WO11 / 109400), IMP321 (anti-LAG-3), urelumab / BMS-66351 3 and PF-05082566 (anti-CD137 / 4-1BB), varlilumab / CDX-1127 (anti-CD27), MEDI-6383 and MEDI-6469 (anti-OX40), RG-7888 (anti-OX40L - WO06 / 029879), Atacicept (anti-TACI), Muromonab-CD3 (anti-CD3), ipilumumab (anti-CTLA-4).

[0223] Other molecules that can be combined with agonist anti-huCD40 antibodies for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, agonist anti-huCD40 antibodies can be combined with antagonists of KIR (e.g., lirilumab).

[0224] Other drugs for 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, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or FPA-008 (WO 11 / 140249; WO 13 / 169264; WO 14 / 036357).

[0225] In general, the agonist anti-huCD40 antibodies described herein can be used with one or more of one or more agonists that bind 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., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), deplete or inhibit Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab)) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes (such as IDO), or reverse / prevent T cell anergy or exhaustion), and agents that cause innate immune activation and / or inflammation at the tumor site.

[0226] Provided herein are methods of stimulating an immune response in a subject, comprising administering to the subject a CD40 agonist, such as an antibody, and one or more additional immunostimulatory antibodies, such as PD-1 antagonists (e.g., antagonist antibodies), PD-L1 antagonists (e.g., antagonist antibodies), CTLA-4 antagonists (e.g., antagonist antibodies), and / or LAG3 antagonists (e.g., antagonist antibodies), thereby stimulating the subject's immune response, for example, to inhibit tumor growth or stimulate an antiviral response. In one embodiment, an agonist anti-huCD40 antibody and an antagonist anti-PD-1 antibody are administered to the subject. In one embodiment, an agonist anti-huCD40 antibody and an antagonist anti-PD-L1 antibody are administered to the subject. In one embodiment, an agonist anti-huCD40 antibody and an antagonist anti-CTLA-4 antibody are administered to the subject. In one embodiment, at least one additional immunostimulatory antibody (e.g., antagonist anti-PD-1, antagonist anti-PD-L1, antagonist anti-CTLA-4, and / or antagonist anti-LAG3 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from mouse anti-PD-1, anti-PD-L1, anti-CTLA-4 and / or anti-LAG3 antibodies).

[0227] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer), comprising administering to a subject an agonist anti-huCD40 antibody and an antagonist PD-1 antibody. In certain embodiments, the agonist anti-huCD40 antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose, wherein the subtherapeutic design references the use of a monotherapy of the agent to be considered. Also provided herein are methods for altering adverse events associated with the use of an immunostimulant to treat a hyperproliferative disease, comprising administering to a subject an agonist anti-huCD40 antibody and a subtherapeutic dose of an anti-PD-1 antibody. In certain embodiments, the subject is human. In certain embodiments, the anti-PD-1 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD40 antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

[0228] 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, such as an Fc fusion protein, such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

[0229] An exemplary anti-PD-1 antibody is / nivolumab (BMS-936558) or an antibody comprising the CDRs or variable regions of one of the antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 ( / pembrolizumab / formerly lambrolizumab); AMP-514 / MEDI-0680 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. Patent Publication No. 2009 / 0317368. Any of the anti-PD-1 antibodies disclosed in WO2013 / 173223 may also be used.Anti-PD-1 antibodies that compete with one of these antibodies for binding, and / or bind to the same epitope on PD-1 as one of these antibodies may also be used in combination therapy.

[0230] In certain embodiments, the anti-PD-1 antibody is administered at 5×10 -8 M or smaller K D Binding to human PD-1, 1×10 -8 M or smaller K D Bind to human PD-1, 5×10 -9 M or smaller K D Combined with human PD-1, or 1×10 -8 M to 1×10 -10 M or smaller between K D Binds to human PD-1.

[0231] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer), comprising administering to a subject an agonist anti-huCD40 antibody and an antagonist PD-L1 antibody. In certain embodiments, the agonist anti-huCD40 antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering adverse events associated with the treatment of hyperproliferative diseases with an immunostimulant, the method comprising administering to a subject an agonist anti-huCD40 antibody and a subtherapeutic dose of an anti-PD-L1 antibody. In certain embodiments, the subject is human. In certain embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD40 antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

[0232] In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as 12A4 in WO 2007 / 005874 and U.S. Pat. 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. Pat. No. 7,943,743. In certain embodiments, the anti-PD-L1 antibody is MEDI4736 (also referred to as anti-B7-H1) or MPDL3280A (also referred to as RG7446). Any of the anti-PD-L1 antibodies disclosed in WO 2013 / 173223, WO 2011 / 066389, WO 2012 / 145493, U.S. Pat. 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 and / or bind to the same epitope as any of these antibodies may also be used in combination therapy.

