Antibodies to human B7-H3 and their variants

Abstract

The present application provides antibodies or antigen-binding fragments thereof, such as monoclonal antibodies (mAbs), that specifically recognize B7-H3. The present application further provides pharmaceutical compositions, or methods for making and using the antibodies or antigen-binding fragments thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to PCT Application No. PCT / CN2022 / 078008, filed February 25, 2022, the entire contents of which are incorporated herein by reference for all purposes. [Technical field]

[0002] The present application relates to antibodies or antigen-binding fragments thereof capable of specifically binding to B7-H3 protein and uses of such agents. These antibodies or antigen-binding fragments thereof may be used in the treatment of diseases associated with B7-H3 activity and / or expression. [Background technology]

[0003] B7-H3 is a single-pass type I transmembrane protein discovered in 2001 and belongs to the B7 family [Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains. J Immunol 172(4):2352-2359]. It is widely expressed in various cancers, including non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), breast cancer, and other cancers, but is low or undetectable in normal tissues [B7-H3: an attractive target for antibody-based immunotherapy. Clin Cancer Res]. In addition, B7-H3 has already been found in solid tumors with low or negative PD-L1 expression. More than 20 clinical trials have already been conducted against B7-H3, including monoclonal antibodies (Mabs), bispecific antibodies (BsAbs), antibody-drug conjugates (ADCs), chimeric antigen receptor (CAR)-T and radioimmunotherapy (RIT).

[0004] Two ADCs against B7-H3, MGC018 (NCT03729596) and DS-7300a (NCT04145622), have been tested in phase I / II clinical studies. Although there are ADCs targeting B7-H3, it is still unclear whether they will cause life-threatening off-tumor on-target toxicity due to the low expression of B7-H3 in normal cells [New B7 family checkpoints in human cancers. Mol Cancer Ther 16(7):1203-1211]. RIT (iodine-131 tagged omburtamab), which targets B7-H3, has been approved as a breakthrough therapy, but to date its indications have mostly been limited to brain tumors.

[0005] MGA271 (Enoblituzumab) and DS-5573a are two antibodies targeting B7-H3. MGA271 (Enoblituzumab, Macrogenics) is an Fc-optimized humanized IgG1 antibody, and its safety profile and efficacy have been tested in three clinical studies (NCT01391143, NCT02923180, NCT02982941), and it has the effect of B7-H3-dependent antibody-dependent cellular cytotoxicity (ADCC). DS-5573a (Daiichi Sankyo Inc) is a humanized anti-B7-H3 IgG1 [Development of DS-5573a: a novel afucosylated mAb directed at B7-H3 with potent antitumor activity. Cancer Sci 107(5):674-681]. The clinical trial of DS-5573a is an open-label Phase I study (NCT021925679) that began in patients with advanced solid tumors in 2014 and was closed for commercial decision in 2017. DS-5573a relies on ADCC and complement-dependent cytotoxicity (CDC) activity to kill tumor cells.

[0006] Avelumab (anti-PD-L1), trastuzumab (anti-HER2) and cetuximab (anti-EGFR) are monoclonal antibodies that mainly rely on ADCC to kill cells. These approved drugs support the therapeutic potential of ADCC-related drugs in solid tumor treatment. Also, according to the clinical results of NCT02923180 and NCT02982941, MGA271 (enoblitumbap) showed remission in patients suffering from recurrent or refractory solid tumors expressing B7-H3.

[0007] There is a need to develop antibodies that target B7-H3, which can kill tumors such as NSCLC and HNSCC that are dependent on ADCC and CDC, and have superior efficacy to the positive controls MGA271 (enoblituzumab) and DS-5573a. Summary of the Invention

[0008] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: I) 1) a heavy chain determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, and 74; 2) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75; 3) a heavy chain variable domain (VH) comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76; II) 1) a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, and 78; 2) a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79; and 3) a light chain variable domain (VL) comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80; wherein said antibody or antigen-binding fragment thereof is capable of specifically binding to B7-H3, preferably human B7-H3.

[0009] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: 1) the VH comprises heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the VL comprises light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively; 2) the VH comprises heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and the VL comprises light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; and 3) the VH comprises heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively, and the VL comprises light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 2) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 30, 31 and 32, respectively; 5) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively; 6) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: and the VL comprises a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 42, 43 and 44, respectively, and the VL comprises a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; 7) the VH comprises a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and the VL comprises a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 54, 55 and 56, respectively; and 8) the VH comprises a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs:and the VL comprises a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 62, 63 and 64, respectively; 9) the VH comprises a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively, and the VL comprises a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 70, 71 and 72, respectively; or 10) the VH comprises a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively, and the VL comprises a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: It comprises light chain CDR1, CDR2 and CDR3 having amino acid sequences of 78, 79 and 80, respectively.

[0010] In some embodiments, the VH comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, and 73, and the VL comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, and 77, respectively.

[0011] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, and 73, or the VH includes at most about three variants thereof with amino acid substitutions, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, and 77, or the VL includes at most about three variants thereof with amino acid substitutions.

[0012] In some embodiments, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of: 1) the VH comprises the amino acid sequence of SEQ ID NO:1, and the VL comprises the amino acid sequence of SEQ ID NO:5; 2) the VH comprises the amino acid sequence of SEQ ID NO:9, and the VL comprises the amino acid sequence of SEQ ID NO:13; 3) the VH comprises the amino acid sequence of SEQ ID NO:17, and the VL comprises the amino acid sequence of SEQ ID NO:21; 4) the VH comprises the amino acid sequence of SEQ ID NO:25, and the VL comprises the amino acid sequence of SEQ ID NO:29; 5) the VH comprises the amino acid sequence of SEQ ID NO:33, and the VL comprises the amino acid sequence of SEQ ID NO:37; and 6) the VH comprises the amino acid sequence of SEQ ID NO:41, and the VL comprises the amino acid sequence of SEQ ID NO:42. 4) the VH comprises the amino acid sequence of SEQ ID NO:49 and the VL comprises the amino acid sequence of SEQ ID NO:53; 8) the VH comprises the amino acid sequence of SEQ ID NO:57 and the VL comprises the amino acid sequence of SEQ ID NO:61; 9) the VH comprises the amino acid sequence of SEQ ID NO:65 and the VL comprises the amino acid sequence of SEQ ID NO:69; or 10) the VH comprises the amino acid sequence of SEQ ID NO:73 and the VL comprises the amino acid sequence of SEQ ID NO:77.

[0013] In some embodiments, the EC of the binding of the isolated antibody or antigen-binding fragment thereof to a cell expressing B7-H3. 50 is 10 -7 M~about 10 -12 M, preferably about 10 -8 M~about 10 -12 M, more preferably about 10 -9 M~about 10 -12 It's M.

[0014] In some embodiments, the binding of the isolated antibody or antigen-binding fragment thereof to a cell expressing B7-H3 has a lower EC 50 has.

[0015] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0016] In some embodiments, the humanized antibody comprises a VH and a VL, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-91.

[0017] In some embodiments, the humanized antibody comprises: 1) a VH comprising the amino acid sequence of SEQ ID NO:81 and a VL comprising the amino acid sequence of SEQ ID NO:88; 2) a VH comprising the amino acid sequence of SEQ ID NO:82 and a VL comprising the amino acid sequence of SEQ ID NO:88; 3) a VH comprising the amino acid sequence of SEQ ID NO:83 and a VL comprising the amino acid sequence of SEQ ID NO:89; 4) a VH comprising the amino acid sequence of SEQ ID NO:84 and a VL comprising the amino acid sequence of SEQ ID NO:89; 5) a VH comprising the amino acid sequence of SEQ ID NO:85 and a VL comprising the amino acid sequence of SEQ ID NO:90; 6) a VH comprising the amino acid sequence of SEQ ID NO:86 and a VL comprising the amino acid sequence of SEQ ID NO:91; or 7) a VH comprising the amino acid sequence of SEQ ID NO:86 and a VL comprising the amino acid sequence of SEQ ID NO:91.

[0018] In some embodiments, the VH is fused to an immunoglobulin heavy chain constant region.

[0019] In some embodiments, the heavy chain constant region is from human IgG1.

[0020] In some embodiments, the heavy chain constant region comprises a modification that enhances the ADCC effect of the antibody.

[0021] In some embodiments, the heavy chain constant region comprises the mutations K214R, L235V, F243L, R292P, Y300L, D356E, L358M and P396L.

[0022] In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 93 or 94.

[0023] In some embodiments, the VL is fused to an immunoglobulin light chain constant region (CL).

[0024] In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO:92.

[0025] In some embodiments, the isolated antibody or antigen-binding fragment thereof is conjugated to a toxin or chemotherapeutic agent.

[0026] In some embodiments, the toxin is Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribotoxin, saporin, calicheamicin, or a botulinum toxin.

[0027] In some embodiments, the chemotherapeutic agent is monomethylauristatin E or a maytansinoid.

[0028] In another aspect, the disclosure provides a bispecific antibody, the bispecific antibody comprising an isolated antibody or antigen-binding fragment thereof and a second antibody portion.

[0029] In some embodiments, the second antibody portion is capable of specifically binding to an antigen other than B7-H3.

[0030] In some embodiments, the antigen other than B7-H3 is CTLA-4, PD-L1, TIM-3, or LAG-3.

[0031] In some embodiments, the second antibody portion is a Fab, a Fab', a (Fab') 2 , Fv, single chain Fv (scFv), scFv-scFv, minibody, diabody, sdAb or antibody mimetic.

[0032] In another aspect, the disclosure provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising said isolated antibody or antigen-binding fragment thereof, or said bispecific antibody, and a pharma- ceutical acceptable carrier agent.

[0033] In some embodiments, the pharmaceutical composition further comprises one or more other anti-cancer agents.

[0034] In some embodiments, the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

[0035] In another aspect, the disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of said isolated antibody or antigen-binding fragment thereof, said bispecific antibody, or said pharmaceutical composition.

[0036] In some embodiments, the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

[0037] In another aspect, the disclosure provides the use of said isolated antibody or antigen-binding fragment thereof, or said bispecific antibody in the manufacture of a medicament for treating cancer.

[0038] In some embodiments, the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

[0039] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof, a bispecific antibody, or a pharmaceutical composition for use in treating cancer in a subject in need thereof.

[0040] In some embodiments, the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

[0041] In some embodiments, the isolated antibody or antigen-binding fragment thereof, the bispecific antibody or the pharmaceutical composition is for systemic or local administration.

[0042] In some embodiments, the isolated antibody or antigen-binding fragment thereof, the bispecific antibody or pharmaceutical composition is for intravenous or intratumoral administration.

[0043] The antibodies according to the present invention are capable of killing tumors such as NSCLC and HNSCC in a manner dependent on ADCC and CDC, and have superior efficacy to the positive controls MGA271 (enoblituzumab) and DS-5573a. [Brief description of the drawings]

[0044] [Figure 1] 1 shows the results of mouse mAb FACS binding analysis. [Diagram 2] CDC induction analysis results of chimeric antibodies. [Diagram 3] 1 shows the results of ADCC induction analysis of chimeric antibodies. [Figure 4] 1 shows the results of FACS binding analysis of humanized antibodies. [Diagram 5] CDC induction analysis of humanized antibodies. [Figure 6-1] 1 shows the results of an ADCC induction analysis of humanized antibodies. [Figure 6-2] 1 shows the results of an ADCC induction analysis of humanized antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present disclosure provides anti-B7-H3 monoclonal antibodies and their applications. The present disclosure provides heavy chain variable domains (V) of mouse anti-B7-H3 monoclonal antibody clones 12C9F1B5, 38F11B2, 34A9C12, 37D11F5B8, 37F11A10, 43H11G12, 47F3F10B4, 47C7E3, 31H11B1, and 38D6A4. H ) and the light chain variable domain (V L The present disclosure further relates to the amino acid sequences of the humanized or post-translationally modified heavy chain variable domains (V H ) and the light chain variable domain (V L The present disclosure relates to methods for producing these anti-B7-H3 monoclonal antibodies.

[0046] The present disclosure provides chimeric anti-B7-H3 monoclonal antibodies by fusing the heavy and light chain variable domains of the disclosed clones with the constant region of human IgG1. The present disclosure provides chimeric anti-B7-H3 monoclonal antibodies by fusing the heavy chain variable domain (V H ) and the light chain variable domain (V L The present invention provides a humanized form of the disclosed anti-B7-H3 monoclonal antibody. The humanized anti-B7-H3 monoclonal antibody is generated by fusing the humanized variable domains of the heavy and light chains of the disclosed clones with the constant region of human IgG1.

[0047] I. Definition Unless specifically indicated to the contrary, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described below for purposes of illustration, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009), Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), DNA Cloning: A Practical Approach, Vols. I and II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., 1984), Nucleic Acid Hybridization (B. Hames & S. Higgins, ed., 1985), Transcription and Translation (B. Hames & S. Higgins, ed., 1984), Animal Cell Culture (R. Freshney, ed., 1986), Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0048] Please note that as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0049] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0050] Throughout the specification and the appended claims, unless the context requires otherwise, the words "comprise" and variations of "comprises" and "comprising" will be understood to include a stated integer or step or group of integers or steps but not to exclude any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the term "containing" or "including," or, as used herein, with the term "having."

[0051] As used herein, "consisting of" does not include any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the preceding terms "comprising," "containing," "including," and "having," when used herein in the context of an aspect or embodiment of this application, can be substituted with the terms "consisting of" or "consisting essentially of" to modify the scope of the disclosure.

[0052] As used herein, the conjunction "and / or" between multiple listed elements should be understood to cover both individual and combined options. For example, when two elements are connected via "and / or", the first option refers to the applicability of the first element in the absence of the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option refers to the combined applicability of both the first element and the second element. Any one of these options should be understood to be included in the meaning and therefore meet the requirements of the term "and / or" as used herein. The combined applicability of one or more of these options should also be understood to be included in the meaning and therefore meet the requirements of the term "and / or".

[0053] Unless otherwise stated, any numerical value (concentration or concentration range described herein) should be understood in each case to be modified by the term "about". Thus, numerical values ​​generally include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1 mg / mL to 10 mg / mL includes 0.9 mg / mL to 11 mg / mL. As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, and includes integers and fractions of values ​​within such ranges unless the context clearly indicates otherwise.

[0054] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. The difference between conformational and nonconformational epitopes is that the binding of the former but not the latter is lost in the presence of denaturing solvents.

[0055] As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired result (including a clinical result). For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms of a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or attenuating the progression of the disease, improving the condition of the disease, providing palliation (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving the quality of life, and / or prolonging survival. "Treatment" further covers reducing the pathological consequences of the disease. The methods of the present invention contemplate any one or more of these therapeutic aspects.

[0056] The term "effective amount" as used herein refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition or disease, such as improving, alleviating, ameliorating and / or delaying one or more symptoms thereof. For cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount refers to an amount sufficient to delay progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and preferably terminate, cancer cell invasion into surrounding organs; (iv) inhibit (i.e., slow to some extent, and preferably terminate) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and / or (vii) relieve to some extent one or more symptoms associated with cancer.

[0057] "Antibody," "antibody portion," or "antibody construct" is used in its broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, which may exhibit the desired antigen-binding activity.

[0058] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, and contain 10 antigen-binding sites, whereas IgA antibodies contain two to five basic four-chain units, which can polymerize to form multivalent aggregates that combine with the J chain. In the case of IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to the H chain through one covalent disulfide bond, whereas the two H chains are linked to each other through one or more disulfide bonds depending on the H chain isotype. Each H and L chain further has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V) at the N-terminus. H ), followed by three constant domains C H (for each chain in the α and γ chains) and four C H Each L chain has a variable domain (V) at the N-terminus, followed by a variable domain (V for μ and ε isotypes). L ) followed by a constant domain at its other end. L is V H Aligned with and C L is the first constant region of the heavy chain (C H 1). Certain amino acid residues are believed to form an interface between the light and heavy chain variable regions. H and V Lpair together to form a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and chapter 6. The L chain from any vertebrate species may be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. The heavy chain constant region (C H Depending on the amino acid sequence of the C-terminal tail, immunoglobulins can be designated as different classes or isotypes. There are five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which contain heavy chains designated as α, δ, ε, γ, and μ, respectively. H Depending on the relatively minor differences in sequence and function, gamma and alpha are further divided into subclasses; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0059] "Antibody having only heavy chains" or "HCAbs" refers to a functional antibody that contains a heavy chain but lacks the light chains normally found in four-chain antibodies. Camelids (e.g., camels, llamas, and alpacas) are known to produce HCAbs.

[0060] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). A single sdAb can bind to an antigen but does not pair with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid HCAbs and their heavy chain variable domains are referred to herein as "V H The heavy chain variable domain of a heavy chain antibody is called "H" (heavy chain variable domain of a heavy chain antibody). HH are also called nanobodies. Camelid sdAbs are among the smallest antigen-binding antibody fragments known (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). The basic V H H has the structure, from the N-terminus to the C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0061] An "isolated" antibody is an antibody that has been identified, isolated and / or recovered from a component (e.g., natural or recombinant) of its production environment. Preferably, an isolated polypeptide is free of association with all other components in its production environment. Contaminating components of its production environment, e.g., produced from recombinant transfected cells, are substances that typically interfere with research, diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is purified (1) to greater than 95% by weight of the antibody, and in some examples, greater than 99% by weight, as determined, e.g., by the Lowry method; (2) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to achieve homogeneity by SDS-PAGE under non-reducing or reducing conditions with Coomassie blue or, preferably, silver staining. An isolated antibody includes the antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will be absent. However, an isolated polypeptide, antibody or construct will ordinarily be prepared by at least one purification step.

[0062] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy and light chain variable domains are respectively referred to as "V H " and "V L These domains are usually the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding sites. Antibodies with only heavy chains in camelid species are called "V H The VH chain has a single heavy chain variable region, called H H is a special type of V H It is.

[0063] The term "variable" refers to the fact that the sequences of several segments of the variable domain in antibodies vary widely. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed over the entire span of the variable domain. Instead, it is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, which use a predominantly β-sheet configuration and are connected through the three CDRs to form loops that connect the β-sheet structure and in some cases form loops that connect parts of the β-sheet structure. The CDRs in each chain are tightly associated through the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0064] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogenous antibody population, i.e., each antibody forming the population is identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In stark contrast to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized in a hybridoma culture and are not contaminated by other immunoglobulins. "Monoclonal" indicates the character of the antibody obtained from a substantially homogenous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present application can be produced by a variety of techniques, including, for example, the hybridoma method (see, e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1997)). (1992), Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004), Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004), Fellouse, Proc. Nat'l. Acad. Sci.USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Nat'l. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93. (1995)).

[0065] The terms "full length antibody," "complete antibody," or "whole antibody" are used interchangeably to refer to an antibody in substantially its complete form, as opposed to an antibody fragment. In particular, full length four-chain antibodies include those antibodies having a heavy chain and a light chain, including an Fc region. Antibodies having only a full length heavy chain include those antibodies having only a heavy chain (e.g., V H H) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0066] An "antibody fragment" includes a portion of an intact antibody, preferably including the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments, diabodies, linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ), single chain antibody molecules, single domain antibodies (e.g., V H Examples of antibodies include, but are not limited to, multispecific antibodies consisting of an L chain and an antibody fragment. Papain digests antibodies to produce two identical antigen-binding fragments (called "Fab" fragments) and a residual "Fc" fragment, the name reflecting its ability to crystallize easily. The Fab fragment contains the entire L chain and the variable region domain of the H chain (V H ) and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin processes the antibody to produce a large single F(ab') fragment. 2 A fragment is obtained, which roughly corresponds to a Fab fragment linked by two disulfide bonds with different antigen binding activity and is still capable of cross-linking antigens. The difference between the Fab′ fragment and the Fab fragment is that H F(ab') has several additional residues at the carboxyl terminus of one domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. 2 Antibody fragments are initially produced as Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0067] The Fc fragment is the carboxyl-terminal portion of two H chains bound via disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) expressed on several types of cells.

[0068] The term "constant domain region" or "constant region" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin, i.e., the variable domain, which contains the antigen binding site. The constant domain is the C of the heavy chain. H 1. C H 2 and C H It contains three domains (collectively referred to as CH) and a light chain CHL (or CL) domain.

[0069] The "light chains" of the antibody (immunoglobulin) of any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of their constant domain.

[0070] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of the two domains produces six hypervariable loops (three each in the H and L chains), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs with specificity for an antigen) has the ability to recognize and bind to an antigen, although with a lower affinity than the entire binding site.

[0071] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is a VFv that is linked to a single polypeptide chain. H and V L An sFv polypeptide is an antibody fragment that contains an antibody domain. H and V LIt further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun, Vol. 113, The Pharmacology of Monoclonal Antibodies, Eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0072] A "functional fragment" of an antibody as described herein comprises a portion of a complete antibody, typically comprising the antigen-binding or variable region of the complete antibody, or the Fc region of the antibody that retains or has altered FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies made up of antibody fragments.

[0073] The term "diabody" refers to H Domain and V L This refers to mini-antibody fragments that are produced in such a way that sFv fragments (see previous paragraph) have a short linker (about 5-10 residues) between the V domains, thereby achieving interchain rather than intrachain pairing of the V domains, producing a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies H Domain and V L The domains are present on different polypeptide chains. Diabodies are described in detail, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Nat'l. Acad. Sci. USA 90: 6444-6448 (1993).

[0074] The monoclonal antibodies of the present specification specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984)). "Humanized antibodies" are used as a subset of "chimeric antibodies".

[0075] "Humanized" forms of non-human (e.g., llama or camel) antibodies are antibodies that contain minimal sequence from non-human immunoglobulin. In some examples, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient CDR (defined below) are replaced by residues from a CDR (donor antibody) from a non-human species (e.g., mouse, rat, rabbit, camel, llama, alpaca, or non-human primate) having the desired specificity, affinity, and capacity. In some cases, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further improve antibody performance, e.g., binding affinity. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR regions may contain one or more single FR residue substitutions which improve antibody performance, e.g., binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically is six or fewer in the H chain and three or fewer in the L chain. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0076] A "human antibody" is an antibody that has an amino acid sequence corresponding to an antibody produced from a human and / or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for producing human monoclonal antibodies are Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be produced by administering antigen to transgenic animals (e.g., immunized xenomice) that have been modified to produce such antibodies in response to antigen challenge but whose endogenous loci have been disabled (e.g., XENOMOUSE TM (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related techniques.) See also, e.g., human antibodies produced by human B cell hybridoma technology in Li et al., Proc. Nat'l. Acad. Sci. USA, 103:3557-3562 (2006).

[0077] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is highly variable in sequence and / or forms a loop that is structurally defined. Typically, a single domain antibody comprises three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2) and HVR3 (or CDR3). HVR3 (or CDR3) represents the greatest diversity of the three HVRs and is believed to play a unique role in conferring fine specificity to the antibody. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0078] The term "complementarity determining region" or "CDR" is intended to refer to hypervariable regions defined according to the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0079] Many HVR descriptions are in use and are covered herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis against available complex crystal structures. The individual residues in these HVRs are shown in Table 1 below.

[0080] [Table 1]

[0081] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable region residues are numbered according to Kabat et al., supra.

[0082] Single domain antibodies (e.g., V HThe amino acid residues of VH are numbered according to the general numbering system for VH domains proposed by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), e.g., in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195, for VH domains from the family Camelidae. H According to the numbering, V H FR1 of H contains amino acid residues 1 to 30, and V H CDR1 of H contains amino acid residues 31 to 35, and V H FR2 of H contains amino acid residues 36 to 49, and V H CDR2 of H contains amino acid residues 50 to 65, and V H FR3 of H contains amino acid residues 66 to 94, and V H CDR3 of H comprises amino acid residues 95 to 102, and H FR4 of H comprises amino acid residues 103 to 113. H Domain and V H It should be noted that, as is well known for H domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering).

[0083] The phrases "variable domain residue numbering, e.g., in Kabat" or "amino acid position numbering, e.g., in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody assembler of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a variable domain FR or HVR. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (based on residue 52a of Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., based on residues 82a, 82b and 82c, etc. of Kabat). The Kabat numbering of residues of a given antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbering sequence at the regions of homology.

[0084] In some embodiments, antibodies may also be analyzed using the IMGT information system, which was created in 1989 by Marie-Paule Lefranc and is currently used as a comprehensive reference for immunogenetics and immunoinformatics (see, e.g., Lefranc MP, The Immunologist 7: 132-136 (1999) and Lefranc MP et al., Nucleic Acids Res 27: 209-212 (1999)). The IMGT numbering scheme is summarized in Table 2 (see https: / / www.imgt.org / IMGTinformation / ).

[0085] [Table 2] (*) +0, 1 or 2 nucleotides before the heptamer.

[0086] Unless otherwise stated herein, the numbering of residues in immunoglobulin heavy chains is that of the EU index as set forth in Kabat et al., supra. The "EU index as set forth in Kabat" is the residue numbering of the human IgG1 EU antibody.

[0087] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0088] The "human consensus framework" or "receptor human framework" refers to the human immunoglobulin V L Or V H A framework that represents the most commonly occurring amino acid residues during the selection of framework sequences. Typically, human immunoglobulin V L Or V H The sequences are selected from a subgroup of variable domain sequences. Typically, the sequence subgroups are subgroups such as those in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). L For example, for VH, the subgroup may be subgroup kappa I, kappa II, kappa III, or kappa IV as described in Kabat et al., supra. For VH, the subgroup may be subgroup I, subgroup II, or subgroup III as described in Kabat et al. Alternatively, the human consensus framework may be derived from the specific residues described above, where the human framework residues are selected based on their homology with the donor framework, for example, by aligning the donor framework sequence with a collection of different human framework sequences. A recipient human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0089] An "affinity matured" antibody is an antibody with one or more modifications in one or more CDRs thereof that result in an improvement in the affinity of the antibody for an antigen, compared to a parent antibody lacking those modifications. In some embodiments, an affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are generated by methods known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity matured antibodies that are capable of binding to a target antigen. H -Domain and V L -Affinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues is described, for example, in Barbas et al. Proc Nat'l. Acad. Sci. USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0090] As used herein, the terms "specifically bind," "specifically recognize," or "have specificity for" refer to a measurable and reproducible interaction, e.g., binding, between a target and an antigen-binding protein (e.g., mAb), which determines the presence of the target in the presence of a heteromolecular (including biomolecule) population. For example, an antigen-binding protein (e.g., mAb) that specifically binds to a target (which may be an epitope) is an antigen-binding protein (e.g., mAb) that binds to the target with greater affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In some examples, the extent of binding of an antigen-binding protein (e.g., mAb) to an unrelated target is about 10% less than the binding of the antigen-binding protein (e.g., mAb) to the target, as measured, for example, by radioimmunoassay (RIA). In some examples, the dissociation constant (K D ) is ≦10 -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M or ≦10 -12 M. In some embodiments, the antigen binding protein specifically binds to an epitope in a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but is not limited to, exclusive binding.

[0091] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding protein (e.g., a mAb). For example, natural antibodies are monospecific. As used herein, the term "multispecificity" indicates that an antigen-binding protein has specificity for multiple epitopes (i.e., can specifically bind to two, three or more epitopes in one biomolecule or specifically bind to epitopes in two, three or more different biomolecules). As used herein, "bispecificity" indicates that an antigen-binding protein has two different antigen-binding specificities. Unless otherwise indicated, the order of antigens bound by a recited bispecific antibody is arbitrary. Thus, for example, the terms "anti-B7-H3 / PD-L1", "anti-PD-L1 / B7-H3", "B7-H3×PD-L1", "PD-L1×B7-H3", "PD-L1-B7-H3" and "B7-H3-PD-L1" are used interchangeably to refer to a bispecific antibody that specifically binds to both B7-H3 and PD-L1. As used herein, the term "monospecific" refers to an antigen-binding protein (e.g., mAb) that has one or more binding sites, each of which binds to the same epitope on the same antigen.

[0092] The term "valent" as used herein indicates that there is a designated number of binding sites on an antigen-binding protein. For example, a native or full-length antibody has two binding sites and is bivalent. Thus, "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that there are two, three, four, five, and six binding sites on an antigen-binding protein, respectively.

[0093] "Antibody effector function" refers to a biological activity attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) and which varies with the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. A "reduced or minimized" antibody effector function refers to an antibody effector function that is reduced by at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) compared to a wild-type or unmodified antibody. The determination of antibody effector function can be readily determined and measured by one of skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity, and antibody-dependent cellular cytotoxicity are affected. In some embodiments, effector function is eliminated by eliminating glycosylated mutations (e.g., "effector null mutations") in the constant region. In one embodiment, the effector null mutations are H In the two regions, it contains N297A or DANA mutations (D265A and / or N297A). Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively, other mutations that cause reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated by production techniques, such as expression in host cells (e.g., E. coli.) that are not glycosylated or that cause changes in glycosylation mode that are ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003)).

[0094] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binding to Fc receptors (FcR) present on some cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies are required to "arm" the cytotoxic cells and kill the target cells by this mechanism. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991) page 464. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as the assays described in U.S. Patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc. Nat'l. Acad. Sci. USA 95:652-656 (1998).

[0095] The term "Fc region" as used herein is intended to define the C-terminal region of an immunoglobulin heavy chain and includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined as the segment of the chain from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed during production or purification of the antibody or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies that contain and do not contain the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0096] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, with the primary distinction being in their intracellular domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its intracellular domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991), Capel et al., Immunomethods 4: 25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term "FcR" herein is intended to cover other FcRs, including those identified in the future.

[0097] The term "Fc receptor" or "FcR" further includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The in vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with mutant Fc regions. WO 2004 / 42072 (Presta) describes antibody mutants that improve or weaken binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0098] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of those patent publications are expressly incorporated herein by reference. See also Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0099] "Binding affinity" refers generally to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. Binding affinity is measured using the K D , K off , K on Or K a As used herein, "K off The term "dissociation rate constant" refers to the dissociation rate constant for an antibody (or antigen-binding domain) to dissociate from an antibody / antigen complex, as determined by a kinetic selection set, and is expressed in units of s -1 As used herein, "K on " refers to the association rate constant at which an antibody (or antigen-binding domain) associates with an antigen to form an antibody / antigen complex, and is expressed in units M -1 s -1 As used herein, the equilibrium dissociation constant "K D The term "dissociation constant" refers to the dissociation constant of a particular antibody-antigen interaction and describes the antigen concentration required to occupy half of all antibody binding domains present in a solution of antibody molecules at equilibrium, and K off / K on and is expressed in units of M. D Measurement of EC assumes that all binding agents are in solution. If the antibody is bound to the cell wall, for example in a yeast expression system, the corresponding equilibrium rate constant is EC 50 It is expressed as K D The affinity constant K a is the dissociation constant K D It is the reciprocal of and has the unit M -1 It is expressed as:

[0100] Dissociation constant (K D) is used as an index showing the affinity of an antibody to an antigen. For example, by using antibodies labeled with various labeling agents by the Scatchard method, analysis can be easily performed using BiacoreX (manufactured by Amersham Biosciences) or a similar set as a non-prescription measurement set (kit), following the manual and experimental operation method attached to the set. The K obtained by these methods can be easily analyzed using BiacoreX (manufactured by Amersham Biosciences) or a similar set as a non-prescription measurement set (kit), following the manual and experimental operation method attached to the set. D Values ​​are expressed in units of M (moles / liter). An antibody or antigen-binding fragment thereof that specifically binds to a target has, for example, a titer of ≦10 -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M or ≦10 -12 Dissociation constant of M (K D ).