[0233] In yet another embodiment, an agonist anti-huCD40 antibody of the invention is 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, such as an anti-GITR or anti-OX40 antibody disclosed herein.

[0234] In another aspect, the immuno-oncology 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).

[0235] In another aspect, the immuno-oncology 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).

[0236] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer), comprising administering to a subject an agonist anti-huCD40 antibody and a CTLA-4 antagonist antibody as described herein. In certain embodiments, the agonist anti-huCD40 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose, wherein the subtherapeutic design references the use of a monotherapy of the agent to be considered. Provided herein are methods for altering adverse events associated with the use of an immunostimulant to treat a hyperproliferative disease, the method comprising administering to a subject an agonist anti-huCD40 antibody and a subtherapeutic dose of an anti-CTLA-4 antibody. In certain embodiments, the subject is human. In certain embodiments, the anti-CTLA-4 antibody is an antibody selected from the following: (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. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145):Abstract No.2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed in WO 2013 / 173223 may also be used.

[0237] Provided herein are methods for treating hyperproliferative diseases (e.g., cancer) comprising administering to a subject an agonist anti-huCD40 antibody and an anti-LAG-3 antibody. In further embodiments, the agonist anti-huCD40 antibody is administered at a subtherapeutic dose, the anti-LAG-3 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. Provided herein are methods for altering adverse events associated with the use of an immunostimulant to treat a hyperproliferative disease, comprising administering to a subject an agonist anti-huCD40 antibody and a subtherapeutic dose of an anti-LAG-3 antibody. In certain embodiments, the subject is human. In certain embodiments, the anti-LAG-3 antibody is a human sequence monoclonal antibody, and the agonist anti-huCD40 antibody is a humanized monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein. Examples of anti-LAG3 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 can be used include IMP731 described in US 2011 / 007023. IMP-321 can 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.

[0238] In certain embodiments, the anti-LAG-3 antibody is administered at 5×10 -8 M or smaller K D Combined with human LAG-3, 1×10 -8 M or smaller K D Combined with human LAG-3, 5×10 -9 M or smaller K D Combined with human LAG-3, or at 1×10 -8 M to 1×10 -10 M or smaller between K D Binds human LAG-3.

[0239] Administration of an agonist anti-huCD40 antibody described herein and an antagonist (e.g., antagonist antibody) to 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 a patient. Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are typically responsive to immunotherapy. Representative examples of cancers treated with the combination therapies of the present disclosure include those specifically listed above in the discussion of monotherapy with agonist anti-huCD40 antibodies.

[0240] 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, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an agonist anti-huCD40 antibody can be administered sequentially, such as first administering the anti-CTLA-4 antibody and then administering the agonist anti-huCD40 antibody, or first administering the agonist anti-huCD40 antibody and then the anti-CTLA-4 antibody. Additionally or alternatively, an anti-PD-1 antibody and an agonist anti-huCD40 antibody can be administered sequentially, such as first administering the anti-PD-1 antibody and then administering the agonist anti-huCD40 antibody; or first administering the agonist anti-huCD40 antibody and then the anti-PD-1 antibody. Additionally or alternatively, the anti-PD-L1 antibody and the agonist anti-huCD40 antibody can be administered sequentially, e.g., the anti-PD-L1 antibody is administered first, followed by the agonist anti-huCD40 antibody, or the agonist anti-huCD40 antibody is administered first, followed by the anti-PD-L1 antibody. Additionally or alternatively, the anti-LAG-3 antibody and the agonist anti-huCD40 antibody can be administered sequentially, e.g., the anti-LAG-3 antibody is administered first, followed by the agonist anti-huCD40 antibody, or the agonist anti-huCD40 antibody is administered first, followed by the anti-LAG-3 antibody.