[0101] The binding specificity of an antibody or an antigen-binding domain thereof may be determined experimentally by methods known in the art, including but not limited to Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore assays and peptide scanning.

[0102] Half-maximal inhibitory concentration (IC 50 IC ) is a measure of the effectiveness of a substance (e.g., an antibody) in inhibiting a particular biological or biochemical function. It indicates how much of a particular drug or other substance (an inhibitor such as an antibody) is needed to inhibit half of a given biological process (e.g., the binding of PD-L1 to B7-1, or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism). The value is typically expressed as a molar concentration. IC 50 is the EC value of an agonist drug or other substance (e.g., an antibody). 50 Equivalent to EC 50 Also represents the plasma concentration required to obtain 50% maximal effect in vivo. As used herein, "IC 50" is intended to indicate the effective concentration of an antibody (e.g., anti-PD-L1 mAb) required to neutralize 50% of the biological activity of an antigen (e.g., the biological activity of PD-L1) in vitro. IC 50 or EC 50 can be measured by biological assays, for example measuring inhibition of ligand binding by FACS analysis (competitive binding analysis), cell-based cytokine release assays or amplified luminescence proximity homogeneous assay (AlphaLISA).

[0103] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in a particular peptide or polypeptide sequence, aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes of determining percent amino acid sequence identity, alignment can be achieved in a variety of ways in the art, for example, using BLAST, BLAST-2, ALIGN, or MEGALIGN. TM Publicly available computer software such as (DNASTAR) software is used. Those skilled in the art can determine appropriate parameters to be used for the measured alignment, including any algorithm that achieves maximal alignment within the full length of the sequences being compared.

[0104] An "isolated" nucleic acid molecule encoding a construct, antibody or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule normally associated with it in its production environment. Preferably, an isolated nucleic acid is free of association with all components of the production environment. The format of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from the naturally occurring format or background. Thus, an isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0105] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which the vector has already been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0106] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with heterologous nucleic acid. Such cells include the original target cell and its progeny.

[0107] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, without regard for the number of passages. The nucleic acid content of the progeny may not be completely identical to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0108] The term "drug formulation" of "pharmaceutical composition" refers to a formulation, the form of which allows the biological activity of the active ingredient to be effective, and which does not contain other ingredients that are unacceptably toxic to the subject to which it is administered. Such a formulation is sterile. A "sterile" formulation is one that has been disinfected or is free of living microorganisms and their spores.

[0109] It should be understood that embodiments of the invention described herein include "consisting of an embodiment" and / or "consisting essentially of an embodiment."

[0110] As used herein, reference to "about" a value or parameter includes (and describes) the variation relative to the value or parameter itself. For example, a statement about "about X" includes a statement of "X."

[0111] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0112] II. Anti-B7-H3 construct Anti-B7-H3 monoclonal antibody The isolated anti-B7-H3 constructs described herein comprise a monoclonal antibody (mAb) portion that specifically recognizes or binds to B7-H3 (or an "anti-B7-H3 mAb"). In some embodiments of the invention, the isolated anti-B7-H3 construct is a full-length IgG.

[0113] B7 superfamily and B7-H3 As used herein, the term "B7-H3" refers to a member of the human B7 protein family, which is a type I membrane protein with an Ig-like domain, also known as CD276.

[0114] Studies of ligands for the CD28 receptor have already produced the characterization of a group of related molecules called the B7 superfamily. B7 family members are members of the immunoglobulin superfamily that have immunoglobulin-V-like and immunoglobulin-C-like domains. The IgV and IgC domains of B7 family members are each encoded by a single exon, with separate exons encoding the leader sequence, transmembrane domain, and intracellular domain. The intracellular domain is short, ranging in length from 19 to 62 amino acid residues, and may be encoded by multiple exons. What is unique about B7-H3 is that the predominant human form contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC) (Collins, M. et al. (2005), Genome Biol. 6:223.1-223.7). Members of the B7 family are predicted to form back-to-back non-covalent homodimers at the cell surface, and such dimers have already been found for B7-1 (CD80) and B7-2 (CD86).

[0115] Initially thought to contain only two Ig domains (IgV-IgC) (Chapoval, A. et al. (2001), Nature Immunol. 2:269-274; Sun, M. et al. (2002), J. Immunol. 168:6294-6297), a variant of the four immunoglobulin extracellular domains ("4Ig-B7-H3") has already been identified and found to be the more common human form of the protein (Sharpe, AH et al. (2002), Nature Rev. Immunol. 2:116-126). No functional differences have yet been observed between the native mouse form (2Ig) and the human 4Ig form, as these two forms display similar functions (Hofmeyer, K. et al. (2008), Proc. Natl. Acad. Sci. USA, 105(30):10277-10278). The 4Ig-B7-H3 molecule inhibits natural killer cell-mediated cancer cell lysis (Castriconi, R. et al., Proc. Natl. Acad. Sci. USA, 101(34): 12640-12645). Human B7-H3 (2Ig form) was previously found to promote T cell activation and IFN-γ production by binding to a putative receptor on activated T cells (Chapoval, A. et al. (2001), Nature Immunol. 2:269-274; Xu, H. et al. (2009), Cancer Res. 69(15):5275-6281). When expressed on tumor cells, both B7-H4 and B7-H1 are effective immune function inhibitors (Flies, DB et al. (2007), J. Immunother. 30(3):251-260).

[0116] The pattern of action of B7-H3 is complex, as the protein mediates both costimulation and co-inhibition of T cells (Hofmeyer, K. et al. (2008), Proc. Natl. Acad. Sci. USA, 105(30):10277-10278; Martin-Orozco, N. et al. (2007), Semin. Cancer Biol. 17(4):288-298; Subudhi, SK et al. (2005), J. Mol. Med. 83:193-202). B7-H3 binds to (TREM)-like transcript 2 (TLT-2) and co-stimulates T cell activation, but also binds to one or more as yet unidentified receptors and mediates co-inhibition of T cells. B7-H3 is also an inhibitor of natural killer cells and osteoblasts through interaction with an unknown receptor (Hofmeyer, K. et al. (2008), Proc. Natl. Acad. Sci. USA, 105(30):10277-10278). The inhibition can be exerted through interaction with members of major signaling pathways (e.g., NFTA, NF-κB, or AP-1 factors) through which the T cell receptor (TCR) regulates gene transcription.

[0117] B7-H3 co-stimulates the proliferation of CD4+ and CD8+ T cells. B7-H3 also stimulates the production of IFN-γ and the lytic activity of CD8+ (Chapoval, A. et al. (2001), Nature Immunol. 2:269-274; Sharpe, AH et al. (2002), Nature Rev. Immunol. 2:116-126). However, the protein can also act through NFAT (nuclear factor of activated T cells), NF-κB (nuclear factor κB) and AP-1 (activated protein-1) factors to inhibit T cell activation (Yi. KH et al. (2009), Immunol. Rev. 229:145-151). Furthermore, B7-H3 is believed to inhibit Th1, Th2 or Th17 in vivo (Prasad, DV et al. (2004), J. Immunol. 173:2500-2506; Fukushima, A. et al. (2007), Immunol. Lett. 113:52-57; Yi. KH et al. (2009), Immunol. Rev. 229:145-151). Several independent studies have already shown that human malignant tumor cells exhibit a significant increase in B7-H3 protein expression and that such increased expression correlates with increased disease severity (Zang, X. et al. (2007), Clin. Cancer Res. 13:5271-5279), suggesting that tumors exploit B7-H3 as an immune evasion pathway (Hofmeyer, K. et al. (2008), Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).

[0118] Molecules that block the ability of B7 molecules to bind to T cell receptors (e.g., CD28) inhibit the immune system and have been proposed as treatments for autoimmune diseases (Linsley, PS et al. (2009), Immunolog. Rev. 229:307-321). Neuroblastoma cells expressing 4Ig-B7-H3 treated with anti-4Ig-B7-H3 antibodies are more susceptible to NK cells. However, it is unclear whether such activity is solely due to antibodies against the 4Ig-B7-H3 form, since all reported antibodies raised against 4Ig-B7-H3 also bind to the two Ig-like forms of B7-H3 (Steinberger, P. et al. (2004), J. Immunol. 172(4): 2352-2359 and Castriconi et al. (2004), Proc. Natl. Acad. Sci. USA, 101(34): 12640-12645).

[0119] B7-H3 is not expressed on resting B or T cells, monocytes or dendritic cells, but is induced by IFN-γ in dendritic cells and by GM-CSF in monocytes (Sharpe, AH et al. (2002), Nature Rev. Immunol. 2:116-126). The receptors that bind B7-H3 have not yet been fully characterized. Early studies have shown that one such receptor is required for rapid, transient upregulation in T cells after activation (Loke, P. et al. (2004), Arthritis Res. Ther. 6:208-214).

[0120] In addition to being expressed in neuroblastoma cells, human B7-H3 is also known to be expressed in a variety of other cancer cells (e.g., gastric cancer, ovarian cancer, and non-small cell lung cancer). Expression of B7-H3 protein has already been detected by immunohistochemistry in tumor cell lines (Chapoval, A. et al. (2001), Nature Immunol. 2:269-274; Saatian, B. et al. (2004), Amer. J. Physiol. Lung Cell. Mol. Physiol. 287:L217-L225; Castriconi et al. (2004), Proc. Natl. Acad. Sci. USA, 101(34):12640-12645); Sun, M. et al. (2002), J. Immunol. 168:6294-6297). mRNA expression has already been found in heart, kidney, testis, lung, liver, pancreas, prostate, colon and osteoblasts (Collins, M. et al. (2005), Genome Biol. 6:223.1-223.7). At the protein level, B7-H3 has been found in human liver, lung, bladder, testis, prostate, mammary gland, placenta and lymphoid organs (Hofmeyer, K. et al. (2008), Proc. Natl. Acad. Sci. USA, 105(30):10277-10278).

[0121] In some embodiments, the anti-B7-H3 mAbs of the present disclosure can cross-react with B7-H3 from species other than human, or with other proteins structurally related to B7-H3, hi some embodiments, the anti-B7-H3 mAbs of the present application are completely specific for human B7-H3 and do not exhibit species or other types of cross-reactivity.

[0122] antibody affinity The binding specificity of an antibody or an antigen-binding domain thereof may be determined experimentally by methods known in the art, including but not limited to Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore assays and peptide scanning.

[0123] In some embodiments, the K D is about 10 -5 M~about 10 -6 M, about 10 -6 M~about 10 -7 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -5 M~about 10 -12 M, about 10 -6 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -5 M~about 10 -11 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -5 M~about 10 -10 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M, about 10 -5 M~about 10 -9 M, about 10 -7 M~about 10 -9 M, about 10 -5 M~about 10 -8 M or about 10 -6 M~about 10 -8 It's M.

[0124] In some embodiments, the Kon is about 10 2 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 2 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 6 M -1 s -1 Or about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 It is.

[0125] In some embodiments, the K off takes about 1s -1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10-4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 Or about 10 -3 s -1 ~about 10 -5 s -1 It is.

[0126] In some examples, FACS studies of binding of anti-B7-H3 mAbs to B7-H3 expressing CHO-K1 cells showed that the E 50 In some embodiments, the EC 50 is less than 1 nM.

[0127] Chimeric or humanized antibodies In some embodiments, the anti-B7-H3 antibody according to the present invention is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region from a camelid species such as a llama) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed compared to the class or subclass of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0128] In some embodiments, a chimeric antibody is a humanized antibody. Typically, humanization is performed on a non-human antibody to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, where the HVRs, e.g., CDRs, (or portions thereof) are from a non-human antibody, while the FRs (or portions thereof) are from human antibody sequences. The humanized antibody may optionally further comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the non-human antibody (e.g., the antibody originating from the HVR residues), e.g., to restore or improve antibody specificity or affinity.

[0129] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described in, e.g., Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (wherein SDR(a-CDR) grafting is described), Padlan, Mol. Immunol. 28:489-498. (1991) (describing "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" method for FR shuffling).

[0130] Human framework regions that may be used for humanization include framework regions selected by the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions from the shared sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Nat'l. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0131] In some embodiments, modifications such as humanization are performed on mAbs to reduce their immunogenicity to heterologous species without compromising the natural affinity of the domains for the antigen. For example, the amino acid residues of the antibody heavy and light chain variable domains (VH and VL) can be defined, and one or more mouse amino acids, for example in the framework regions, are replaced with their human counterparts found in the human shared sequence, but the polypeptide does not lose its typical characteristics, i.e., humanization does not significantly affect the antigen-binding ability of the resulting polypeptide. Humanization of mouse monoclonal antibodies requires the introduction and mutagenesis of a limited amount of amino acids in the two chains (light and heavy chains) and the retention of the assembly of the two chains.

[0132] Human antibodies In some embodiments, the anti-B7-H3 antibodies, particularly mAbs, according to the present invention are human antibodies. Human antibodies may be produced using different techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Transgenic mice or rats capable of producing fully human single domain antibodies are known in the art. See, for example, US 20090307787 A1, US Patent No. 8,754,287, US 20150289489 A1, US 20100122358 A1, and WO 2004049794.

[0133] Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals usually contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci or are present on the chromosome or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, e.g., XENOMOUSE, et al. TM See U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HuMab™ technology, 5,770,429, which describes HuMab™ technology, 7,041,870, which describes KM MOUSE™ technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes VELOCIMOUSE™ technology. The human variable regions from whole antibodies generated by such animals may be further modified (e.g., by conjugation to a different human constant region).

[0134] Human antibodies may be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987); Boerner et al., J. Immunol., 147: 86 (1991). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Nat'l. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0135] Human antibodies may be generated by isolating Fv clone variable domain sequences selected from a human phage display library. These variable domain sequences may then be combined with desired human constant domains. A description of techniques for selecting human antibodies from antibody libraries follows.

[0136] Antibodies from the library The antibodies of the present application can be isolated by screening for antibodies with the desired activity in a combinatorial library, for example, many methods are known in the art for making phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and also described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004), Fellouse, Proc. Nat'l. Acad. Sci. USA 101(34): 12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004). Methods for constructing single domain antibody libraries have been described, see, e.g., U.S. Patent No. 7,371,849.