[0241] In addition, if more than one dose of the combination therapy is administered sequentially, the order of sequential administration may be reversed or remain the same at each administration time point, sequential administration may be combined with simultaneous administration, or any combination thereof. For example, the first administration of the combination of an anti-CTLA-4 antibody and an agonist anti-huCD40 antibody may be performed simultaneously, the second administration may be sequentially anti-CTLA-4 antibody first and then agonist anti-huCD40 antibody, the third administration may be sequentially agonist anti-huCD40 antibody first and then anti-CTLA-4 antibody, and so on. Additionally or alternatively, the first administration of the combination of an anti-PD-1 antibody and an agonist anti-uCD40 antibody may be performed simultaneously, the second administration may be sequentially anti-PD-1 antibody first and then agonist anti-huCD40 antibody, and the third administration may be sequentially agonist anti-huCD40 antibody first and then anti-PD-1 antibody, and so on. Additionally or alternatively, the first administration of the combination of anti-PD-L1 antibody and agonist anti-huCD40 antibody may be performed simultaneously, the second administration may be sequentially anti-PD-L1 antibody first and then agonist anti-huCD40 antibody, and the third administration may be sequentially agonist anti-huCD40 antibody first and then anti-PD-L1 antibody, etc. Additionally or alternatively, the first administration of the combination of anti-LAG-3 antibody and agonist anti-huCD40 antibody may be performed simultaneously, the second administration may be sequentially anti-LAG-3 antibody first and then agonist anti-huCD40 antibody, and the third administration may be sequentially agonist anti-huCD40 antibody first and then anti-LAG-3 antibody, etc. Another representative dosing regimen may involve a first administration in the order of agonist anti-huCD40 first and then anti-CTLA-4 antibody (and / or anti-PD-1 antibody and / or anti-PD-L1 antibody and / or anti-LAG-3 antibody), and subsequent administrations may be simultaneous. Optionally, agonist anti-huCD40 as the sole immunotherapeutic agent, or a combination of agonist anti-huCD40 antibodies 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 an immunogenic agent, 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).CD40 agonists 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, CD40 agonists 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 chemotherapy regimens. In these cases, it is possible to reduce the dose of other chemotherapeutic agents administered with the combination of the present disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of a CD40 agonist antibody 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 combined with dacarbazine for the treatment of melanoma. Another example is a combination of an agonist anti-huCD40 antibody with or without an anti-CTLA-4 antibody and / or an anti-PD-1 antibody and / or an anti-PD-L1 antibody and / or an anti-LAG-3 antibody, further combined 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 can result in synergy through cell death with CD40 agonists 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 regimens produces a source of tumor antigens in the host. Angiogenesis inhibitors may also be combined with a combined CD40 agonist and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocker. Inhibition of angiogenesis leads to tumor cell death, which may be a source of tumor antigens that enter the host antigen presentation pathways.

[0242] Agonist anti-huCD40 antibodies as the sole immunotherapeutic, or a combination of a CD40 agonist and a CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking antibody can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells. See, for example, U.S. Pat. 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 can be enhanced by using a combined CD40 agonist and a CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking agent.

[0243] In another example, an agonistic anti-CD40 antibody as the sole immunotherapeutic agent or a combination of an anti-CD40 antibody and an additional immunostimulatory agent (e.g., an anti-CTLA-4 antibody and / or an anti-PD-1 antibody and / or an anti-PD-L1 antibody and / or a LAG-3 agent (e.g., an antibody)) can be administered in combination with an anti-tumor antibody (e.g., (rituximab), (Trastuzumab), (tositumomab), (ibritumomab tiuxetan), (Alemtuzumab), (eprtuzumab), (bevacizumab) and (erlotinib), etc.). For example, and not wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can result in cancer cell (e.g., tumor cell) death, which will enhance the immune response mediated by an immunostimulatory agent (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) may include an anti-cancer agent (e.g., an antibody) in combination with an agonist anti-huCD40 antibody and optionally an additional immunostimulatory agent (e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 agent, such as an antibody) that is capable of enhancing the host's anti-tumor immune response, which is administered simultaneously or sequentially or in any combination thereof.

[0244] Provided herein are methods for altering adverse events associated with treatment of hyperproliferative diseases (e.g., cancer) using immunostimulants, comprising administering to a subject an agonist anti-huCD40 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 methods for reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. As used herein, "non-absorbable steroids" are glucocorticoids that exhibit 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 locally acting glucocorticoid that is extensively metabolized primarily by the liver after oral administration. (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon. It is approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and / or ascending colon. The usual oral dose for the treatment of Crohn's disease is 6 to 9 mg / day. It is released in the intestine before being absorbed and retained in the intestinal mucosa. Once it passes through the intestinal mucosal target tissue, Budesonide is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (about 10%). The low bioavailability of budesonide leads to an improved therapeutic rate compared to other glucocorticoids with a smaller extent of first-pass metabolism. Compared with systemically acting corticosteroids, budesonide produces fewer side effects, including less hypothalamic-pituitary suppression. However, long-term use Leads to systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR 58th edition 2004; 608-610.

[0245] In additional embodiments, CD40 agonists (i.e., immunostimulatory therapeutic antibodies directed against CD40 and optionally anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies) with or without CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockers in combination with non-absorbable steroids may be further combined with salicylates. Salicylates include 5-ASA agents, such as sulfasalazine ( Pharmacia & UpJohn); Osalazine ( Pharmacia & UpJohn); balsalazide ( Salix Pharmaceuticals, Inc.); and mesalamine ( Procter & Gamble Pharmaceuticals; Shire US; Axcan Scandipharm, Inc.; Solvay).

[0246] According to the methods described herein, salicylate is administered in combination with an agonist anti-huCD40 antibody with or without an anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or LAG-3 antibody and a non-absorbable steroid, and any overlapping or sequential administration of salicylate and non-absorbable steroids may be included for the purpose of reducing the incidence of colitis induced by the immunostimulatory antibodies. Thus, for example, a method for reducing the incidence of colitis induced by the immunostimulatory antibodies described herein comprises administering salicylate and non-absorbable steroids simultaneously or sequentially (e.g., administering salicylate 6 hours after non-absorbable steroids) or a combination thereof. In addition, salicylate and non-absorbable steroids may be administered by the same route (e.g., both are administered orally) or by a different route (e.g., salicylate is administered orally and non-absorbable steroids are administered rectally) that is different from the route used to administer anti-huCD40 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies.

[0247] The agonist anti-huCD40 antibodies and combination antibody therapies described herein can also be used in combination with other well-known therapies selected for their specific use for the indication being treated (e.g., cancer). The combinations of agonist anti-huCD40 antibodies described herein can be used sequentially with one or more known pharmaceutically acceptable agents.

[0248] For example, the agonist anti-huCD40 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 combo), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-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 (apoptotic protein antagonists (e.g., smac7, smac, small molecule smac mimetics, synthetic smac peptides (see Fulda et al., Nat Med 2002; 8: 808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., ), synthetic triterpenoids (see Hyer et al., Cancer Research 2005; 65: 4799-808), c-FLIP (cellular FLICE inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARγ (peroxisome proliferator-activated receptor γ)), 5809354 or 5569100), kinase inhibitors (e.g., sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors (e.g., rapamycin and temsirolimus), bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3β inhibitors, IAP inhibitors and / or genotoxic drugs.

[0249] The agonist anti-huCD40 antibodies and combination antibody therapies described herein can be further used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that can be used as anti-proliferative cytotoxic agents include, but are not limited to, the following:

[0250] Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard, chlormethine, cyclophosphamide (CYTOXANTM )fosfamide, melphalan, chlorambucil, pipobroman, triethylene melamine, triethylene thiophosphamide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, and temozolomide.

[0251] 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.

[0252] Suitable antiproliferative agents for use in combination with agonist anti-huCD40 antibodies include, but are not limited to, taxanes, paclitaxel (paclitaxel can be used as TAXOL TMcommercially available), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanyl epothilone D, deoxy epothilone B1,

[17] -dehydrodeoxy epothilone B,

[18] dehydrodeoxy epothilone B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethyl epothilone B, epothilone B10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584 , ZK-EPO, ABJ-789, XAA296A (soblidotin), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, maytansine immunoconjugate (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, ST A-5312, eleutherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discinolide, cryptothilone 1, in addition to other microtubule stabilizers known in the art.

[0253] Where it is desired to render abnormally proliferative cells quiescent in conjunction with or prior to treatment with an agonist anti-huCD40 antibody described herein, hormones and steroids (including synthetic analogs), such as 17a-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorethoxyquin, hydroxyprogesterone, aminoglutethimide, estramustine, methoxyprogesterone formate, leuprolide, flutamide, toremifene, ZOLADEX TM It can also be used for administration to patients. When using the methods or compositions described herein, other agents used in clinical settings to regulate tumor growth or metastasis, such as anti-mimetic drugs, can also be administered as needed.

[0254] Methods for safely and effectively administering chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in Physicians' Desk Reference (PDR), such as the 1996 edition (Medical Economics Company, Montvale, NJ07645-1742, USA), the disclosure of which is incorporated herein by reference.

[0255] One or more chemotherapeutic agents and / or radiotherapy can be administered according to treatment regimens 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 radiotherapy can vary according to the disease being treated and the known effects of one or more chemotherapeutic agents and / or radiotherapy. Also, according to the knowledge of a skilled clinician, the treatment regimen (e.g., the dosage and time of administration) can be changed in view of the observed effects of the administered therapeutic agent on the patient and in view of the observed response of the disease to the administered therapeutic agent.