[0137] In some phage display methods, VH and VL gene libraries may be cloned by polymerase chain reaction (PCR), respectively, randomly recombined in a phage library, and screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments in the form of single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to immunogens without constructing hybridomas. Alternatively, the original library can be cloned (e.g., from humans) to provide a single source of antibodies against various non-self and self antigens without the need for any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, the original library can be further synthesized by cloning unrearranged V gene fragments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and complete in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0138] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0139] Anti-B7-H3 antibody In some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb having the sequence set forth in SEQ ID NO: 2 ( DHYMH ), SEQ ID NO: 10( GMN ), SEQ ID NO: 18( GIN ), SEQ ID NO: 26( DFGMN ), SEQ ID NO: 34( NNGMN ), SEQ ID NO: 42( NYGMN ), SEQ ID NO: 50( DFGMN), SEQ ID NO: 58( DYGMN ), SEQ ID NO: 66( NFGMN ) and SEQ ID NO: 74( SYWMH 3, SEQ ID NO: 4, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions) (for a CDR having three amino acid residues, the variant may comprise at most about two (e.g., one or two) amino acid substitutions). YIFPNNGVSGYNQNFKD ), SEQ ID NO: 11( WINPYSGVPTYADDFKG ), SEQ ID NO: 19( WINPYSGVPTYADDFKG ), SEQ ID NO: 27( WINPYSGVPTYADEFKG ), SEQ ID NO: 35( WINPYSGVPTYADDFKG ), SEQ ID NO: 43( WINPYSGVPTYADDFKG ), SEQ ID NO: 51( WLNSYSGVPTYADDFKG ), SEQ ID NO: 59( WINIYSGVPTYADDFKG ), SEQ ID NO: 67( WIYTPSGVPIYADDFKG ) and SEQ ID NO: 75( HINPNSGSTNKNQNAKFKN ), or a variant thereof, wherein the variant comprises a heavy chain CDR2 having at most about three amino acid substitutions (e.g., at most about one, two or three amino acid substitutions), and a heavy chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 4 ( RQLGLGTMDY ), SEQ ID NO: 12( DDGFHYTMDY ), SEQ ID NO: 20( DDGFHYTMDY ), SEQ ID NO: 28( DDGYHYTMDY ), SEQ ID NO: 36( DDGYHYTMDF ), SEQ ID NO: 44( DDGFHYTMDY ), SEQ ID NO: 52( DDGYNYTMNY), SEQ ID NO: 60( DDGYHYTMNY ), SEQ ID NO: 68( DDGYYYTMDY ) and SEQ ID NO: 76( SGSNYRRNYFDY 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 RASKSVSTSGYNYMH ), SEQ ID NO: 14( RSSQNLVHSNGITYLH ), SEQ ID NO: 22( RSSQNLVHSNGITYLH ), SEQ ID NO: 30( RSSQSLIHSNGITYLH ), SEQ ID NO: 38( RSSQSLVHSNGITYLH ), SEQ ID NO: 46( RSSQSLLHSNGITYLH ), SEQ ID NO: 54( RSSQSLVHSNGITYLH ), SEQ ID NO: 62( RSSQSLVHSNGITYLH ), SEQ ID NO: 70( RSSQSLVHSNGDTYLH ) and SEQ ID NO: 78( KASDHINNWLA 7, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions); LASNLES ), SEQ ID NO: 15( KVSNRFS ), SEQ ID NO: 23( KVSNRFS ), SEQ ID NO: 31( KVSNRFS ), SEQ ID NO: 39( KVSHRFS ), SEQ ID NO: 47( KVSHRFS ), SEQ ID NO: 55( KVSNRFS ), SEQ ID NO: 63( KVSNRFS ), SEQ ID NO: 71( KVSNRFS ) and SEQ ID NO: 79( GATSLET), or a variant thereof, wherein the variant comprises a light chain CDR2 having at most about three amino acid substitutions (e.g., at most about one, two or three amino acid substitutions), and a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 8 ( QHSKELPWT ), SEQ ID NO: 16( SQSTLVPWT ), SEQ ID NO: 24( SQSTLVPWT ), SEQ ID NO: 32( SQSTHVPWT ), SEQ ID NO: 40( SQSTHVPWT ), SEQ ID NO: 48( SQSTHVPWT ), SEQ ID NO: 56( SQSTHVPWT ), SEQ ID NO: 64( SQSTHVPWT ), SEQ ID NO: 72( SQSTHVPWT ) and SEQ ID NO: 80( QQYWSTPLT ), or a variant thereof, wherein the variant comprises a light chain variable domain (VL) having a light chain CDR3 comprising at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions). In some embodiments, the K D is about 10 -5 M~about 10 -12 M (e.g., about 10 -7 M~about 10 -12 M or about 10 -8 M~about 10 -12 In some embodiments, the anti-B7-H3 antibody is rodent, chimeric, human, partially humanized, or fully humanized.

[0140] In some embodiments, the anti-B7-H3 mAb comprises a VH CDR3 and a VL CDR3, wherein the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76, and the VL CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80, and the amino acid substitutions are in CDR1 and / or CDR2 of the VH domain and the VL domain.

[0141] Thus, in some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, and 74, or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of about one, two, or three amino acid substitutions); a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75, or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of about one, two, or three amino acid substitutions); and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: a heavy chain variable domain (VH) having a CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76; a CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, and 78 or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions); a CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79 or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions); and a light chain variable domain (VL) having a CDR3 comprising any one of the amino acid sequences of 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80. In some embodiments, the K D is about 10 -5 M~about 10 -12 M (e.g., about 10 -7 M~about 10 -12 M or about 10 -8 M~about 10 -12M) or less. In some embodiments, the anti-B7-H3 mAb is rodent, chimeric, human, partially humanized, or fully humanized.

[0142] In some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb comprising a heavy chain variable domain (VH) having a CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, and 74, a CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75, and a CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76; and a light chain variable domain (VL) having a CDR2 comprising any one of the amino acids of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79, and a CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80. In some embodiments, the K D is about 10 -5 M~about 10 -12 M (e.g., about 10 -7 M~about 10 -12 M or about 10 -8 M~about 10 -12 M). In some embodiments, the anti-B7-H3 mAb is rodent, chimeric, human, partially humanized or fully humanized.

[0143] In some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb comprising: 1) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively; 2) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; 3) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 2) a VH comprising a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and a VL comprising a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 30, 31 and 32, respectively; 5) a VH comprising a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, and a VL comprising a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively; 6) a VH comprising a heavy chain CDR1, CDR2 and CDR3 sequence having the amino acid sequences of SEQ ID NOs: 42, 43 and 44, respectively, and a VL comprising a light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 6) a VL comprising light chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; 7) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 54, 55 and 56, respectively; 8) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 58, 59 and 60, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs:8) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 70, 71 and 72, respectively; or 9) a VH comprising heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 74, 75 and 76, respectively, and a VL comprising light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively.

[0144] In some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb being selected from the group consisting of: 1) a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid sequence of SEQ ID NO: 5 (47F3F10B4); 2) a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 13 (12C9F1B5); 3) a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 21 (34A9C12); 4) a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 29 (38D6A4); 5) a VH comprising the amino acid sequence of SEQ ID NO: 33 and a VL comprising the amino acid sequence of SEQ ID NO: 37 (37D11F5B8); 6) a VH comprising the amino acid sequence of SEQ ID NO: 65 and a VL comprising the amino acid sequence of SEQ ID NO: 69 (47C7E3); or 7) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 77 (31H11B1).

[0145] CDRs can be combined in various pairwise combinational ways to form many humanized anti-B7-H3 antibodies. Humanizing substitutions will be apparent to one of skill in the art. For example, potentially useful humanizing substitutions can be identified by comparing the FR sequence of a naturally occurring VH or VL with the corresponding FR sequence of one or more closely related human VH or VL, and introducing one or more such potentially useful humanizing substitutions into said VH or VL using methods known in the art (also described herein). The humanized heavy and light chains are paired. The resulting humanized antibodies can be tested for B7-H3 binding affinity, stability, ease and level of expression, and / or other desired properties. The anti-B7-H3 mAbs described herein may be partially or fully humanized. Preferably, the resulting humanized antibodies, e.g., humanized mAbs or antigen-binding fragments thereof, are humanized according to the K- and K-specific nucleotide sequences described herein. D , K on , K off and binds to B7-H3.

[0146] In some embodiments, an anti-B7-H3 humanized mAb or antigen-binding fragment thereof is provided, the anti-B7-H3 humanized mAb or antigen-binding fragment thereof comprising a VH domain comprising the amino acid sequence of any one of SEQ ID NOs: 81-87 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 81-87, and a VH domain comprising the amino acid sequence of any one of SEQ ID NOs: 88-91 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 81-87, and a VL domain comprising an amino acid sequence of any one of SEQ ID NOs: 81-87 or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 81-87 or a variant thereof. In some embodiments, an anti-B7-H3 mAb is provided, the anti-B7-H3 mAb comprising a VH domain comprising an amino acid sequence of any one of SEQ ID NOs: 81-87 or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of 1, 2, or 3 amino acid substitutions) in the VH domain, and a VL domain comprising an amino acid sequence of any one of SEQ ID NOs: 88-91 or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of 1, 2, or 3 amino acid substitutions) in the VL domain. In some embodiments, the anti-B7-H3 mAb or antigen-binding fragment thereof comprises a mutant VH domain having the amino acid sequence of any one of SEQ ID NOs: 81-87, the mutant VH domain comprising an amino acid substitution in a CDR of the VH, e.g., CDR1 and / or CDR2 and / or CDR3, and a mutant VL domain having the amino acid sequence of any one of SEQ ID NOs: 88-91, the mutant VL domain comprising an amino acid substitution in a CDR of the VL, e.g., CDR1 and / or CDR2 and / or CDR3.In some embodiments, the anti-B7-H3 mAb or antigen-binding fragment thereof comprises a mutant VH domain having the amino acid sequence of any one of SEQ ID NOs: 81-87, the mutant VH domain comprising an amino acid substitution in any one of the FRs of the VH, e.g., FR1 and / or FR2 and / or FR3 and / or FR4, and a mutant VL domain having the amino acid sequence of any one of SEQ ID NOs: 88-91, the mutant VL domain comprising an amino acid substitution in a FR, e.g., FR1 and / or FR2 and / or FR3 and / or FR4.

[0147] In some embodiments, an anti-B7-H3 humanized mAb or antigen-binding fragment thereof is provided, the anti-B7-H3 humanized mAb or antigen-binding fragment thereof comprising: 1) a VH comprising the amino acid sequence of SEQ ID NO: 81 and a VL comprising the amino acid sequence of SEQ ID NO: 88 (47F3F10B4-VH1-VL4); 2) a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO: 88 (47F3F10B4-VH2-VL4); 3) a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising the amino acid sequence of SEQ ID NO: 89 (12C9F1B5-VH2.1-VL2); 4) a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 89 (12C9F1B5-VH4-VL2); 5) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 91 (31H11B1-VH1-VL3); or 6) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 91 (31H11B1-VH1.1-VL3).

[0148] In some embodiments, an anti-B7-H3 antibody, e.g., a mAb (hereinafter referred to as a "competitive anti-B7-H3 antibody or competitive anti-B7-H3 mAb") or antigen-binding fragment thereof is provided that specifically binds to B7-H3 in competition with any one of the anti-B7-H3 mAbs described herein. In some embodiments, competitive binding can be determined by an ELISA assay. In some embodiments, the competitive anti-B7-H3 antibody and the anti-B7-H3 antibody bind to the same epitope on B7-H3.

[0149] Constructs containing anti-B7-H3 mAb The anti-B7-H3 constructs comprising anti-B7-H3 mAbs may be in any possible form.

[0150] In some embodiments, the anti-B7-H3 construct comprising the anti-B7-H3 mAb may further comprise additional polypeptide sequences, e.g., one or more antibody moieties. Such additional polypeptide sequences may or may not change or otherwise affect the (biological) properties of the anti-B7-H3 mAb and may or may not increase other functions to the anti-B7-H3 mAb described herein. In some embodiments, the additional polypeptide sequences confer one or more desired properties or functions to the anti-B7-H3 mAb of the present application. In some embodiments, the anti-B7-H3 construct is a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, the extracellular antigen binding domain comprising one or more anti-B7-H3 binding moieties described herein.

[0151] In some examples, the additional polypeptide sequence may be a second antibody portion (e.g., sdAb, scFv) that specifically recognizes a second antigen. In some examples, the second antigen is not B7-H3. In some examples, the second antibody portion and the anti-B7-H3 mAb described herein specifically recognize the same epitope on B7-H3. In some examples, the second antibody portion and the anti-B7-H3 mAb described herein specifically recognize different epitopes on B7-H3.

[0152] In some examples, the additional polypeptide sequence may increase the molecular stability, solubility or absorption of the anti-B7-H3 constructs of the invention, reduce immunogenicity or toxicity, eliminate or attenuate undesirable side effects, and / or confer other advantageous properties and / or reduce undesirable properties to the anti-B7-H3 constructs of the invention, relative to the anti-B7-H3 mAb itself described herein.

[0153] Full-length IgG In some embodiments, the anti-B7-H3 mAb is a full-length IgG. In some embodiments, the anti-B7-H3 mAb comprises a constant region of an IgG (e.g., any one of IgG1, IgG2, IgG3, or IgG4). In some embodiments, the constant region is a human constant region. In some embodiments, the constant region is a human IgG1 constant region.

[0154] Thus, in some embodiments, an anti-B7-H3 full length IgG is provided, the anti-B7-H3 full length IgG having a variable region (VH) comprising a CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, and 74, or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of 1, 2, or 3 amino acid substitutions); and a CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75, or a variant thereof, the variant comprising at most about three amino acid substitutions (e.g., at most about any one of 1, 2, or 3 amino acid substitutions); and a CDR3 comprising any one of the amino acid sequences of 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76, or a variant thereof, wherein the variant comprises a CDR3 comprising at most about three amino acid substitutions (e.g., any one of about one, two, or three amino acid substitutions), and wherein the VH comprises an immunoglobulin heavy chain constant region (hinge region, C H 1. C H 2 and C H3), and a variable region (VL) comprising a CDR1 having an amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, and 78, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions), and a CDR2 having an amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions), and a CDR2 having an amino acid sequence of any one of SEQ ID NOs: and a CDR3 comprising any one of the amino acid sequences of 8, 16, 24, 32, 40, 48, 56, 64, 72 and 80, or a variant thereof, wherein the variant comprises a CDR3 comprising at most about three amino acid substitutions (e.g., any one of about one, two or three amino acid substitutions), and wherein the VL comprises a light chain fused to an immunoglobulin light chain constant region (CL). In some embodiments, an anti-B7-H3 full length IgG is provided, the anti-B7-H3 full length IgG having a variable region (VH) comprising a CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, and 74, a CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75, and a CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions), and wherein the VH is a CDR1 comprising an immunoglobulin heavy chain constant region (e.g., hinge region, CDR2), a CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions). H 1. C H 2 and C Hand a light chain variable region (VL) comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, and 78, a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79, and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80, or a variant thereof, wherein the variant comprises at most about three amino acid substitutions (e.g., at most about one, two, or three amino acid substitutions), and wherein the VL is fused to an immunoglobulin light chain constant region (CL). In some embodiments, the full-length anti-B7-H3 mAb comprises a VH and a VL, wherein the VH comprises any one of the amino acid sequences of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, and 73, and the VL comprises any one of the amino acid sequences of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, and 77.

[0155] In some embodiments, the K D is about 10 -5 M~about 10 -12 M (e.g., about 10 -7 M~about 10 -12 M or about 10 -8 M~about 10 -12 M) or less. In some embodiments, the full-length anti-B7-H3 IgG is rodent, chimeric, human, partially humanized, or fully humanized.

[0156] Multivalent and / or multispecific antibodies In some embodiments, an anti-B7-H3 construct comprises an anti-B7-H3 mAb described herein fused to one or more other antibody moieties (e.g., an antibody moiety that specifically recognizes another antigen), which may be any antibody or antibody fragment form, such as an sdAb, full length antibody, Fab, Fab', (Fab') 2, Fv, single chain Fv (scFv), scFv-scFv, minibody or diabody. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthuen, Vol. 113, The Pharmacology of Monoclonal Antibodies, Eds. Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994), and see WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain rescue receptor binding epitope residues and have extended in vivo half-lives. 2For a discussion of fragments, see U.S. Patent No. 5,869,046. For a review of multispecific antibodies, see Weidle et al., Cancer Genomics Proteomics, 10(1):1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015; Stamova et al., Antibodies, 1(2):172-198, 2012. Diabodies are antibody fragments that have two antigen binding sites and can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Nat'l. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Antibody fragments may be produced by several techniques, including, but not limited to, proteolytic digestion of whole antibodies and production in recombinant host cells (e.g., E. coli or phage), as described herein. In some embodiments, the one or more other antibody moieties are antibody mimetics, small engineered proteins that contain antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from traditional human scaffold proteins and contain multiple polypeptides. Exemplary antibody mimetics that may be included in the anti-B7-H3 constructs described herein may be, but are not limited to, designed ankyrin repeat proteins (DARPins, containing 3-5 fully synthetic ankyrin repeat sequences flanking N-terminal and C-terminal Cap domains), avimers (high affinity proteins containing multiple A-domains, each domain having a low affinity for the target) or Anticalins (lipocalin-based scaffold, with 4 accessible loops, where the sequence of each loop may be randomized).