[0256] X. Specific agonist anti-CD40 antibodies

[0257] The agonist anti-CD40 antibodies of the present invention with improved humanized heavy and light chain variable region sequences are derived from the anti-CD40 antibodies described in WO 2017 / 004006. The variable domain and CDR sequence regions of the exemplary antibodies described in WO 2017 / 004006 are provided in the sequence table and summarized in Table 2. For all antibodies derived from the same original clone, the variable domain and CDR region numbering of the improved anti-CD40 antibodies of the present invention is the same, that is, the humanized variants provided herein do not include any insertions or deletions, except for the improved modified mAb12D6 light chain variable region sequences provided in SEQ ID NOs: 47-51 and the heavy chain variable region sequences provided in SEQ ID NOs: 52-54.

[0258] Table 2

[0259] Antibody variable domains and CDRs

[0260]

[0261] The present 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. Example

[0262] Example 1

[0263] Characterization of a humanized monoclonal antibody against human CD40

[0264] The agonist anti-CD40 antibodies of the present invention, such as antibody 12D6-24, are produced by transient transfection in HEK293 cells. The yield and percentage of monomers are determined to assess their feasibility for development into therapeutic proteins. TM The antibody 12D6-24 (comprising heavy and light chain variable regions consisting of residues 1-119 of SEQ ID NO: 11 and residues 1-112 of SEQ ID NO: 8, respectively) had a titer of 18 mg / L after 5 days in the protein expression system (Thermo Fisher Scientific, Waltham Mass., USA), with only 67% of the material in monomeric form. Other similar antibodies are typically expressed at 50-100 mg / L and >95% monomeric. Low yield and tendency to aggregate are undesirable properties of therapeutic antibodies.

[0265] It is assumed that the light chain that causes chain imbalance is poorly expressed, causing the formation of heavy chain dimers and high molecular weight species. Further experiments show that increasing the ratio of DNA encoding light chain to DNA encoding heavy chain improves yield and monomer fraction. See Table 3.

[0266] Table 3

[0267] Properties of Unmodified 12D6-24

[0268]

[0269] These results confirm the hypothesis that proper chain balancing is critical to obtain reasonable yields and high percentages of monomers (reduced aggregation) of mAb 12D6-24.

[0270] Example 2

[0271] Modified heavy and light chain variable regions for humanized anti-huCD40 antibody 12D6-24

[0272] In view of the poor clinical manufacturing characteristics identified in Example 1, including low expression levels and aggregation tendency, both the heavy and light chain variable domains of the agonist anti-huCD40 mAb 12D6-24 were reengineered to generate new light chain variable region sequences L2 (SEQ ID NO: 47), L3 (SEQ ID NO: 48), L4 (SEQ ID NO: 49) and L5 (SEQ ID NO: 50) and new heavy chain variable region sequences H2 (SEQ ID NO: 52) and H3 (SEQ ID NO: 53).

[0273] To prepare an expression vector for human IgG light chain, DNA encoding the light variable domain was genetically fused to DNA encoding human kappa constant region in an expression vector under the control of a CMV promoter and a human IgG secretory leader sequence. Similarly, to prepare an expression vector for human IgG heavy chain, DNA encoding the heavy variable domain was genetically fused to DNA encoding human CH1, hinge, and human IgG1 Fc in an expression vector under the control of a CMV promoter and a human IgG secretory leader sequence.

[0274] To prepare unique antibodies, expression vectors for each of the unique light chains were co-transfected with each of the unique heavy chains into HEK293 cells. After 5 days of incubation, the concentration of each antibody in the culture medium was measured, and the unique antibodies were purified by MAB Select chromatography. After purification, the aggregation of the antibodies was assessed by size exclusion chromatography, and the binding affinity of the antibodies was assessed by ELISA. See Table 4.

[0275] Table 4

[0276] Improved properties of the 12D6-24 antibody

[0277]

[0278] Additional heavy chain (H4) and light chain (L6) variable regions were designed by reverting some of the framework sequences to germline, as shown in SEQ ID NOs: 54 and 51, respectively. Expression vectors were prepared and evaluated for expression, aggregation potential, and antigen binding. See Table 5.

[0279] Table 5

[0280] Additional Improved Properties of the 12D6-24 Antibody

[0281]

[0282] Combining L2 or L3 with H1, H2, or H3 resulted in increased expression and purification yield, but the resulting antibodies lost detectable binding to human CD40. L5 paired with H1, H2, or H3 reduced expression and binding of the resulting antibodies. Combining L6 with H1, H2, or H4 resulted in significantly lower expression and low purification yields, so these antibodies were not analyzed further. In contrast, pairing H4 with L1 or L4 resulted in higher expression and higher monomer percentages after protein A purification.

[0283] Taking all the data into account, L4 improved the manufacturability of the antibody when paired with H1, H2 or H4. In addition, when comparing the contribution of the heavy chains, it was found that heavy chain 4 resulted in significantly more robust expression than the parent antibody and significantly reduced the tendency to aggregate.