[0157] Techniques for making multispecific antibodies include, but are not limited to, recombinant expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Nat'l. Acad. Sci. USA, 90:6444-6448 (1993)), using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1993)), and the like. (1994)), for example, by making trispecific antibodies as described in Tutt et al. J. Immunol. 147: 60 (1991), by producing polypeptides that include tandem single domain antibodies (see, e.g., U.S. Patent Application No. 20110028695, and Conrath et al. J. Biol. Chem., 2001, 276(10): 7346-50). The present specification further includes engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576 A1).

[0158] Peptide Linker In some embodiments, two or more antibody moieties of the anti-B7-H3 construct in vivo may be optionally linked via a peptide linker. The length, flexibility and / or other properties of the peptide linker used in the anti-B7-H3 construct may have some effect on the properties, including but not limited to the affinity, specificity or avidity for one or more specific antigens or epitopes. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, the peptide linker includes flexible residues (e.g., glycine and serine), allowing adjacent domains to move freely relative to each other. For example, a glycine-serine dyad may be a suitable peptide linker.

[0159] The peptide linker may have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is less than or equal to about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is any one of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0160] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, the sequence of a hinge region from an antibody having only a heavy chain may be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a mutated human IgG1 hinge (EPKSSDKTHTSPPSP, SEQ ID NO: 95). In some embodiments, the peptide linker is a flexible linker. An exemplary flexible linker is a glycine polymer (G n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , (GGGS) n and (GGGGS) n where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGS (SEQ ID NO: 96). In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 97 (GGGGSGGGGSGGGGS).

[0161] bispecific antibody In some examples, the isolated antibody or antigen-binding fragment thereof of the present application is a bispecific or multispecific antibody comprising an anti-B7-H3 IgG as described herein fused to a second antibody moiety, where the second antibody moiety specifically binds to another antigen, preferably another inhibitory immune checkpoint molecule.

[0162] In one example, the other antigen is CTLA-4, and the second antibody portion comprises an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4, e.g., an anti-CTLA-4 mAb, preferably an anti-CTLA-4 sdAb. An isolated antibody or antigen-binding fragment thereof having dual specificity for B7-H3 and CTLA-4 may be referred to hereinafter as an "anti-B7-H3 / CTLA-4 antibody," an "anti-B7-H3 / CTLA-4 construct," or a "B7-H3xCTLA-4 antibody."

[0163] In one example, the other antigen is PD-L1 and the second antibody portion comprises an antibody or antigen-binding fragment thereof that specifically binds to PD-L1, such as an anti-PD-L1 mAb, preferably an anti-PD-L1 sdAb.An isolated antibody or antigen-binding fragment thereof with dual specificity for B7-H3 and PD-L1 may be referred to hereinafter as an "anti-B7-H3 / PD-L1 antibody," an "anti-B7-H3 / PD-L1 construct," or a "B7-H3xPD-L1 antibody."

[0164] In one example, the other antigen is TIM-3, and the second antibody portion comprises an antibody or antigen-binding fragment thereof that specifically binds to TIM-3, e.g., an anti-TIM-3 mAb, preferably an anti-TIM-3 sdAb. An isolated antibody or antigen-binding fragment thereof with dual specificity for B7-H3 and TIM-3 may be referred to hereinafter as an "anti-B7-H3 / TIM-3 antibody," an "anti-B7-H3 / TIM-3 construct," or a "B7-H3xTIM-3 antibody."

[0165] In one example, the other antigen is LAG-3, and the second antibody portion comprises an antibody or antigen-binding fragment thereof that specifically binds to LAG-3, e.g., an anti-LAG-3 mAb, preferably an anti-LAG-3 sdAb. An isolated antibody or antigen-binding fragment thereof having dual specificity for B7-H3 and LAG-3 may be referred to hereinafter as an "anti-B7-H3 / LAG-3 antibody," an "anti-B7-H3 / LAG-3 construct," or a "B7-H3xLAG-3 antibody."

[0166] CTLA-4, PD-L1, TIM-3 and LAG-3 are inhibitory immune checkpoint molecules.

[0167] Conjugates Human monoclonal antibodies or antigen-binding fragments thereof with specificity for B7-H3 can be conjugated to agents such as effector molecules or detectable markers using a variety of methods known to those of skill in the art. Covalent and non-covalent linking methods may be used. Conjugates include, but are not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment thereof that specifically binds to B7-H3. Those of skill in the art will appreciate that conjugation of such agents with chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents (e.g., 125 I, 32 P, 14 C. 3 H and 35 It will be appreciated that a variety of effector molecules and detectable markers can be used, including, but not limited to, S) and other tags, targeting moieties and ligands.

[0168] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell, and the desired biological effect, so, for example, the effector molecule may be a cytotoxin to cause the death of a particular target cell (e.g., a tumor cell).

[0169] Effector molecules and detectable markers can be linked to the antibody of interest or its antigen-binding fragment using any method known to those of skill in the art. Covalent and non-covalent linking methods may be used. The procedure for attaching an effector molecule or detectable marker to an antibody or its antigen-binding fragment will vary depending on the chemical structure of the effector. Polypeptides usually contain a variety of functional groups, e.g., carboxylic acids (COOH), free amines (-NH 2) or mercapto (-SH) groups, which can be used to react with appropriate functional groups on the antibody to effector molecule or detectable marker attachment. Alternatively, the antibody or antigen-binding fragment thereof is derivatized to expose or attach another reactive functional group. The derivatization can involve attachment to any one of a variety of known linker molecules, such as those available from Pierce Chemical Company, Rockford, Ill. The linker can be any molecule for linking the antibody or antigen-binding fragment thereof to the effector molecule or detectable marker. The linker can form covalent bonds with both the antibody or antigen-binding fragment thereof and the effector molecule or detectable marker. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment thereof and the effector molecule or detectable marker are polypeptides, the linker may be linked to the constituent amino acids through its pendant groups (e.g., through a disulfide bond with cysteine) or may be contiguous to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0170] In some embodiments, the linker may also include a spacer element, which, if present, increases the size of the linker and increases the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment thereof. Exemplary spacers are known to those of skill in the art and are described in U.S. Patents 7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,28 Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414 and U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated by reference in its entirety.

[0171] Thus, in some embodiments, the conjugate comprises a linker that links the effector molecule or detectable marker to the B7-H3 specific antibody or antigen-binding fragment thereof. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment thereof in the intracellular environment. In other embodiments, the linker is non-cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or pit). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids in length or at least three amino acids in length. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acids in length. Proteases may include cathepsin B and D and plasmin, both of which are known to hydrolyze dipeptide drug derivatives, causing the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker that can be cleaved by a thiol-dependent protease, i.e., cathepsin-B, may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). Other examples of such linkers are described, for example, in US Pat. No. 6,214,345, which is incorporated herein by reference.In one specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin having a valine-citrulline linker).

[0172] In another embodiment, the cleavable linker is pH sensitive, i.e., susceptible to hydrolysis at some pH values. Typically, the pH sensitive linker is hydrolyzable under acidic conditions. For example, an acid labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in the lysosome may be used. (See, e.g., U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0 (the approximate pH of the lysosome). In some examples, the hydrolyzable linker is a thioether linker (eg, a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0173] In other examples, the linker (e.g., a disulfide linker) is cleavable under reducing conditions. Many disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene)-, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (ed. C.W. Vogel, Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008. See also U.S. Pat. No. 4,880,935.)

[0174] In other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0175] In other examples, the linker is non-cleavable and the effector molecule or detectable marker is released by antibody degradation. (See US Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety.)

[0176] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linker is cleaved in a conjugate sample. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating a conjugate containing a desired linker with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and quantifying the amount of free effector molecule or detectable marker present in the plasma. Various exemplary linkers that can be used in the conjugates are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 2005 / 0238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0177] The antibodies or antigen-binding fragments thereof disclosed herein can be derivatized, for example, by crosslinking two or more antibodies (of the same type or of different types, e.g., to produce bispecific antibodies). Suitable crosslinkers include crosslinkers that are heterobifunctional and have two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional crosslinkers (e.g., disuccinimidyl suberate). Such linkers are commercially available.

[0178] Considering that numerous methods have been reported for conjugating numerous radioactive diagnostic compounds, radioactive therapeutic compounds, tags (e.g., enzymes or fluorescent molecules), toxins, and other agents to antibodies, one of skill in the art can determine the appropriate method for conjugating a given agent to an antibody or antigen-binding fragment thereof or other polypeptide. For example, an antibody or antigen-binding fragment thereof can be conjugated with a low molecular weight drug such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), maytansine, maytansine derivatives (including a maytansine derivative designated DM1, also called mertansine), or other chemotherapeutic agents to produce an antibody drug conjugate (ADC). In some examples, various chemotherapeutic agents described herein can be conjugated to the provided antibodies to generate conjugates.

[0179] In some embodiments, conjugates of an antibody or antigen-binding fragment thereof and one or more small molecule toxins are provided, such as calicheamicins, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, as well as toxic derivatives of these toxins.

[0180] Maytansine compounds suitable for use as maytansinoid toxin moieties are commercially available and can be isolated from natural sources and produced by genetic engineering techniques according to known methods (see Yu et al. (2002) PNAS 99:7968-7973), or synthesized according to known methods to produce maytansinol and maytansinol analogues. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from an East African shrub, namely Maytenus serrata (U.S. Pat. No. 3,896,111). It was subsequently discovered that several microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated by reference. Conjugates containing maytansinoids, their preparation methods and their therapeutic uses are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064, 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated by reference.

[0181] Anti-B7-H3 antibody variant In some embodiments, amino acid sequence variants of the antibodies according to the present disclosure are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be made by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0182] a) Substitutions, insertions, deletions and mutations In some examples, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 3, entitled "Preferred Substitutions." More substantial changes are provided in Table 3, entitled "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0183] [Table 3]

[0184] Amino acids can be grouped according to common side chain properties.

[0185] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0186] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0187] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). The resulting variants are usually selected for further study and are modified (e.g., improved) with some biological property (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or substantially retain some biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be readily generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0188] Modifications (e.g., substitutions) can be made in HVRs to, for example, improve the affinity of the antibody. Such modifications can be made in HVR "hot points", i.e., residues encoded by codons frequently mutated during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in SDRs (a-CDRs), where the binding affinity of the resulting variant VH or VL is tested. For example, Hoogenboom et al. have previously described affinity maturation by construction and reselection from secondary libraries in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) introduces diversity into the variable genes selected for maturation. A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves the HVR orientation method, in which a number of HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are routinely targeted.

[0189] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (e.g., conservative substitutions according to the present disclosure) may be made in HVRs that do not significantly reduce binding affinity. Such modifications may be outside of HVR "hot points" or CDRs. The above mutant Vs provided herein are H In some embodiments of the H sequence, each HVR is unaltered or contains no more than one, two or three amino acid substitutions.

[0190] As described in Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying antibody residues or regions that can undergo targeted mutagenesis is called "alanine scanning mutagenesis". In this method, a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody-antigen interaction is affected. Additional substitutions can be introduced at the amino acid positions to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted or eliminated as substitution candidates. Mutants may be screened to determine whether they contain the desired properties.

[0191] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., in the case of ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0192] b) Glycosylation mutants In some embodiments, the anti-B7-H3 constructs provided herein are modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to an antibody can be readily accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0193] If the anti-B7-H3 construct comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced from mammalian cells typically contain branched bicontact angle oligosaccharides, which are usually attached to Asn297 in the Fc region CH2 domain via an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may contain a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose attached to GlcNAc in the "stem" of the bicontact angle oligosaccharide structure. In some embodiments, modifications may be made to the oligosaccharides in the anti-B7-H3 constructs of the present application to generate antibody variants with some improved properties.

[0194] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., positions 294-300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L, and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107).

[0195] The anti-B7-H3 construct variants further have aliquoted oligosaccharides, for example, where the bi-contact angle oligosaccharides attached to the antibody Fc region are aliquoted by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Further provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0196] c) Fc domain mutants In some examples, an Fc region variant can be generated by introducing one or more amino acid modifications into the Fc region of an anti-B7-H3 construct according to the present invention. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region), which comprises an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0197] In some examples, the present application contemplates anti-B7-H3 construct variants that have some, but not all, effector functions, making them ideal candidates for applications in which the in vivo half-life of the anti-B7-H3 construct is important, but some effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not have FcγR binding capacity (and thus may lack ADCC activity) but can retain FcRn binding capacity. Primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)), 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods (e.g., ACTI for flow cytometry) can be used. TMNon-radioactive cytotoxicity assays (see Cell Technology, Inc. Mountain View, CA, and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, WI)) may also be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the ELISA for C1q to C3c binding in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0198] Antibodies with reduced effector function include antibodies with substitutions for one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions of two or more at amino acid positions 265, 269, 270, 297 and 327, including an Fc mutant designated "DANA" in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0199] Several antibody variants with improved or decreased binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0200] In some embodiments, the anti-B7-H3 construct variant comprises an Fc region with one or more amino acid substitutions, and these substitutions (e.g., substitutions at positions 298, 333 and / or 334 (EU numbering of residues) within the Fc region) improve ADCC.

[0201] In some examples, modifications occur in the Fc region that result in modified (i.e., enhanced or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0202] In some embodiments, anti-B7-H3 constructs (e.g., HCAb) variants are provided that include a variant Fc region, the variant Fc region comprising one or more amino acid substitutions that extend half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and are described in US 2005 / 0014934 A1 (Hinton et al.). These antibodies include an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0203] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0204] Any of the Fc variants described herein or combined anti-B7-H3 constructs (eg, anti-B7-H3 IgG fused to full length IgG or sdAb) are contemplated.

[0205] In some embodiments, the constant region is a human IgG1 constant region. In some embodiments, the heavy chain constant region comprises a modification that enhances ADCC efficacy. In some embodiments, the heavy chain constant region comprises the mutations K214R, L235V, F243L, R292P, Y300L, D356E, L358M and P396L. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 93 or 94. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 92.

[0206] d) Cysteine ​​Engineered Antibody Variants In some examples, it may be desirable to generate cysteine ​​engineered anti-B7-H3 constructs, e.g., "thioMAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues. In certain examples, the replaced residues are located at accessible sites of the antibody. By replacing those residues with cysteines, reactive thiol groups are located at accessible sites of the antibody that may be used to conjugate the antibody with other moieties, e.g., drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In some examples, any one or more of residues A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region may be replaced with cysteine. Cysteine ​​engineered anti-B7-H3 constructs may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0207] e) Antibody derivatives In some embodiments, the anti-B7-H3 constructs according to the present invention may be further modified to contain other non-protein moieties that are known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) with dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one type of polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy for a defined condition, etc.

[0208] In some embodiments, a conjugate of an anti-B7-H3 construct with a non-protein is provided, where the non-protein moiety can be selectively heated by exposure to radiation. In some embodiments, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Nat'l. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells, but heat the non-protein moiety to a temperature where cells adjacent to the antibody-non-protein moiety are killed.

[0209] In some embodiments, an anti-B7-H3 construct according to the present invention (e.g., an anti-B7-H3 IgG, an anti-B7-H3 / CTLA-4 bispecific antibody, an anti-B7-H3 / PD-L1 bispecific antibody, an anti-B7-H3 / TIM-3 bispecific antibody, or an anti-B7-H3 / LAG-3 bispecific antibody) may be further modified to contain one or more biologically active proteins, polypeptides, or fragments thereof. As used herein, "biologically active" or "having biological activity" refers to exhibiting a biological activity in vivo that performs a specific function. For example, it can mean binding to a specific biomolecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of such a biomolecule. In some embodiments, the biologically active protein or fragment thereof has an immunostimulatory / immunomodulatory activity, a membrane transport activity, or an enzymatic activity.