[0284] Further experiments were performed on the most promising antibodies comprising the improved heavy and light chain variable region sequences of the invention. See Table 6. L4H1 comprises the light chain variable region and heavy chain variable region of SEQ ID NO: 49 and residues 1-119 of SEQ ID NO: 11, respectively. L4H2 comprises the light chain and heavy chain variable regions of SEQ ID NO: 49 and SEQ ID NO: 52, respectively. L4H4 comprises the light chain and heavy chain variable regions of SEQ ID NO: 49 and SEQ ID NO: 54, respectively. The antibodies (including the unmodified mAb 12D6-24) were otherwise identical.

[0285] Table 6

[0286] Properties of anti-CD40 antibodies with improved heavy and light chain variable regions

[0287]

[0288] It is evident that the improved forms of antibody 12D6, particularly L4H1 and L4H4, exhibited improved yields and greatly increased monomer percentages (reduced aggregation).

[0289] ELISA was performed to confirm that the improved antibodies retained affinity for CD40. See Table 6. All of these improved anti-CD40 antibodies retained affinity to the original mAb 12D6-24 (K d =0.24 μM) with similar binding.

[0290] Example 3

[0291] Biological activity of anti-CD40 antibodies with improved heavy and light chain variable regions

[0292] Additional experiments were performed to evaluate the biological activity of the improved anti-CD40 (mAb 12D6) antibodies of the present invention. In the first experiment, human monocytes (CD14 + ). Monocytes were cultured with 100 ng / mL GM-CSF (Miltenyi Biotec) and 100 ng / mL IL-4 (Miltenyi Biotec). Half of the culture medium was removed and replenished on days 2 and 5. Immature dendritic cells were harvested on days 6-7. DCs were incubated with the indicated concentrations of antibodies overnight at 37°C. Cell culture supernatants were collected and assayed for human TNF-α production. See Figure 1 .

[0293] Growth medium alone (AIMV) and control human IgG1 antibody did not induce TNF-α production. The middle curve represents unmodified mAb 12D6-24 and improved version of L4H4, while the upper curve represents improved version of L4H1 (performed using DC from two donors 6P1 and 6P2). Clearly, the anti-CD40 antibodies of the present invention with improved heavy and / or light chain variable regions retain similar biological activity to unmodified mAb 12D6-24 in this assay.

[0294] A second experiment was performed to measure the ability of the improved agonist anti-CD40 antibodies of the invention to activate dendritic cells, as measured by the induction of cell surface markers CD83 and CD86. Immature dendritic cells (isolated as described in the previous paragraph) were measured in vitro by plating the cells in a 96-well plate, adding the indicated antibodies, and then incubating overnight at 37°C. The cells were then harvested and stained with fluorescent anti-CD83 antibodies and fluorescent anti-CD83 antibodies, in each case detected by fluorescence activated cell sorting (FACS). The results are expressed as mean fluorescence intensity (MFI). The fluorescent anti-CD83 antibody used for FACS was an excitation wavelength of 647 nm. 647 anti-human CD83 antibody, fluorescent anti-CD86 antibody is excited at 488nm 488 anti-human CD86 antibody (both from BioLegend, San Diego Calif., USA). Figure 2A Data obtained with excitation at 647 nM are presented, thus showing predominantly CD83 staining (light grey bars), whereas Figure 2B Data obtained with excitation at 647 nM are presented, thus showing predominant staining for CD86 (dark grey bars). Control experiments were performed in the absence of staining antibody (black bars).

[0295] The data demonstrate that the improved forms (L4H1 and L4H4) of the agonist anti-CD40 mAb 12D6 of the present invention are at least as effective as the unmodified mAb 12D6 in inducing CD83 and CD86 expression on the surface of immature dendritic cells. Because antibodies L4H1 and L4H4 retain CD40 binding affinity and biological activity, and still show improved yield and purity, they represent excellent candidates for clinical development.

[0296] Table 7

[0297] SEQUENCE LISTING SUMMARY

[0298]

[0299]

[0300] The sequence listing provides the sequences of mature heavy and light chains (i.e., the sequences do not include the signal peptide). A signal sequence for producing the antibodies of the invention, e.g., in human cells, is provided in SEQ ID NO:46. As used herein, "residues 1-119 of SEQ ID NO:54" is synonymous with "SEQ ID NO:54" because SEQ ID NO:54 consists of 119 residues. Residue numbers are sometimes included for SEQ ID NO:54 simply to facilitate referring to the variable region sequence and the full-length heavy or light chain sequence in the same phrase or claim element.