[0210] In some embodiments, biologically active proteins or fragments thereof that can be fused to the anti-B7-H3 constructs described herein are ligands such as lymphokines and cytokines that interact with specific cell receptors. Lymphokines are low molecular weight proteins secreted by T cells when an antigen or lectin stimulates the growth of the T cells. Examples of lymphokines include, but are not limited to, interferon-α, interferon-γ, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), tumor necrosis factor (TNF), colony stimulating factors (e.g., CSF-1, G-CSF, or GM-CSF), chemokines, macrophage migration inhibitory factor (MIF), macrophage activating factor (MAF), NK cell activating factor, T cell replacement factor, leukocyte inhibitory factor (LIF), lymphotoxin, osteoclast activating factor (OAF), soluble immune response inhibitor (SIRS), growth stimulating factors, targeted growth factors, and the like. Cytokines that can be incorporated into anti-B7-H3 fusion proteins of the invention include, but are not limited to, tumor necrosis factor alpha (TNFα), interferon (IFN), and nerve growth factor (NGF).

[0211] III. Pharmaceutical Compositions The present application further provides pharmaceutical compositions, which include any one of the anti-B7-H3 constructs (e.g., anti-B7-H3 IgG, full-length anti-B7-H3 IgG, anti-B7-H3 / CTLA-4 bispecific antibody, anti-B7-H3 / PD-L1 bispecific antibody, anti-B7-H3 / TIM-3 bispecific antibody, or anti-B7-H3 / LAG-3 bispecific antibody) and an optional pharma- ceutical acceptable carrier agent. The pharmaceutical composition can be prepared by mixing the anti-B7-H3 constructs having the desired purity described herein with an optional pharma- ceutical acceptable carrier agent, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)), and is in the form of a lyophilized formulation or an aqueous solution.

[0212] The pharmaceutical composition is preferably stable, where the anti-B7-H3 construct comprising the anti-B7-H3 mAb described herein substantially retains its physical and chemical stability and integrity upon storage. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. If the formulation is stored at 2-8°C, typically the formulation should be stable at 30°C or 40°C for at least 1 month, and / or at 2-8°C for at least 2 years. When a formulation is stored at 30° C., typically the formulation should be stable for at least 2 years at 30° C. and / or at least 6 months at 40° C. For example, the degree of aggregation over storage time can be used as an indicator of protein stability. In some examples, a stable formulation of an anti-B7-H3 construct described herein may contain less than about 10% (preferably less than about 5%) of the anti-B7-H3 construct present in the formulation as aggregates.

[0213] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonicity agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., Zn-protein complexes), chelating agents such as EDTA, and / or non-ionic surfactants.

[0214] Examples of physiologically acceptable carrier agents include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine acid, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (about 10 residues) glycerides, and the like. groups), proteins such as polypeptides, serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt forming counterions such as sodium ions, metal complexes (e.g., Zn-protein complexes), and / or TWEEN. TM , Polyethylene Glycol (PEG) and PLURONICS TM or non-ionic surfactants such as polyethylene glycol (PEG).

[0215] The buffering agent is for controlling pH within a range that optimizes therapeutic efficacy, especially when stability is pH dependent. The buffering agent is preferably present at a concentration of about 50 mM to about 250 mM. Buffering agents suitable for the present application include organic acids, inorganic acids and their salts, such as citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, and acetates. Buffering agents may also include histidine salts and trimethylamine salts, such as Tris.

[0216] Preservatives are added to retard microbial growth, and are usually present in the range of 0.2% to 1.0% (w / v). The addition of a preservative can, for example, facilitate the manufacture of multi-use (multi-dose) formulations. Preservatives suitable for this application include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium halides (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzethonium chloride, thimerosal, phenol, butanol or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

[0217] Tonicity agents (sometimes called "stabilizers") may be present to adjust or maintain the liquid tension in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often called "stabilizers" because they can reduce the possibility of inter- and intra-molecular interactions by interacting with the charged groups of the amino acid side chains. Taking into account the relative amounts of other ingredients, tonicity agents can be present in any amount from 0.1% to 25% by weight, preferably 1% to 5% by weight. Preferred tonicity agents include polyhydroxy sugar alcohols, preferably trihydroxy or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0218] Additional excipients include agents that may be used as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or sticking to container walls. Such excipients include polyhydroxy sugar alcohols (listed above), amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like, and sugars such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), These include organic sugars or sugar alcohols such as polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0219] A non-ionic surfactant or detergent (also called a "wetting agent") may be present to facilitate dissolution of the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, thereby allowing the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in the range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0220] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC™ polyols, TRITON™, polyoxyethylene sorbitan monoethers (TWEEN™-20, TWEEN™-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Usable anionic detergents include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0221] The pharmaceutical composition must be sterile for use in in vivo administration. The pharmaceutical composition can be sterilized by filtration through a sterile filtration membrane. The pharmaceutical composition herein is usually placed into a container with a sterile inlet, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0222] The route of administration is in accordance with known and acceptable methods, such as single or multiple bolus injections or injection or infusion by suitable routes, e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation, or prolonged infusion by sustained or delayed release modes. In some embodiments, the pharmaceutical composition is administered locally, e.g., intratumorally.

[0223] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, these matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT TM (injectable microspheres of poly(lactic-co-glycolic acid) and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyrate.

[0224] The pharmaceutical compositions herein may further comprise one or more active compounds as required for the particular indication being treated, preferably with those active compounds having complementary activities that do not adversely affect each other. Alternatively or additionally, the compositions may comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant agent or a growth inhibitory agent. Such molecules are present in any suitable combination in amounts effective for the desired purpose.

[0225] The active ingredient may also be embedded in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Edition.

[0226] In some embodiments, the pharmaceutical composition is contained in a single-use vial, e.g., a sealed single-use vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a container in bulk. In some embodiments, the pharmaceutical composition is stored frozen.

[0227] IV. Method of Use or Application The anti-B7-H3 constructs described herein, including mAbs that specifically recognize B7-H3 (e.g., anti-B7-H3 full length IgG, anti-B7-H3 / CTLA-4 bispecific antibody, anti-B7-H3 / PD-L1 bispecific antibody, anti-B7-H3 / TIM-3 bispecific antibody, or anti-B7-H3 / LAG-3 bispecific antibody), and compositions thereof (e.g., pharmaceutical compositions) can be used in a variety of applications, such as diagnostics, molecular assays, and therapy.

[0228] In one aspect of the invention, there is provided a method of treating a B7-H3-associated disease or condition in an individual in need thereof, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-B7-H3 construct as described herein. In some embodiments, the B7-H3-associated disease is cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), or breast cancer.

[0229] The present application contemplates, in part, a protein construct (e.g., an anti-B7-H3 full length IgG, an anti-B7-H3 / CTLA-4 bispecific antibody, an anti-B7-H3 / PD-L1 bispecific antibody, an anti-B7-H3 / TIM-3 bispecific antibody, or an anti-B7-H3 / LAG-3 bispecific antibody), a nucleic acid molecule and / or vector encoding the protein construct, and a host cell comprising a nucleic acid molecule and / or vector encoding the protein construct, which may be administered alone or in combination with another therapy, and in at least some embodiments, may be administered with a pharma- ceutically acceptable carrier agent or excipient. In some embodiments, prior to administration of the anti-B7-H3 constructs, they may be combined with suitable drug carrier agents and excipients well known in the art. Compositions prepared according to the present disclosure may be used to treat or delay the progression of cancer.

[0230] In some embodiments, a method of treating cancer is provided, the method comprising administering to an individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising an isolated anti-B7-H3 construct (e.g., an anti-B7-H3 full length IgG, an anti-B7-H3 / CTLA-4 bispecific antibody, an anti-B7-H3 / PD-L1 bispecific antibody, an anti-B7-H3 / TIM-3 bispecific antibody, or an anti-B7-H3 / LAG-3 bispecific antibody) comprising a mAb that specifically recognizes B7-H3. In some embodiments, the cancer is a solid tumor (e.g., lung cancer). In some embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously). In some embodiments, the pharmaceutical composition is administered locally (e.g., intratumorally). In some embodiments, the method further comprises administering to the individual an additional cancer therapy (e.g., surgery, radiation, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof). In some embodiments, the individual is a human. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing), (2) inhibiting cancer cell proliferation, (3) inducing an immune response in a tumor, (4) reducing tumor size, (5) alleviating one or more symptoms of an individual with cancer, (6) inhibiting tumor metastasis, (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the method of cancer cell killing mediated by the pharmaceutical compositions described herein can achieve any one of at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher percent tumor cell mortality. In some embodiments, the cancer cell killing methods mediated by the pharmaceutical compositions described herein can achieve at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher percent mortality of bystander tumor cells (not infected by the oncolytic cell VV).In some embodiments, the methods of reducing tumor size mediated by the pharmaceutical compositions described herein can reduce tumor size by at least about 10% (e.g., including at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, the methods of inhibiting tumor metastasis mediated by the pharmaceutical compositions described herein can inhibit metastasis by at least about 10% (e.g., including at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, the methods of extending individual (e.g., human) survival mediated by the pharmaceutical compositions described herein can extend the survival of the individual by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months. In some embodiments, the methods of increasing the time to cancer progression mediated by the pharmaceutical compositions described herein can increase the time to cancer progression by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0231] The methods described herein may be used to treat a variety of cancers, including both solid and liquid cancers. These methods are suitable for all stages of cancer, including early stage, non-metastatic, primary, advanced, locally advanced, metastatic, or remission. The methods described herein may be used in adjuvant or novel adjuvant treatments, as a combination therapy with first therapy, second therapy, third therapy, or other types of cancer therapy known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (e.g., T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc. (i.e., the method may be performed before the main / definitive therapy). In some embodiments, the method is for treating a previously treated individual. In some embodiments, the cancer has become refractory to a previous therapy. In some embodiments, the method is for treating a previously untreated individual.

[0232] In some embodiments, the methods are suitable for treating cancers with aberrant B7-H3 expression, activity and / or signaling, non-limiting example methods include bladder cancer, cervical cancer, colon cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, breast cancer, head and neck cancer, skin cancer, sarcoma, brain cancer, brain and spinal cord cancer, adrenal cancer, uterine cancer, neuroblastoma, small round cell tumor, peripheral nerve sheath tumor, bone cancer, rhabdomyoma, lymphoma, multiple myeloma, leukemia, neuroendocrine tumors, and melanoma.

[0233] Thus, in some embodiments, a method is provided for treating an immunotherapy-responsive solid tumor (e.g., a carcinoma or adenocarcinoma, e.g., a cancer having aberrant B7-H3 expression, activity and / or signaling), the method comprising administering to an individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising an isolated anti-B7-H3 construct (e.g., an anti-B7-H3 full length IgG, an anti-B7-H3 / CTLA-4 bispecific antibody, an anti-B7-H3 / PD-L1 bispecific antibody, an anti-B7-H3 / TIM-3 bispecific antibody, or an anti-B7-H3 / LAG-3 bispecific antibody) that contains a monoclonal antibody that specifically recognizes B7-H3. In some embodiments, the cancer is a solid tumor (e.g., lung cancer). In some embodiments, the pharmaceutical composition is administered systemically (e.g., intravenously). In some embodiments, the pharmaceutical composition is administered locally (e.g., intratumorally). In some embodiments, the method further comprises administering additional cancer therapy (e.g., surgery, radiation, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof) to the individual. In some embodiments, the individual is a human. In some embodiments, the method of treating cancer has one or more biological activities of: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing an immune response in the tumor; (4) reducing tumor size; (5) alleviating one or more symptoms of an individual with cancer; (6) inhibiting tumor metastasis; (7) increasing survival time; (8) increasing the time to cancer progression; and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the method of cancer cell killing mediated by the pharmaceutical compositions described herein can achieve any one of at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher percent tumor cell mortality. In some embodiments, the cancer cell killing methods mediated by the pharmaceutical compositions described herein can achieve at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher percent mortality of bystander tumor cells (not infected by the oncolytic cell VV).In some embodiments, the methods of reducing tumor size mediated by the pharmaceutical compositions described herein can reduce tumor size by at least about 10% (e.g., including at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, the methods of inhibiting tumor metastasis mediated by the pharmaceutical compositions described herein can inhibit metastasis by at least about 10% (e.g., including at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, the methods of extending individual (e.g., human) survival mediated by the pharmaceutical compositions described herein can extend the survival of the individual by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months. In some embodiments, the methods of increasing the time to cancer progression mediated by the pharmaceutical compositions described herein can increase the time to cancer progression by at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0234] The dosage and desired drug concentration of the pharmaceutical composition of the present application may vary depending on the specific intended use. The determination of the appropriate dosage or route of administration is within the skill of the art. Animal experiments provide reliable guidance for determining effective dosages for human treatment. Interspecies analogy of effective dosages may be performed according to the principles set forth in Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., eds., Pergamon Press, New York 1989, pages 42-46.

[0235] For in vivo administration of anti-B7-H3 constructs comprising anti-B7-H3 mAbs as described herein, normal dosages may vary from about 10 ng / kg to about 100 mg / kg of mammalian body weight per day or more, preferably about 1 mg / kg / day to 10 mg / kg / day, e.g., about 1-3 mg / kg / day, about 2-4 mg / kg / day, about 3-5 mg / kg / day, about 4-6 mg / kg / day, about 5-7 mg / kg / day, about 6-8 mg / kg / day, about 6-6.5 mg / kg / day, about 6.5-7 mg / kg / day, about 7-9 mg / kg / day, or about 8-10 mg / kg / day, depending on the route of administration. Within the scope of this application, different formulations are effective for different treatments and different diseases, and administrations aimed at treating a particular organ or tissue may need to be delivered in a different manner than delivery to another organ or tissue. The doses can then be administered separately, one or more times, or by continuous infusion. For repeated administration over several days or longer, depending on the condition, treatment is continued until a desired suppression of disease symptoms occurs. However, other dosage schemes may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0236] In some embodiments, the pharmaceutical composition is administered once (e.g., bolus injection). In some embodiments, the pharmaceutical composition is administered multiple times (e.g., any one of 2, 3, 4, 5, 6 or more times). In the case of multiple administrations, they may be administered by the same or different routes and may be administered at the same site or at alternative sites. The pharmaceutical composition may be administered twice a week, three times a week, four times a week, five times a week, once a day, once a day without interruption, once a week, once a week without interruption, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once a year. The interval between doses may be any one of about 24h to 48h, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 3 months, 3 months to 6 months, or 6 months to 1 year. The interval may be irregular (e.g., after tumor progression). In some embodiments, the dosing regimen is not interrupted. In some embodiments, the pharmaceutical composition is administered four times every four days. The optimal dosage and treatment scheme for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's disease symptoms and adjusting the treatment accordingly.

[0237] The pharmaceutical compositions of the present application (including but not limited to reconstituted and liquid formulations) are administered to an individual, preferably a human, in need of treatment with the anti-B7-H3 constructs described herein according to known methods, such as intravenous administration as a bolus or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal fluid, subcutaneous, intravenous (iv), intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. Reconstituted formulations may be prepared by dissolving the lyophilized anti-B7-H3 constructs described herein in a diluent and dispersing the protein therein. Exemplary pharma- ceutically acceptable (safe and non-toxic for administration to humans) diluents suitable for the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution or glucose solution, or aqueous solutions of salts and / or buffers.

[0238] In some embodiments, the pharmaceutical composition is administered to an individual by subcutaneous (i.e., under the skin) administration. For such purposes, the pharmaceutical composition may be injected with a syringe. However, other devices for administering the pharmaceutical composition are available, such as injection devices, injection pens, autoinjector devices, needle-free devices, and subcutaneous patch delivery systems.

[0239] In some embodiments, the pharmaceutical composition is administered to the individual intravenously. In some embodiments, the pharmaceutical composition is administered to the individual by injection (e.g., intravenous injection). Injection techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)).

[0240] The anti-B7-H3 constructs described herein, including mAbs that specifically recognize B7-H3 (e.g., anti-B7-H3 full length IgG, anti-B7-H3 / CTLA-4 bispecific antibody, anti-B7-H3 / PD-L1 bispecific antibody, anti-B7-H3 / TIM-3 bispecific antibody, or anti-B7-H3 / LAG-3 bispecific antibody), and compositions thereof (e.g., pharmaceutical compositions) may also be used in diagnostic or molecular assays. For example, the antibodies or antigen-binding fragments thereof may be used to detect or monitor the progression or treatment of a disease associated with B7-H3 (e.g., the diseases described above) by detecting or quantifying B7-H3 in a biological sample.

[0241] Administration of the antibody, antigen-binding fragment, conjugate, bispecific antibody, or composition may be accompanied by administration of other anti-cancer or anti-angiogenic agents or therapeutic treatments (e.g., surgical removal of the tumor or radiation therapy). For example, the subject may receive one or more additional therapies before, during, or after administration of a therapeutic amount of the antibody or conjugate. In one example, before administering a therapeutic amount of one or more agents for treating a tumor, the subject receives one or more therapies to eliminate or reduce the tumor. For example, the additional agents may include, but are not limited to, chemotherapeutic agents, anti-angiogenic agents, or combinations thereof. In another example, at least a portion of the tumor is surgically or otherwise removed or reduced in size or volume before administering a therapeutic amount of the antibody or antigen-binding fragment thereof, or conjugate.

[0242] Specific examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenesis agent can be administered in combination with the antibodies, conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and can be determined by a skilled clinician. In one example, the chemotherapeutic agent includes 5-FU or IRT, or both 5-FU and IRT.

[0243] V. Manufacturing method The anti-B7-H3 constructs described herein (eg, anti-B7-H3 monoclonal antibodies) can be produced by any method known to one of skill in the art or described herein.

[0244] Rodent monoclonal antibodies can be obtained by methods known to those skilled in the art, such as by immunizing rodent species (e.g., mouse or rat) and obtaining hybridomas therefrom, or by cloning Fab fragment or single chain Fc (scFv) libraries by molecular biology techniques known in the art, followed by selection of single clones of unselected libraries by ELISA, or by using phage display.

[0245] To produce a monoclonal antibody recombinantly, the nucleic acid encoding the monoclonal antibody is isolated or synthesized and inserted into a replicable vector for further cloning (DNA amplification) or expression. DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to antibody-encoding heavy and light chain genes). Many vectors are available. The choice of vector depends on the host cell to be used. Usually, the preferred host cells are of prokaryotic or eukaryotic (usually mammalian) origin.

[0246] example The following examples are intended to be merely illustrative of the invention and should not be construed as limiting the invention in any manner. The following specific examples and detailed description are offered by way of illustration and not by way of limitation.

[0247] Example 1: Production of anti-B7-H3 monoclonal antibodies (mAbs) immunity A / J mice were subcutaneously immunized under the abdominal skin with 25 μg of human B7-H3 protein immunization (AcroBiosystems, Cat. No. B7B-H52E7), which was prepared as an emulsion with adjuvants (CFA, complete Freund's adjuvant used for primary immunization, IFA, incomplete Freund's adjuvant used for booster immunization) in a volume of 200 μl. Each mouse received 3-5 doses. Seven days after each time point of immunization (except the last immunization), 20 μl blood samples were collected from the animals and antiserum titers were monitored in an ELISA assay using immobilized B7-H3-His protein until fusion criteria were met.

[0248] Selection of hybridomas secreting anti-human B7-H3 antibodies Three days after the last immunization, spleen cells from selected mice were extracted and fused with SP2 / 0 cells according to a standard hybridoma generation scheme in a sterile environment. The fused cells were cultured in DMEM medium (supplemented with 10% FBS) containing 1x HAT (hypoxanthine-aminopterin-thymidine) for 7 days. The hybridoma supernatants were analyzed for their binding ability to human B7-H3, cynomolgus monkey B7-H3 or mouse B7-H3 by ELISA, respectively. Positive hybridoma clones were subcloned by finite dilution and cultured in DMEM medium (supplemented with 10% FBS) containing 1x HT (hypoxanthine-thymidine). The cells were cultured for one week and new screening was performed until a positive monoclonal was obtained. Each clone was used to produce 0.5 mg of antibody, which was purified using protein A chromatography and used for further characterization. Antibody isotypes were tested using the Clone Typing System-HRP, Southern Biotech.

[0249] Example 2: In vitro characterization of B7-H3 mouse antibody CHO-K1 / B7-H3 cell binding measured by FACS Purified antibodies from positive clones were used in FACS binding assays against the CHO-K1 / B7-H3 cell line (Probio Biotech), where the binding EC 50 Values ​​(Figure 1) were generated. Positive clones include 12C9F1B5, 38F11B2, 34A9C12, 37D11F5B8, 37F11A10, 43H11G12, 47F3F10B4, 47C7E3, 31H11B1, 38D6A4, MGA271. Mouse IgG was used as an isotype control.

[0250] EC of FACS binding measurements 50 The values ​​are shown in Table 4. All positive clones have higher binding affinity than the reference antibody (enoblitumb) for MGA271.

[0251] [Table 4]

[0252] Example 3: Production and analysis of chimeric antibodies Construction of chimeric antibodies for characterization The variable region coding sequences of the ten mAbs were optimized for human codon bias expression using the GenScript online tool. The variable DNA fragments were synthesized and fused with human IgG1 heavy chain domains (CH1-hinge-CH2-CH3) and light chain kappa constant region (CL) and used for transient expression in chimeric form. The heavy and light chain expression constructs were cloned into a single pTT5-based plasmid and used for secretory expression after signal peptide synthesis.

[0253] Chimeric antibodies were expressed in HEK293-6E cells transfected with plasmids carrying antibody heavy / light chain pairs using PEImax 40,000 (Polysciences). After 24 hours, expression / secretion was enhanced with Tryptone N-1 supplement (Kirgen Bioscience). Incubated at 37°C, 5% CO 2 After incubation for 5 days with shaking under reduced pressure, the supernatant was collected and the antibody content was purified with Protein-A beads (Genscript). The chimeric antibody product was stored in PBS and used for analysis.

[0254] CDC analysis of anti-B7-H3 antibody induction CDC assay was performed on chimeric antibodies. Target cell lines (human B7-H3 overexpressing CHO-K1) were cultured and harvested, and the target cell lines were seeded in 384-well plates (5E3 cells / well) at a specific cell density. Antibody samples were added to the plates accordingly, and the plates were incubated at 37°C / 5% CO 2The plates were incubated under low-temperature for 30 min. Purified normal human serum complement (NHSC, SIGMA-ALDRICH) was then added to the plates and the plates were incubated for an additional 4 h. The plates were removed from the incubator and cell viability was analyzed with the Cell Titer-Glo™ Assay Kit (Promega). Luminescence data was captured by PheraStar (BMG) and used for cell viability analysis. The CDC assay results, expressed as % target cell lysis versus candidate antibody concentration, are shown in Figure 2.

[0255] EC of all candidate chimeric antibodies from CDC assay 50 The values ​​are summarized in Table 5. Clone 43H11G12-hIgG1 had the lowest EC 50 The value is 0.1217 μg / ml. The EC of the self-synthesized positive control with the same amino acid sequence as DS-5573a (patent number: US 2013 / 0078234 A1) 50 The values ​​were used to calculate the relative activity of each candidate antibody (% relative activity = (EC of positive control) 50 / EC of sample 50 )*100%). All chimeric mAb samples showed relative activity greater than 100%.

[0256] [Table 5]

[0257] Anti-B7-H3 antibody-induced ADCC assay The assay procedure involved culturing and harvesting a target cell line (human B7-H3 overexpressing CHO-K1) (Probio Biotech), seeding the target cell line at a specific cell density into a 96-well plate. A chimeric antibody sample or positive control, which is synthesized in-house and has the same amino acid sequence as MGA-271 (enoblituzumab) (patent number: US 2012 / 0294796 A1), was added to the plate, and the plate was incubated at 37°C / 5% CO. 2The plates were incubated for 30 min under 5% CO. NK92 / CD16a-VV cells were used as effector cells, added to the plates and incubated for 6 h under the same conditions. The assay plates were removed and briefly centrifuged. Supernatants were collected and transferred to new plates for LDH activity assay according to the manufacturer's instructions (Roche). Absorbance data were captured by PheraStar (BMG) and analyzed with GraphPad Prism 6.0. The ADCC effects of all chimeric antibodies were compared. The ADCC assay results, expressed as % target cell lysis versus candidate antibody concentration, are shown in Figure 5.

[0258] Table 6 shows the ECs of the following candidate chimeric antibodies from the ADCC assay (two plates in one experiment): 50 The values ​​are summarized. Clone 43H11G12-hIgG1 had the lowest EC 50 The EC value is 0.001615 μg / ml. The EC value of the in-house synthesized positive control, which has the same amino acid sequence as MGA-271 (enoblitutuzumab) (patent number: US 2012 / 0294796 A1), was 50 The values ​​were used to determine the relative activity (% relative activity = (EC of positive control)) of each candidate antibody in each assay plate. 50 / EC of sample 50 )*100) was calculated. All chimeric mAb samples showed relative activity greater than 300%.

[0259] [Table 6]

[0260] Example 4. Production and analysis of humanized antibodies Humanized design of candidate antibodies Based on the effectiveness of the antibodies in functional assays and their variable domain sequences, modeled structures of mAbs 47F3F10B4, 12C9F1B5, 43H11G12 and 31H11B1 were obtained by analyzing CDRs and FRs and performing homology modeling. The solvent accessible surface area of ​​framework residues was calculated. Based on the results, buried framework residues (i.e., solvent accessible surface area <15%) were identified. Human receptors with high sequence identity to their mouse counterparts, VH and VL, were selected. The sequences of grafted antibodies without any back mutations were obtained by directly grafting the CDRs of mouse antibodies into the human receptor framework. Post-translational modifications and chemical degradations, including deamidation, isomerization oxidation and glycosylation, in the grafted sequences were analyzed by developability assessment. PTM hot points, such as N-glycosylation sites, abnormal proline residues, deamidation sites, isomerization sites, oxidation sites and unpaired cysteine ​​residues, were identified, which may affect the binding activity and manufacturability of the grafted antibodies. The DNA sequences encoding the humanized light and heavy chains were synthesized. The antibody characteristics were compared to select the best candidate antibodies. These humanized molecules were successfully produced.

[0261] Humanized antibodies The humanized heavy and light chains of the antibodies were combined and used to produce the antibodies. Detailed information on each heavy and light chain combination is shown in Table 7. In addition, a heavy chain mutant of human IgG1 called IgG1m (K214R, L235V, F243L, R292P, Y300L, D356E, L358M and P396L) was used to enhance the ADCC effect.

[0262] [Table 7]

[0263] CHO-K1 / B7-H3 cell binding measured by FACS To verify the cell surface antigen binding of the humanized antibodies, CHO-K1 cells expressing human B7-H3 were harvested and incubated with anti-B7-H3 mAb, followed by incubation with goat anti-human IgG (H+L) secondary antibody (Invitrogen). Samples were then analyzed by flow cytometry. A typical binding diagram is shown in Figure 4.

[0264] FACS binding ECs 50 The values ​​are summarized in Table 8. All humanized antibodies showed much higher binding affinity than the reference antibodies MGA271 (Patent No: US 2012 / 0294796 A1, where the heavy chain is IgG1m as above) and MGA271-hIgG1 (where the heavy chain is wild type human IgG1 and other amino acid sequences are the same as MGA271).

[0265] [Table 8]

[0266] CDC assay of anti-B7-H3 antibody induction CDC assay was performed on humanized antibodies. Target cell lines (human B7-H3 overexpressing CHO-K1) were cultured and harvested, and the target cell lines were seeded in 96-well plates (5E3 cells / well) at a specific cell density. Antibody samples were added to the plates accordingly, and the plates were incubated at 37°C / 5% CO 2 The plates were incubated under low temperature for 30 minutes. Purified normal human serum complement (NHSC, SIGMA-ALDRICH) was then added to the plates and the plates were incubated for an additional 4 hours. The plates were removed from the incubator and cell viability was analyzed with a Cell Titer-Glo™ Assay Kit. Luminescence data was captured by PheraStar (BMG) and used for cell viability analysis. The CDC assay results, expressed as % target cell lysis versus candidate antibody concentration, are shown in Figure 5.

[0267] Table 9 shows the EC values ​​of all candidate humanized antibodies from the CDC assay (two plates in one experiment). 50The EC values ​​are summarized. The EC values ​​of the positive control synthesized in-house, which has the same amino acid sequence as DS-5573a (patent number: US 2013 / 0078234 A1), 50 The values ​​were used to determine the relative activity (% relative activity = (EC of positive control)) of each candidate antibody in each assay plate. 50 / EC of sample 50 )*100%). Clones 31H11B1-VH1-VL3 (IgG1m), 31H11B1-VH1.1-VL3 (IgG1m), and 47F3F10B4-VH1-VL4 (IgG1m) showed relative activity of over 300%.

[0268] [Table 9]

[0269] Anti-B7-H3 antibody-induced ADCC assay In the assay procedure, the target cell lines A549 (Plate 01 and Plate 02) and Detroit 562 (Plate 03 and Plate 04) were cultured, harvested, and seeded into 96-well plates at a specific cell density (1E4 cells / well). A humanized antibody sample or positive control with the same amino acid sequence as MGA-271 (enoblituzumab) (Patent No.: US 2012 / 0294796 A1), synthesized in-house, was added to the plate, and the plate was incubated at 37°C / 5% CO. 2The plates were incubated at 4°C for 30 min. An internally constructed ADCC reporter cell line (GS-J2C / CD16A) was used as effector cells, which was added to the plate and incubated under the same conditions for 6 h. The assay plate was removed and the activation of the ADCC reporter cell line was analyzed with Bio-Glo™ Assay Kit (Promega). Luminescence data was captured by PheraStar (BMG) and used for cell viability analysis. Absorbance data was captured by PheraStar (BMG) and analyzed with GraphPad Prism 6.0. The ADCC effect of humanized antibodies was compared. The results of the ADCC assay are shown in Figure 6A and B.

[0270] EC of the following humanized antibodies from ADCC assay 50 The values ​​are summarized in Table 10. Clone 47F3F10B4-VH1-VL4 (IgG1m) had the lowest EC 50 The EC values ​​were shown for the in-house synthesized positive control with the same amino acid sequence as MGA-271 (enoblitutuzumab) (patent number: US 2012 / 0294796 A1). 50 The values ​​were used to determine the relative activity (% relative activity = (EC of positive control)) of each candidate antibody in each assay plate. 50 / EC of sample 50 )*100) was calculated. 31H11B1-VH1-VL3(IgG1m), 31H11B1-VH1.1-VL3(IgG1m) and 47F3F10B4-VH1-VL4(IgG1m) showed greater than 1000% relative activity targeting A549 cells and greater than 600% relative activity targeting Detroit 562 cells in the ADCC assay.

[0271] These in vitro assay results provide a possible treatment for solid tumors (HNSCC, NSCLC), and the safety and efficacy of these antibodies need to be proven in further preclinical and clinical studies.

[0272] [Table 10]

[0273] Listed below are some of the amino acid and nucleic acid sequences referred to herein. The underlined amino acid sequences are CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, respectively.