[0301] Equivalent:

[0302] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments disclosed herein. Such equivalents are intended to be encompassed by the appended claims. Sequence Listing <110> Bristol-Myers Squibb Cytomex Therapeutics Inc. <120> Anti-CD40 antibodies and uses thereof <130> 3338.112PC01 / ELE / PAC / DKC <150> US 62 / 610,642 <151> 2017-12-27 <160> 54 <170> PatentIn Version 3.5 <210> 1 <211> 277 <212> PRT <213> Homo sapiens <220> <221> Signal <222> (1)..(20) <220> <221> Peptides <222> (21)..(277) <220> <221> domain <222> (21)..(193) <223> Extracellular domain <220> <221> domain <222> (194)..(215) <223> Transmembrane domain <220> <221> Domain <222> (216)..(277) <223> Intracellular domain <400> 1 Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser 260 265 270 Val Gln Glu Arg Gln 275 <210> 2 <211> 261 <212> PRT <213> Homo sapiens <400> 2 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 3 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(119) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_feature <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(108) <223> CDRH3 <400> 3 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 4 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 4 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Asn Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(119) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(108) <223> CDRH3 <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 6 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 6 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 7 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(119) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(108) <223> CDRH3 <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 8 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_feature <222> (94)..(102) <223> CDRL3 <400> 8 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 9 <211> 448 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(119) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(108) <223> CDRH3 <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 10 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 10 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 11 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(119) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(108) <223> CDRH3 <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 12 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(111) <223> Variable fields <220> <221> MISC_Features <222> (24)..(38) <223> CDRL1 <220> <221> MISC_Features <222> (54)..(60) <223> CDRL2 <220> <221> MISC_Features <222> (93)..(101) <223> CDRL3 <400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Asn Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Gly Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 13 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(117) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(106) <223> CDRH3 <400> 13 Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Leu 20 25 30 Ser Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Thr Pro Ser Ser Gly Tyr Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Ile Leu Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 14 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(117) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(106) <223> CDRH3 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Leu 20 25 30 Ser Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Thr Pro Ser Ser Gly Tyr Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Ile Leu Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 15 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(111) <223> Variable fields <220> <221> MISC_Features <222> (24)..(38) <223> CDRL1 <220> <221> MISC_Features <222> (54)..(60) <223> CDRL2 <220> <221> MISC_Features <222> (93)..(101) <223> CDRL3 <400> 15 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Asn Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 16 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(117) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(106) <223> CDRH3 <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Leu 20 25 30 Ser Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Thr Pro Ser Ser Gly Tyr Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Ile Leu Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 17 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(111) <223> Variable fields <220> <221> MISC_Features <222> (24)..(38) <223> CDRL1 <220> <221> MISC_Features <222> (54)..(60) <223> CDRL2 <220> <221> MISC_Features <222> (93)..(101) <223> CDRL3 <400> 17 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Asn Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 18 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(117) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(106) <223> CDRH3 <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Thr Pro Ser Ser Gly Tyr Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Ile Leu Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 19 <211> 218 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(111) <223> Variable fields <220> <221> MISC_Features <222> (24)..(38) <223> CDRL1 <220> <221> MISC_Features <222> (54)..(60) <223> CDRL2 <220> <221> MISC_feature <222> (93)..(101) <223> CDRL3 <400> 19 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Asn Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95 Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 20 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(111) <223> CDRH3 <400> 20 Gln Val Gln Phe Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Ser Ser Ile Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Asp Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 21 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> twenty one Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(111) <223> CDRH3 <400> twenty two Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Asp Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> twenty three <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> twenty three Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> twenty four <211> 451 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(111) <223> CDRH3 <400> twenty four Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 25 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_feature <222> (55)..(61) <223> CDRL2 <220> <221> MISC_feature <222> (94)..(102) <223> CDRL3 <400> 25 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 26 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(111) <223> CDRH3 <400> 26 Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 27 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 27 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 28 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(111) <223> CDRH3 <400> 28 Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Ser Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 29 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 29 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 30 <211> 451 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(122) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(111) <223> CDRH3 <400> 30 Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asp Ser Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Leu Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser Leu Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 <210> 31 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_feature <222> (55)..(61) <223> CDRL2 <220> <221> MISC_feature <222> (94)..(102) <223> CDRL3 <400> 31 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg 20 25 30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 32 <211> 442 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(113) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(102) <223> CDRH3 <400> 32 Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Lys Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Lys Asn Gly Gly Thr Ile Tyr Asn Leu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Met Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 33 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(107) <223> Variable fields <220> <221> MISC_Features <222> (24)..(34) <223> CDRL1 <220> <221> MISC_Features <222> (50)..(56) <223> CDRL2 <220> <221> MISC_Features <222> (89)..(97) <223> CDRL3 <400> 33 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Ile Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 34 <211> 442 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> Region <222> (1)..(113) <223> Variable region <220> <221> MISC_feature <222> (31)..(35) <223> CDRH1 <220> <221> MISC_feature <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(102) <223> CDRH3 <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asn Pro Lys Asn Gly Gly Thr Ile Tyr Asn Leu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 35 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(107) <223> Variable fields <220> <221> MISC_Features <222> (24)..(34) <223> CDRL1 <220> <221> MISC_Features <222> (50)..(56) <223> CDRL2 <220> <221> MISC_Features <222> (89)..(97) <223> CDRL3 <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ile Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 36 <211> 442 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(113) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(102) <223> CDRH3 <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asn Pro Lys Asn Ala Gly Thr Ile Tyr Asn Leu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 37 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(107) <223> Variable fields <220> <221> MISC_Features <222> (24)..(34) <223> CDRL1 <220> <221> MISC_Features <222> (50)..(56) <223> CDRL2 <220> <221> MISC_Features <222> (89)..(97) <223> CDRL3 <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ile Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 38 <211> 441 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(101) <223> CDRH3 <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ser Arg Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 39 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Chimeric antibodies with mouse variable domains and human constant domains <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_feature <222> (55)..(61) <223> CDRL2 <220> <221> MISC_feature <222> (94)..(102) <223> CDRL3 <400> 39 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 40 <211> 441 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_feature <222> (99)..(101) <223> CDRH3 <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Arg Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 41 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 41 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 42 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (31)..(35) <223> CDRH1 <220> <221> MISC_Features <222> (50)..(66) <223> CDRH2 <220> <221> MISC_Features <222> (99)..(101) <223> CDRH3 <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Arg Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 43 <211> 219 <212> PRT <213> Artificial sequence <220> <223> Humanized antibodies with mouse CDRs and human framework and constant regions <220> <221> domain <222> (1)..(112) <223> Variable fields <220> <221> MISC_Features <222> (24)..(39) <223> CDRL1 <220> <221> MISC_Features <222> (55)..(61) <223> CDRL2 <220> <221> MISC_Features <222> (94)..(102) <223> CDRL3 <400> 43 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Gln Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 44 <211> 329 <212> PRT <213> Homo sapiens <400> 44 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 45 <211> 107 <212> PRT <213> Homo sapiens <400> 45 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 46 <211> 17 <212> PRT <213> Homo sapiens <400> 46 Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala <210> 47 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 47 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Lys Leu Thr Asn Arg Phe Phe Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ile His Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 48 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 48 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu 65 70 75 80 Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ile His Val Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 49 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 50 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 50 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ile 85 90 95 His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 51 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 51 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Ala Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Leu Thr Asn Arg Phe Phe Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser 85 90 95 Ile His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 52 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse antibody variable region <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 53 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized Mouse Antibody Variable Region <400> 53 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 54 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized Mouse Antibody Variable Region <400> 54 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Thr Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115

Claims

1. An isolated antibody or antigen-binding portion thereof that specifically binds to human CD40, comprising: a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: (i) residues 1-119 of SEQ ID NO: 11, (ii) SEQ ID NO: 54 and (iii) SEQ ID NO: 52; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

49.

2. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein: a) the heavy chain variable region comprises the amino acid sequence of residues 1-119 of SEQ ID NO: 11; and b) The light chain variable region comprises the amino acid sequence of SEQ ID NO:

49.

3. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein: a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54; and b) The light chain variable region comprises the amino acid sequence of SEQ ID NO:

49.

4. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein: a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 52; and b) The light chain variable region comprises the amino acid sequence of SEQ ID NO:

49.

5. A nucleic acid encoding the heavy chain variable region and light chain variable region of the antibody or antigen binding portion thereof according to any one of claims 1 to 4. An expression vector comprising the nucleic acid according to claim 5 .

7. A cell transformed with (i) the expression vector of claim 6 or (ii) an expression vector comprising a nucleic acid encoding the heavy chain variable region of the antibody or antigen binding portion thereof of any one of claims 1-4 and an expression vector comprising a nucleic acid encoding the light chain variable region of the antibody or antigen binding portion thereof of any one of claims 1-4.

8. A method for preparing an anti-human CD40 antibody or an antigen-binding portion thereof, comprising: a) expressing the antibody or antigen-binding portion thereof in the cell of claim 7; and b) isolating the antibody or antigen-binding portion thereof from the cell.

9. A pharmaceutical composition comprising: a) the antibody or antigen-binding portion thereof of any one of claims 1 to 4; and b) Vector.

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