[0274] >47F3F10B4-VH(SEQ ID NO:1) EVQLQQSGPELVKPGASVKMSCKASGYTFT DHYMH WVKQSHGKSLEWIG YIFPNNG VSGYNQNFKD KATLTVDKSSSTAYMDLRSLTPEDSAVYYCTR RQLGLGTMDY WGQGTSVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 2, 3 and 4, respectively) >47F3F10B4-VL(SEQ ID NO:5) DIVLTQSPASLAVSLGQRATISC RASKSVSTSGYNYMH WYQQKPGQPPKLLIY LASNL ES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSKELPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 6, 7 and 8, respectively) >12C9F1B5-VH(SEQ ID NO:9) QGQIQLVQSGPELKKPGETVKISCKASGFTNY GMN WVKQAPGKGLKWMG WINPYSG VPTYADDFKG RFAFSLETSASTAYLQINNLKNEDTATYFCAR DDGFHYTMDYWGQGT SVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 10, 11 and 12, respectively) >12C9F1B5-VL(SEQ ID NO:13) DVVLTQTPLSLPVSLGDQASISC RSSQNLVHSNGITYLH WYLQKPGQSPKLLIY KVSNR FS GVPDRFSGSGSGTDFTLTISRVEAEDLGVYFC SQSTLVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 14, 15 and 16, respectively) >34A9C12-VH(SEQ ID NO:17) QGQIQLVQSGPELKKSGETVKISCKASGFTNY GIN WVKQAPGKGLKWMG WINPYSGV PTYADDFKG RFAFSLETSASTAYLQINNLKNEDSATYFCAR DDGFHYTMDY WGQGTS VTVSS (underlined CDR amino acid sequences are designated as SEQ ID NOs: 18, 19 and 20, respectively) >34A9C12-VL(SEQ ID NO: 21) DVVLIQTPLSLPVSLGDQASISC RSSQNLVHSNGITYLH WYLQKPGQSPKLLIY KVSNR FS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTLVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 22, 23 and 24, respectively) >38D6A4-VH(SEQ ID NO: 25) QIQLVQSGPELKKPGEAVKISKASGSTFT DFGMN WVRQAPGKGLKWMG WINPYSG VPTYADEFKG RFVFSLETSARVAYMQINDLKNEDTATYFCAR DDGYHYTMDY WGQGTSVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 26, 27 and 28, respectively) >38D6A4-VL(SEQ ID NO:29) DVVMTQTPLPLPVSLGDQASISC RSSQSLIHSNGITYLH WYLQKPGQSPKLLIY KVSNR FS GVPDRFTGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGSKLEIR (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 30, 31 and 32, respectively) >37D11F5B8-VH(SEQ ID NO: 33) QIQLVQSGPELKKPGETVKISCKASGDTFR NNGMN WVKQAPGKGLKWMG WINPYSG VPTYADDFKG RFAFSLETSASTAFLQINNLKNEDTATYFCAR DDGYHYTMDF WGLGT SVTVSS (underlined CDR amino acid sequences are designated as SEQ ID NOs: 34, 35 and 36, respectively) >37D11F5B8-VL(SEQ ID NO:37) DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGITYLH WYLQKPGQSPKLLTY KVSH RFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 38, 39 and 40, respectively) >37F11A10-VH(SEQ ID NO: 41) QIQLVQSGPELKKPGETVKISCKASGDTFR NYGMN WVKQAPGKGLKWMG WINPYSG VPTYADDFKG RFAFSLETSASTAFLQINNLKNEDTATYFCAR DDGFHYTMDY WGLGT SVTVSS (underlined CDR amino acid sequences are designated as SEQ ID NOs: 42, 43 and 44, respectively) >37F11A10-VL(SEQ ID NO: 45) DVVMTQTPLSLPVSLGDQASISC RSSQSLLHSNGITYLH WYLQKPGQSPKLLTY KVSH RFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIR (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 46, 47 and 48, respectively) >43H11G12-VH(SEQ ID NO:49) QIQLVQSGPELKKPGETVKISCKASGYNFR DFGMN WVKQAPGKGLKWMG WLNSYSG VPTYADDFKG RFVLSLETSASTAYLQINNLINEDTATYFCGR DDGYNYTMNY WGQGT SVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 50, 51 and 52, respectively) >43H11G12-VL(SEQ ID NO:53) DVVMTQAPLSLPVSLGDQASISC RSSQSLVHSNGITYLH WYLQRAGQSPQLLIY KVSN RFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 54, 55 and 56, respectively) >38F11B2-VH(SEQ ID NO: 57) QIQLVQSGPELKKPGETVKISCKASGYTFI DYGMN WVKQAPGKGLKWMG WINIYSGV PTYADDFKG RFAFSLETSANTAYLQINNLKNEDTATYFCAR DDGYHYTMNY WGQGT SVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 58, 59 and 60, respectively) >38F11B2-VL(SEQ ID NO: 61) DIVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGITYLH WYLQKSGQSPKLLIY KVSNR FS GVPDRFSGSGSGTAFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 62, 63 and 64, respectively) >47C7E3-VH(SEQ ID NO: 65) QIQLVQSGPELKKPGETVKISCKASGYTFT NFGMN WVKQAPGKGLKWMG WIYTPSG VPIYADDFKG RFAVSLETSASTAYLQINNLKNEDTATYFCAR DDGYYYTMDY WGQGT SVTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 66, 67 and 68, respectively) >47C7E3-VL(SEQ ID NO: 69) DDVMTQTPLSLPVSLGDHASISC RSSQSLVHSNGDTYLH WYLQKPGQSPKLLIY KVSN RFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 70, 71 and 72, respectively) >31H11B1-VH(SEQ ID NO: 73) QVQLQQPGAELGKPGTSVKLSCKASGYTFT SYWMH WVKQRPGQGLEWIG HINPNSG STNQNAKFKN KATLTVDESSSTAYMQLSTLTSEDSAVYYCTR SGSNYRRNYFDY WGQ GTTLTVSS (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 74, 75 and 76, respectively) >31H11B1-VL(SEQ ID NO:77) DIQMTQSSSYLSVSLGGRVTITC KASDHINNWLA WYQQKPGNAPRLLIS GATSLET GV PSRFSGSGSGKDYTLSITSLQTEDVATYYC QQYWSTPLT FGAGTKLELK (the underlined CDR amino acid sequences are designated as SEQ ID NOs: 78, 79 and 80, respectively) The following protein sequences are humanized mAb sequences.

[0275] >47F3F10B4-VH1(SEQ ID NO: 81) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DHYMH WVRQAPGQGLEWMG YIFPNN GVSGYNQNFKD RVTTMTRDTTSISTAYMELSRLRSDDTAVYYCAR RQLGLGTMDY WGQ GTLVTVSS >47F3F10B4-VH2(SEQ ID NO: 82) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DHYMH WVRQAPGQGLEWMG YIFPNNGVSGYNQNFKD RVTTMTRDTTSTSTVYMELSSLRSEDTAVYYCAR RQLGLGTMDY WGQGTLVTVSS >12C9F1B5-VH2.1(SEQ ID NO: 83) QVQLVQSGAEVKKPGASVKVSCKASGFTNY GMN WVRQAPGQGLEWMG WINPYSGVPTYADDFKG RVTTMTRDTTSISTAYMELSRLRSDDTAVYYCAR DDSFHYTMDY WGQGT LVTVSS >12C9F1B5-VH4(SEQ ID NO: 84) QVQLVQSGAEVKKPGASVKVSCKASGFTNY GMN WVRQAPGQRLEWMG WINPYSGVPTYADDFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR DDGFHYTMDY WGQGT LVTVSS >43H11G12-VH4(SEQ ID NO: 85) QVQLVQSGAEVKKPGASVKVSCKASGYNFR DFGMN WVRQAPGQRLEWMG WLNSYSGVPTYADDFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR DDGYNYTMNY WG QGTLVTVSS >31H11B1-VH1(SEQ ID NO: 86) QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG HINPNSGSTNQNAKFKN RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR SGSNYRRNYFDY W GQGTLVTVSS >31H11B1-VH1.1(SEQ ID NO: 87) QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG HINPNSGSTNQNAKFKN RVTMTRDTSISTAYMELSRLRSDDTAVYYCTR SGSNYRRNYFDY W GQGTLVTVSS >47F3F10B4-VL4(SEQ ID NO: 88) DIQMTQSPSSLSASVGDRVTITC RASKSVSTSGYNYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHSKELPWT FGQGTKVEIK >12C9F1B5-VL2(SEQ ID NO: 89) DIVMTQSPLSLPVTPGEPASISC RSSQNLVHSNGITYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTLVPWT FGQGTKVEIK >43H11G12-VL2(SEQ ID NO: 90) DIVMTQSPLSLPVTPGEPASISC RSSQSLVHSNGITYLH WYLQKPGQSPQLLIY KVSNRF S GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPWT FGQGTKVEIK >31H11B1-VL3(SEQ ID NO: 91) DIQMTQSPSSLSASVGDRVTITC KASDHINNWLA WYQQKPGKAPKLLIY GATSLET GV PSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYWSTPLTFGGGTKLEIK Human κ light chain constant region (SEQ ID NO: 92): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Human IgG1 heavy chain constant region (SEQ ID NO: 93): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1m sequence (SEQ ID NO: 94): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof, I) 1) a heavy chain determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66 and 74; 2) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, and 75; 3) a heavy chain variable domain (VH) comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, and 76; II) 1) a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, and 78; 2) a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, and 79; 3) a light chain variable domain (VL) comprising a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, and 80; wherein said antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment thereof capable of specifically binding to B7-H3, preferably human B7-H3.

2. 1) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively; 2) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; 3) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively; 4) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 30, 31 and 32, respectively; 5) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 38, 39 and 40, respectively; 6) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 42, 43 and 44, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; 7) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 50, 51 and 52, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 54, 55 and 56, respectively; 8) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 58, 59 and 60, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 62, 63 and 64, respectively; 9) the VH comprises heavy chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 66, 67 and 68, respectively, and the VL comprises light chain CDR1, CDR2 and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 70, 71 and 72, respectively; or 10) The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises heavy chain CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 74, 75, and 76, respectively, and the VL comprises light chain CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 78, 79, and 80, respectively.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VH comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, and 73, and the VL comprises an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, and 77, respectively.

4. 4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, and 73, or the VH includes at most about three variants thereof with amino acid substitutions, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, and 77, or the VL includes at most about three variants thereof with amino acid substitutions.

5. 1) the VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5; 2) the VH comprises the amino acid sequence of SEQ ID NO: 9, and the VL comprises the amino acid sequence of SEQ ID NO: 13; 3) the VH comprises the amino acid sequence of SEQ ID NO: 17, and the VL comprises the amino acid sequence of SEQ ID NO: 21; 4) the VH comprises the amino acid sequence of SEQ ID NO: 25, and the VL comprises the amino acid sequence of SEQ ID NO: 29; 5) the VH comprises the amino acid sequence of SEQ ID NO: 33, and the VL comprises the amino acid sequence of SEQ ID NO: 37; 6) the VH comprises the amino acid sequence of SEQ ID NO: 41, and the VL comprises the amino acid sequence of SEQ ID NO: 45; 7) the VH comprises the amino acid sequence of SEQ ID NO: 49, and the VL comprises the amino acid sequence of SEQ ID NO: 53; 8) the VH comprises the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO: 61; 9) the VH comprises the amino acid sequence of SEQ ID NO: 65, and the VL comprises the amino acid sequence of SEQ ID NO: 69; or 10) The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the VH comprises the amino acid sequence of SEQ ID NO: 73, and the VL comprises the amino acid sequence of SEQ ID NO:

77.

6. EC20 of binding of the isolated antibody or antigen-binding fragment thereof to cells expressing B7-H3. ５０ is 10 －７ M ~ about 10 －１２ M, preferably about 10 －８ M ~ about 10 －１２ M, more preferably about 10 －９ M ~ about 10 －１２ The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein M is

7. The binding of the isolated antibody or antigen-binding fragment thereof to cells expressing B7-H3 is lower than that of the reference antibody MGA271. ５０ The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, having the following structure:

8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

9. 9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the humanized antibody comprises a VH and a VL, the VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-87, and the VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-91.

10. The humanized antibody comprises: 1) a VH comprising the amino acid sequence of SEQ ID NO: 81 and a VL comprising the amino acid sequence of SEQ ID NO: 88; 2) VH comprising the amino acid sequence of SEQ ID NO: 82 and VL comprising the amino acid sequence of SEQ ID NO: 88; 3) VH comprising the amino acid sequence of SEQ ID NO: 83 and VL comprising the amino acid sequence of SEQ ID NO: 89; 4) VH comprising the amino acid sequence of SEQ ID NO: 84 and VL comprising the amino acid sequence of SEQ ID NO: 89; 5) VH comprising the amino acid sequence of SEQ ID NO: 85 and VL comprising the amino acid sequence of SEQ ID NO: 90; 6) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 91; or 7) The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO:

91.

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the VH is fused to an immunoglobulin heavy chain constant region.

12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the heavy chain constant region is from human IgG1.

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the heavy chain constant region comprises a modification that enhances the ADCC effect of the antibody.

14. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the heavy chain constant region comprises the mutations K214R, L235V, F243L, R292P, Y300L, D356E, L358M and P396L.

15. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 93 or 94.

16. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the VL is fused to an immunoglobulin light chain constant region (CL).

17. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

92.

18. 18. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 17, wherein the isolated antibody or antigen-binding fragment thereof is conjugated to a toxin or a chemotherapeutic agent.

19. 19. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 18, wherein the toxin is Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribosomal toxin, saporin, calicheamicin, or botulinum toxin.

20. 20. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 19, wherein the chemotherapeutic agent is monomethylauristatin E or a maytansinoid.

21. A bispecific antibody comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 20 and a second antibody portion.

22. The bispecific antibody of claim 21 , wherein the second antibody portion is capable of specifically binding to an antigen other than B7-H3.

23. The bispecific antibody of claim 21 or 22, wherein the antigen other than B7-H3 is CTLA-4, PD-L1, TIM-3 or LAG-3.

24. The second antibody portion may be a Fab, a Fab', a (Fab') ２ 24. The bispecific antibody of any one of claims 21 to 23, which is an Fv, a single chain Fv (scFv), a scFv-scFv, a minibody, a diabody, a sdAb or an antibody mimetic.

25. A pharmaceutical composition for treating cancer, comprising: An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, or a bispecific antibody according to any one of claims 21 to 24, and A pharmaceutical composition comprising a pharma- ceutically acceptable carrier agent.

26. 26. The pharmaceutical composition of claim 25, further comprising one or more other anti-cancer agents.

27. The pharmaceutical composition according to claim 25 or 26, wherein the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

28. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 20, the bispecific antibody of any one of claims 21 to 24, or the pharmaceutical composition of any one of claims 25 to 27.

29. The method of claim 28, wherein the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

30. 25. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 20 or the bispecific antibody of any one of claims 21 to 24 in the manufacture of a medicament for treating cancer.

31. The use according to claim 30, wherein the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

32. 28. Use of an isolated antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 20, a bispecific antibody according to any one of claims 21 to 24 or a pharmaceutical composition according to any one of claims 25 to 27 in the treatment of cancer in a subject in need thereof.

33. The isolated antibody or antigen-binding fragment thereof, bispecific antibody or pharmaceutical composition for use according to claim 32, wherein the cancer is a B7-H3 expressing cancer, preferably NSCLC, HNSCC or breast cancer.

34. 34. The isolated antibody or antigen-binding fragment thereof, bispecific antibody or pharmaceutical composition for use according to claim 32 or 33, wherein the isolated antibody or antigen-binding fragment thereof, bispecific antibody or pharmaceutical composition is for systemic or local administration.

35. 35. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to any one of claims 32 to 34, wherein the isolated antibody or antigen-binding fragment, the bispecific antibody or the pharmaceutical composition is for intravenous or intratumoral administration.

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